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Regulation of mitochondrial bioenergetics via PTEN (phosphatase and tensin homolog deleted on chromosome 10)/estrogen-related receptor alpha (ERRα) signaling
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Regulation of mitochondrial bioenergetics via PTEN (phosphatase and tensin homolog deleted on chromosome 10)/estrogen-related receptor alpha (ERRα) signaling
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Regulation of Mitochondrial Bioenergetics via PTEN (Phosphatase and
tensin homolog deleted on chromosome 10)/Estrogen-related Receptor alpha
(ERRα) Signaling
By YANG LI
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
October 2014
Copyright 2014 YANG LI
i
Acknowledgement
First and foremost, I want to thank my advisor, Dr. Bangyan Stiles. She is a great
scientist with enthusiasm for her research, and also a wonderful mentor. During my Ph.D.
time, Bangyan not only provided me an incredible research environment, but also helped
me develop critical thinking and scientific writing skills. I appreciate all her contributions
of ideas, guidance and funding to make this study possible.
I am also thankful for the tremendous support from my committee members. Dr.
Enrique Cadenas and Dr. Neil Kaplowitz helped my research with their expertise in the
field of mitochondria and liver disease. Dr. Bogdan Olenyuk significantly contributed to
my polyamide study.
I would like to thank my previous and current colleagues for their constant
support, especially Dr. Ni Zeng, Dr. Jennifer Bayan, Dr. Vivian Medina, Dr. Lina He, Dr.
Indra Mahajan, Dr. Chengyou Jia, Zhechu Peng, Ankeste Kassa, Richa Aggarwal, Jingyu
Chen, Joshua Chen, Yating Guo, Melissa Chung and Kevin Hua.
Finally, I dedicate this dissertation to my beloved parents, Yiru Li and Huimin
Geng, sister, Fan Li and wife, Fan Ding.
ii
Table of Contents
Acknowledgement ............................................................................................................... i
Table of Contents ................................................................................................................ ii
List of Figures .................................................................................................................... iii
Abstract ............................................................................................................................... v
Chapter I. Overviews of transcriptional network for mitochondrial biogenesis and PTEN
signaling pathway ............................................................................................................... 1
I-1: Transcriptional network mediating mitochondrial biogenesis ................................. 1
1.1 Transcriptional Factors ......................................................................................... 2
1.2 Transcriptional Coactivators ................................................................................. 8
I-2: Mitochondrial alterations in cancer ........................................................................ 10
I-3: Introduction of PTEN/PI3K/AKT pathway and its role in cancer ......................... 11
I-4: Rationale of the study............................................................................................. 14
Chapter II: PTEN signaling regulates mitochondrial biogenesis and respiration via
estrogen-related receptor alpha (ERRα) ........................................................................... 17
II-1: Introduction ........................................................................................................... 17
II-2: Results ................................................................................................................... 18
II-3: Discussion ............................................................................................................. 50
II-4 Methods and Materials ........................................................................................... 57
Chapter III: Targeting estrogen-related receptor alpha (ERRα) with pyrrole-imidazole
polyamide in PTEN loss-induced fatty liver and tumorigenesis ...................................... 61
III-1: Introduction ......................................................................................................... 61
III-2: Results ................................................................................................................. 63
III-3: Discussion............................................................................................................ 80
III-4 Methods and Materials ......................................................................................... 85
Chapter IV: Distinct roles of AKT1 and AKT2 in the regulation of hepatic mitochondrial
metabolism. ....................................................................................................................... 89
IV-1: Introduction ......................................................................................................... 89
IV-2: Results ................................................................................................................. 91
IV-3 Discussion ............................................................................................................ 95
IV-4 Methods and Materials ......................................................................................... 97
Chapter V: Overall Discussion ......................................................................................... 99
Chapter VI: Reference .................................................................................................... 107
iii
List of Figures
Figure II-1. Pten deletion leads to increased reactive oxygen species (ROS) production
and higher oxidative stress condition in murine liver and hepatocytes. ........................... 20
Figure II-2. Increased mitochondrial respiration and glycolysis rate in Pten-deficient
hepatocytes. ....................................................................................................................... 23
Figure II-3. PTEN regulates mitochondrial respiration. ................................................... 25
Figure II-4. PI3K/AKT signal regulates mitochondrial respiration and glycolysis. ......... 27
Figure II-5. PTEN/PI3K signal controls mitochondrial mass. .......................................... 30
Figure II-6. PTEN signal regulates mitochondrial function via ERRα. ............................ 34
Figure II-7. PTEN regulates ERRα expression through PI3K/AKT signaling pathway. . 37
Figure II-8. CREB mediates ERRα regulation downstream of PI3K/AKT signaling. ..... 41
Figure II-9. CREB serves as a putative AKT substrate and regulates ERRα level
independent of protein kinase A (PKA)............................................................................ 44
Figure II-10. ERRα regulates ROS production. ................................................................ 46
Figure II-11. ERRα knockdown attenuates colony forming potential in Pten-null
hepatocytes, and PTEN level is negatively correlated with ERRα level in samples from
patients with liver cancer. ................................................................................................. 49
Figure III-1. Pten deletion is associated with elevated ERRα expression in liver and
prostate cancer cell lines. .................................................................................................. 64
Figure III-2. ERRα knockdown reduces cytochrome c expression. ................................. 65
Figure III-3. ERRα knockdown decreases mitochondrial function. ................................. 67
Figure III-4. ERRα knockdown impedes tumor cell proliferation. ................................... 68
iv
Figure III-5. Polyamide targeted to ERRE inhibits ERRα’s transcriptional activity,
mitochondrial function and cell proliferation. .................................................................. 72
Figure III-6. Inhibition of ERRα’ activity by PA prevents fatty liver development induced
by Pten loss. ...................................................................................................................... 75
Figure III-7. ERRα inhibition by PA exhibits anti-tumor activity in xenograft. .............. 77
Figure III-8. Expressions of PTEN and ERRα is negatively correlated in cancer patients
and ERRα level is higher in cancer tissue versus normal counterparts. ........................... 79
Figure IV-1. AKT2 is responsible for up-regulations of ERRα and PGC-1α upon Pten
loss. ................................................................................................................................... 92
Figure IV-2. Increased mtDNA content and oxygen consumption in Pten-deficient
hepatocytes are rescued by AKT2 deletion. ..................................................................... 93
Figure IV-3. Hepatic mitochondrial function did not significantly change by AKT1
deletion. ............................................................................................................................. 94
v
Abstract
Mitochondrial abnormalities are associated with cancer development, yet how
oncogenic signals affect mitochondrial function has not been fully understood. The
purpose of the current study is to investigate whether and how the oncogenic signal,
PTEN (Phosphatase and tensin homolog deleted on chromosome 10)/PI3K
(phosphoinositide 3-kinase)/AKT (Protein kinase B) pathway, might control
mitochondrial biogenesis and function.
Using a isogenic cell system established from mice carrying liver-specific
deletion of Pten, which leads to activation of PI3K/AKT, We showed that PTEN loss
leads to elevated oxidative stress, increased mitochondrial mass and augmented
respiration, accompanied by up-regulation of estrogen-related receptor α (ERRα), an
orphan nuclear receptor known for its role in mitochondrial biogenesis. Our
pharmacological and genetic studies further illustrated that the PI3K/AKT/CREB/PGC-
1α signal axis acts downstream of PTEN to regulate ERRα’s expression. Preliminary
analysis suggests that AKT may directly phosphorylate CREB and controls its activity in
a manner that is independent of PKA. Our data further demonstrated that AKT2, but not
AKT1, is responsible for regulating the expressions of ERRα and PGC-1α, establishing
isoform specific functions for AKT2. ERRα knockdown significantly attenuated
proliferation and colony forming potential in Pten-null hepatocytes as well as human
tumor cell lines where PTEN expression is dimished. Moreover, analysis of datasets from
clinical samples showed a negative correlation between expressions of E R Rα and PTEN
in multiple tumor types. Together, our data established a previously unrecognized link
between a growth signal and mitochondrial metabolism.
vi
To explore the effect of inhibiting ERRα in vivo, a class of synthetic amino acid
oligomers capable of binding to DNA minor groove with sequence specificity,
polyamide, was developed to specifically bind to the DNA sequence that ERRα needs to
bind for its function. This binding thus blocks the binding of ERRα and its
transactivation activity. We show here that inhibiting ERRα by polyamide hampered the
growth of grafted tumors. Furthermore, In vivo polyamide administration in 1.5-month-
old Pten-null mice completely blocked the fatty liver development and led to decreased
expression of key lipogenic enzymes such as Fas (fatty acid synthase) and Acc (acety-
CoA carboxylase).
In summary, data from these studies established for the first time the
PI3K/AKT/CREB/PGC-1α signaling pathway for the regulation of ERRα and
mitochondrial biogenesis. The ERRα up-regulation upon PTEN loss is important for
maintaining the mitochondrial function, ROS production and cell proliferation. As a
major hepatic AKT isoform, AKT2 is found to be responsible for the regulation of the
observed metabolic events induced by Pten loss. By adopting the novel polyamide
strategy to modulate ERRα’s transcriptional activity, we demonstrated the potential of
ERRα inhibition in targeting fatty liver and tumorigenesis.
1
Chapter I. Overviews of transcriptional network for mitochondrial
biogenesis and PTEN signaling pathway
I-1: Transcriptional network mediating mitochondrial biogenesis
Mitochondria are double-membrane subcellular organelles that contribute to many
important biological functions, including pyruvate and fatty acid oxidation, homeostatic
control of calcium signaling and intrinsic cell apoptosis (Balaban, 1990; Green & Reed,
1998; Naon & Scorrano, 2014). Most importantly, mitochondria are well known as major
sites of energy production through the electron transport chain and oxidative
phosphorylation (OXPHOS) (Hatefi, 1985). Mitochondria contain their own genetic
system, which undergoes cytoplasmic inheritance. The mitochondrial DNA (mtDNA) is a
16-kilobase circular double-stranded DNA and encodes 13 genes for essential subunits of
mitochondrial respiratory chain, as well as 22 tRNAs and 2 rRNAs required for mtDNA
transcription and translation (Clayton, 2000). The proteins specified by mitochondrial
genome only account for a small fraction of total proteins necessary for proper
mitochondrial structure and function. Therefore, the majority of protein units required for
maintaining the organelle architecture and respiratory function are specified by nuclear
genome. For example, 39 out of 46 subunits of complex I, NADH dehydrogenase, and
the entire complex II are nucleus-encoded. The nuclear genes also specify 10 out of 11
complex III subunits and 10 out of 13 complex IV subunits (Bonawitz, Clayton, &
Shadel, 2006; Clayton, 2003; Scarpulla, 2011).
The limited encoding capacity of mtDNA necessitates the nuclear origin of
regulatory factors governing mitochondrial genes’ expressions. The key transcription
2
factors for mitochondrial biogenesis can be divided into two classes. The first class
contains nucleus-encoded auxiliary factors that translocate into mitochondria and act
upon mitochondrial genome to facilitate mtDNA’s transcription and translation. The
second class includes nuclear transcription factors that act upon nuclear genome and
directly control respiratory gene expression. In addition to these two classes of
transcription factors, another family of nuclear proteins required to maintain
mitochondrial biogenesis is co-activators, which interact with transcription factors and
induces their activity.
1.1 Transcriptional Factors
Factors acting on mitochondrial genome
The mtDNA transcription is a bi-directional process that is tightly regulated by
nucleus-encoded but mitochondria-localized factors such as POLRMT (DNA-directed
mitochondrial RNA polymerase), Tfam (mitochondrial transcription factor A), TFB1M,
TFB2M (mitochondrial transcription factor B1, B2) and mTERF (mitochondrial
transcription terminator) (Scarpulla, 2011). The mitochondrial RNA polymerase,
POLRMT and the stimulatory factor that unwinds mtDNA, Tfam, are required to activate
the mtDNA transcription (Tiranti et al., 1997; Wang, Cotney, & Shadel, 2007). Two
isoforms of dissociable specificity factor, TFB1M and TFB2M, directly contact with both
POLRMT and Tfam to facilitate the transcription initiation on mtDNA promoters
(McCulloch & Shadel, 2003). High rate of mitochondrial rRNA synthesis relative to
mitochondrial mRNA is an indication of more frequent mtDNA transcription. Such
situation signals mTERF to bind to the specific termination site at the end of the 16 rRNA
and maintain the ratio of mRNA to rRNA (Daga, Micol, Hess, Aebersold, & Attardi,
3
1993). Therefore, these auxiliary factors cooperatively guide mtDNA transcription while
maintaining steady levels of mitochondrial transcripts. The homozygous knockout mice
for these nuclear genes whose products reside in mitochondria have been generated. The
Tfam, POLRMT, TFB1/2M and mTERF knockouts all resulted in embryonic lethality
together with mtDNA depletion, respiratory chain deficiency and abnormal mitochondrial
morphology, showing that these auxiliary factors control proper mitochondrial biogenesis
and function, which are strictly required during development (Larsson et al., 1998;
Metodiev et al., 2009; Park et al., 2007).
Factors acting on nuclear genome
Besides nucleus-encoded, mitochondria-localized regulators, nuclear transcription
factors and coactivators synergistically induce expressions of genes that directly
constitute the mitochondrial respiratory apparatus. In particular, important transcription
factors promoting mitochondrial biogenesis such as nuclear transcription factors 1 and 2
(NRF-1, NRF-2) and estrogen-related receptor α (ERRα), bind to promoters of
respiratory genes involved in mitochondrial function and activate their expressions.
Another protein family of nuclear co-activators, including PGC-1α/β (peroxisome
proliferator-activated receptor gamma, coactivator 1 alpha/beta) and PRC (PGC-1-related
coactivator), do not directly interact with promoters but rather bind to transcription
factors to induce their activity. The interplay between these two nuclear protein families
plays major roles in coordinating mitochondrial biogenesis.
NRF-1
The initial transcription factors implicated in the regulation of mitochondrial
genes are NRF-1 and NRF-2. NRF-1 was identified through a search for transcription
4
factors that control expressions of cytochrome c and cytochrome oxidase (Evans &
Scarpulla, 1990). NRF-1 was found to bind as a homo-dimer to a palindromic sequence
within the cytochrome c promoter and function to induce cytochrome c’s transcription.
Following studies have linked NRF-1 to expressions of a majority of genes that encode
essential subunits of respiratory complexes I to IV, as well as mitochondrial outer
membrane components such as TOMM20 and COX17, which import proteins required
for mitochondrial function (Kelly & Scarpulla, 2004; Takahashi et al., 2002; Truscott,
Brandner, & Pfanner, 2003). In addition, NRF-1 acts upon promoters of Tfam, TFB1/2M
and POLRMT, whose products, as mentioned above, predominantly regulate
mitochondrial transcription (Gleyzer, Vercauteren, & Scarpulla, 2005; Virbasius &
Scarpulla, 1994). A recent study utilizing chromatin immunoprecipitation (ChIP)
followed by microarray analysis has identified approximately 700 genes whose promoters
exhibit NRF-1 occupancy (Cam et al., 2004). NRF-1 exists in the dephosphorylated state
in serum-starved cells. Serum addition promotes phosphorylation of NRF-1 at multiple
serine sites within the amino-terminal domain and enhances its DNA-binding and
transcriptional activities (R. P. Herzig, Scacco, & Scarpulla, 2000). Homozygous NRF-1
knockout in mice resulted in embryonic lethality between E3.5 and 6.5, accompanied by
inability to maintain mitochondrial membrane potential and mtDNA copy number.
However, the early embryonic lethality by NRF-1 knockout might result from affected
expression of NRF-1 target genes involved in regulating cell cycle progression given that
NRF-1 also engages in the regulation of E2F (E2f transcription factor)-responsive genes.
NRF-2
5
The second nuclear respiratory factor, NRF-2, was identified as a transcriptional
activator of cytochrome oxidase IV and the human homolog of mouse GABP (GA-
binding protein) (Carter, Bhat, Basu, & Avadhani, 1992; LaMarco & McKnight, 1989).
NRF-2 is a multi-subunit protein that shares similar DNA-binding domain and trans-
activation domain with NRF-1 (Gugneja, Virbasius, & Scarpulla, 1995). Subsequent
chromatin immunoprecipitation studies revealed that NRF-2 is associated with 10
cytochrome oxidase subunits’ promoters and positively regulates cytochrome oxidase
complex expression (Ongwijitwat & Wong-Riley, 2005). Although the respiratory genes
initially defined as NRF-2 targets do not overlap with NRF-1 targets, a number of
mitochondrial genes are found to utilize both NRFs to activate their expressions. For
example, transcriptions of human mitochondrial cytochrome oxidase subunit V and entire
complex II subunits highly depend on promoter occupancy by both NRF-1 and NRF-2
(Au & Scheffler, 1998; Virbasius, Virbasius, & Scarpulla, 1993). Moreover, functional
NRF-2 sites have been identified on promoters of mitochondria-related factors, including
Tfam, TFB1M and TFB2M (Gleyzer et al., 2005; Larsson et al., 1998). Therefore, like
NRF-1, NRF-2 coordinates the expressions of key respiratory chain components, as well
as mitochondrial transcription machinery.
ERRα
Estrogen-related receptor alpha (ERRα), a member of the orphan nuclear receptor
family, is abundantly expressed in high oxidative organs such as liver, and recently has
been recognized as key regulator of adaptive energy metabolism (Villena & Kralli, 2008).
ERRα was first identified using the DNA-binding domain (DBD) of estrogen receptor α
(ERα) as a screen probe (Giguere, Yang, Segui, & Evans, 1988). Subsequent sequence
6
analysis showed that ERRα shares about 68% homology in DBD and 33% homology in
ligand-binding domain (LBD) with estrogen receptor α (ERα), which might explain the
divergence in ligand binding nature between these two receptors (Stein & McDonnell,
2006).
ERRα, by itself, is a weak transcriptional factor that preferentially binds to a
consensus DNA sequences 5’-TCAAGGTCA-3’, termed estrogen-related response
element (ERRE) (Sladek, Bader, & Giguere, 1997). However, both of its activity and
expression are significantly increased when it interacts with a nuclear co-activator, PGC-
1α. The PGC-1/ERRα complex has been shown to regulate a variety of metabolic
processes taking place in mitochondria such as TCA cycle, fatty acid oxidation and
mitochondrial OXPHOS. Studies using ERRα-null embryonic fibroblasts or tumor cells
with ERRα knockdown showed that expressions of genes encoding mitochondrial
protein, mtDNA level and TCA enzyme activity were significantly decreased in response
to reduced ERRα level (Rangwala et al., 2007). A genome-wide analysis for genomic
locations of transcription factor occupancy revealed that ERRα is bound to promoters of
195 genes, a majority of which is involved in OXPHOS and TCA cycle as reported by
previous studies, including cytochrome c (Cyt c), ATP synthase beta (Atp5b), fumarate
hydratase (Fh1) and succinate dehydrogenase (Sdha). Another subset of promoters
occupied by ERRα drive expressions of genes functioning in fatty acid oxidation,
including Acadm, Slc25a29 (palmitoylcarnitine transporter) and FABP3 (fatty acid
binding protein 3) (Dufour et al., 2007).
Interestingly, NRF-2 was found to be one of the identified genes with ERRα
occupancy at their promoters. Moreover, a recent study discovered that NRF-1 also acts
7
downstream of PGC-1α/ERRα complex in cultured muscle and liver cells (Mootha et al.,
2004). Given the fact that overexpression of NRF-1 itself in muscle cells is not sufficient
to promote mitochondrial biogenesis, ERRα seems to function at upstream of NRF-1 and
NRF-2, which are crucial but relatively late mediators of mitochondrial transcription
paradigm (Baar et al., 2003). Therefore, ERRα predominantly orchestrates mitochondrial
transcription by either directly activating genes composing mitochondrial structure or
indirectly inducing major transcription factors controlling mitochondrial generation.
The in vivo function of ERRα has been comprehensively elaborated by the study
using ERRα knockout mice. Under cold exposure, ERRα knockouts failed to initiate the
adaptive thermogenesis, a process to cope with cold environment, because of reduced
mitochondrial mass together with decreased expression of respiratory chain components
and reduced mitochondrial DNA copy number (Villena et al., 2007). Such phenotypes are
well expected given the dominant role of ERRα in the energy metabolism within
mitochondria. Strikingly, the homozygous Err α-null mice are also found to be fertile and
viable with reduced fat mass in peripheral organs and resistance to high-fat diet-induced
obesity, demonstrating that ERRα controls the lipid homeostasis (Luo et al., 2003).
Recently, several clinical oncology studies showed that the elevated ERRα expression
was significantly associated with unfavorable clinical outcome, hormonal insensitivity
and increased risk of recurrence in human breast cancer, and ERRα might serve as a
potent predictive biomarker of cancer therapy (Ariazi, Clark, & Mertz, 2002; T. Suzuki et
al., 2004).
8
1.2 Transcriptional Coactivators
Transcriptional coactivators are multifunctional nuclear proteins that act upon the
DNA-binding transcription factors to induce their activities and downstream target
expression. The major breakthrough in ERRα regulation research came with the
discovery of PGC-1α, which is a member of a transcriptional coactivator family that also
includes PGC-1β and PGC-1 related coactivator (PRC). The interaction between ERRα
and PGC-1α is mediated by the N-terminal activation domain near leucine-rich LXXLL
motifs on PGC-1α (Scarpulla, 2011). Forced overexpression of PGC-1α not only
potentiates ERRα’s transcriptional activity but also induces its mRNA and protein level
(Mootha et al., 2004). Moreover, PGC-1α is capable of inducing mitochondrial
biogenesis, respiratory capacity and fatty acid oxidation, supported by results using both
cultured cells overexpressing PGC-1α and tissue-conditional PGC-1α transgenic mice
(Puigserver et al., 1998; Russell et al., 2004). Subsequent studies revealed that PGC-1α
mediates such effects via direct interaction with, and activation of, ERRs, NRF-1 and
NRF-2, which are key transcription factors promoting mitochondrial biogenesis as
discussed above (Finck & Kelly, 2006).
Even though the germline PGC-1α deletion in mice exhibits normal mitochondrial
volume and morphology in liver and fat tissues, the PGC-1α knockout mice show
inability to initiate the adaptive thermogenesis and to increase mitochondrial mass and
function in response to physiological stimuli such as cold exposure and exercise (Leone
et al., 2005; Lin et al., 2004). Therefore, even though PGC-1α is not strictly required for
development and maintenance of mitochondria, it serves as a central coordinator to
optimize mitochondrial homeostasis under the stimulation of physiological stress. PGC-
1β, possessing high homology with PGC-1α, has been shown to interact with the same
9
nuclear factors as PGC-1α and therefore, shares a significant amount of downstream
target genes (Lin, Puigserver, Donovan, Tarr, & Spiegelman, 2002).
Similar with PGC-1α-null mice, PGC-1β knockouts showed normal energy
expenditure and respiratory capacity, but mitochondrial dysfunction when challenged by
stress (Sonoda, Mehl, Chong, Nofsinger, & Evans, 2007; Vianna et al., 2006). Expression
of PGC-1α is induced in the brown adipose tissue of PGC-1β knockout mice and this
compensatory induction of PGC-1α failed to rescue the mitochondrial dysfunction in
PGC-1β knockout mice, suggesting there are some extent of non-redundant functions
between these two isoforms. In sharp contrast to individual knockouts, PGC-1α/β double
knockouts died within 24 hours after birth due to cardiac failure, arguing that these two
coactivators play important roles in postnatal cardiac function (Lai et al., 2008).
The third PGC-1 family member, PRC was identified in a search for similarities
to PGC-1α. Molecular cloning of PRC showed similarities with the other two PGC-1
members in several domains, including amino-terminal domain, the LXXLL nuclear
receptor-binding motif, RNA recognition motif and a carboxyl-terminal RS (Ser-Arg)
domain (Andersson & Scarpulla, 2001). Besides binding to, and activate, mitochondria-
associated transcription factors that overlap with PGC-1α/β such as NRF-1 and ERRα,
PRC plays an essential role in early embryonic development upon growth factor
stimulation as the PRC expression peaks during the first day of embryonic body
formation and the germline PRC deletion result in peri-implantation lethality (X. He et
al., 2012; Vercauteren, Gleyzer, & Scarpulla, 2008; Vercauteren, Pasko, Gleyzer,
Marino, & Scarpulla, 2006).
10
I-2: Mitochondrial alterations in cancer
Otto Warburg observed that tumor cells consume a large quantity of glucose and
prefer glycolysis over mitochondrial OXPHOS even in the presence of ample oxygen, a
phenomenon termed “Warburg effect” or “aerobic glycolysis” (Warburg, 1956). This
observation led to the assumption that dysfunction of the mitochondrial respiratory chain
might be the cause of such unique metabolic feature. Later, mitochondrial abnormalities
have been frequently reported to associate with development and progression of cancer
(Pedersen, 1978). These defects include altered expression of mitochondrial respiratory
complexes, mtDNA mutations, abnormal reactive oxygen species (ROS) production, as
well as increased or decreased mitochondrial numbers. The first identified mtDNA
mutation that has functional significance in cancer was the deletion of 294 nucleotides in
NADH dehydrogenase-1 (ND1), a subunit of complex I, in renal adenocarcinoma
patients (Horton et al., 1996). This mutation led to decreased complex I activity,
associated with high metastatic potential. Low expressions of complex III and complex
IV were also reported in different cancer types afterwards (Cavelier et al., 1995;
Dasgupta, Hoque, Upadhyay, & Sidransky, 2008). Since the mitochondrial electron
transport chain (ETC) components, particularly complex I and complex III, are the major
sites for ROS generation, decreased expression or reduced activity of ETC complexes
often results in increased ROS production (Gasparre et al., 2008). ROS is proposed to
induce oncogenic transformation and promote tumor progression and metastasis (Zager,
Johnson, Hanson, & Lund, 2006).
Despite the strong association between mitochondrial defects and cancer, the
current challenge is to determine whether mutated mitochondrial genome contribute to
tumor progression. A study overexpressed a mutated mitochondrial cytochrome b with a
11
21-bp deletion in a mouse carcinoma cell line and showed that the overexpression led to
increased ROS production, increased cell growth and accelerated xenografted tumor
formation (Dasgupta et al., 2008). Interestingly, the mutated cytochrome b
overexpression simultaneously resulted in both elevated glycolysis and oxygen
consumption, which together with recent clinical reports showing mitochondrial function
remain intact or even enhanced in many cancer cells, might refute the hypothesis that the
respiratory defects confer the glycolytic metabolism in tumor cells (Lynam-Lennon et al.,
2014; Pasto et al., 2014a). Furthermore, increased mtDNA copy numbers have been
reported in various tumor types, including head and neck squamous cell carcinoma (Kim
et al., 2004), thyroid carcinoma (Rogounovitch et al., 2002), lung cancer (Bonner et al.,
2009) and liver cancer (Vivekanandan, Daniel, Yeh, & Torbenson, 2010). However,
decreased mtDNA copy number has also been reported in a number of cancer cases
(Mambo et al., 2005). Whether the change in mtDNA copy number is an adaptive
response to mitochondrial dysfunction or is due to dys-regulated nuclear transcriptional
machinery requires further investigation.
I-3: Introduction of PTEN/PI3K/AKT pathway and its role in cancer
Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate a variety of
cellular processes including growth, proliferation, differentiation, survival and motility ,
as well as cellular glucose and lipid metabolism (Vivanco & Sawyers, 2002). PI3K
catalyzes the reaction from Ptdlns (4,5) P2 to Ptdlns (3,4,5) P3. It achieves this task by
phosphorylating the hydroxyl group of the 3
rd
position on the inositol ring of
Phosphatidylinositols (Ptdlns) (Yuan & Cantley, 2008). PI3K is activated in response to
growth factor binding to their receptors. Such binding induces tyrosine phosphorylation
12
on the receptor tyrosine kinases (RTK) as well as downstream scaffolding proteins. The
phospho-tyrosine residues on the receptors or scaffolding proteins, for example, insulin
receptor substrate-1 (IRS-1), then serve as docking site to recruit and activate PI3K. The
regulatory subunit of PI3K, e.g. p85, contains a src-homology (SH2) domain that allows
the PI3K to bind the phospho-tyrosine motif pYXXM on RTK or IRS (Yonezawa et al.,
1992). In the absence of stimulation, PI3K remains in resting status in the cytoplasm as
the inactive p85-p110 complex. In response to signals such as ligand-mediated RTK
activation, the SH2 domain of p85 interacts with consensus phospho-tyrosine residues on
RTK or IRS. This interaction brings PI3K to the cell membrane where the lipid substrates
are located. Moreover, it is believed that the interaction between p85 and RTK or IRS
releases the inhibitory effect of p85 on p110, leading to the induction of PI3K’s catalytic
activity (Yu et al., 1998).
The action of PI3K leads to activation of a number of molecules that contain the
pleckstrin-homology (PH) domain, which is a lipid-binding domain involved in recruiting
proteins to the inner surface of plasma membrane (DiNitto, Cronin, & Lambright, 2003).
One primary effect of PI3K action is activation of AKT, a serine/threonine kinase also
known as Protein Kinase B (PKB). AKT harbors a PH domain at the amino terminal
(Matheny & Adamo, 2009). Following PI3K activation, accumulation of PIP
3
allows
recruitment of AKT via direct interaction with its PH domain (Vivanco & Sawyers,
2002). Meanwhile, another PH domain-containing kinase 3-phosphoinositide-dependent
protein kinase 1 (PDK1) also interacts with PIP
3
and translocates to cell membrane,
where it phosphorylates AKT at Thr308 (Vanhaesebroeck & Alessi, 2000). It has been
shown that AKT is further phosphorylated by mTOR2 at Ser473 (Hresko & Mueckler,
13
2005; Knowles, Platt, Ross, & Hurst, 2009). Phosphorylation on Ser473 is important for
initial activation of AKT whereas Thr308 phosphorylation by PDK1 is required for
maximal AKT activation (Alessi et al., 1997). Activated AKT has a plethora of
downstream functions including the regulations of kinases such as GSK3β (Pap &
Cooper, 1998), IκB kinases (IKKα and IKKβ) (Gustin et al., 2004), apoptotic factors such
as BAD (Datta et al., 1997), MDM2, a ubiquitin ligase for p53 (Ashcroft et al., 2002);
GTPases like Rac and Rho (Liliental et al., 2000); cell cycle inhibitors p21 and p27
(Collado et al., 2000); and transcription factors such as forkhead transcription family
(FoxO) members (Biggs, Meisenhelder, Hunter, Cavenee, & Arden, 1999; Brunet et al.,
1999; Guo et al., 1999).
PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) is
encoded on chromosome 10q23, a region where loss of heterozygosity frequently occurs.
The protein encoded by the PTEN gene is a 403-amino acid protein whose amino-
terminal region shares sequence homology with tensin and auxilin (Stiles, Groszer,
Wang, Jiao, & Wu, 2004)
(D. M. Li & Sun, 1997). Crystal structure analysis for PTEN
has revealed a C2 domain that retains affinity for phospho-lipids on plasma membrane
and a phosphatase domain that displays dual specificity towards both protein and lipid
substrates in vitro (J. O. Lee et al., 1999). As the PIP
3
phosphatase, PTEN antagonizes
the function of PI3K by removing the phosphate from the 3
rd
position on PIP3, leading to
reduced PIP
3
production and the down-regulation of signals downstream of PI3K.
Hyper-activation of PI3K signaling cascade is one of the most frequent events
associated with human cancers. Most of the PI3K hyper-activation cases identified in
tumors are resulted from mutations of the PI3K gene or other genes downstream of PI3K,
14
such as AKT kinases. In addition, mutation, silencing, aberrant transcripts and allelic
imbalance of its negative regulator PTEN (phosphatase and tensin homologue deleted on
chromosome 10) also commonly occur in cancers (Vivanco & Sawyers, 2002). Studies
over the last two decades have poised PI3K and its downstream targets as promising drug
targets for cancer therapy.
I-4: Rationale of the study
As the vital organ for metabolism, liver executes essential functions in the body,
including lipid metabolism, hormone production and detoxification. Liver can suffer from
increased lipid accumulation induced by high fat and calorie diets. This hepatic lipid
accretion can further progress to steatosis and nonalcoholic steatohepatitis (NASH). Even
though the etiology requires further elaboration, emerging evidences suggest
mitochondrial dysfunction plays a pivotal role in the NASH. The mitochondrial defects
not only disrupts lipid metabolic homeostasis but also results in augmented ROS
production, which in an environment enriched with lipid, might trigger lipid peroxidation
and impose substantial oxidative stress on the hepatocytes. These hepatic events that are
associated with mitochondrial dysfunction might also increase the risk of cancer.
Hepatocellular carcinoma (HCC) ranks fifth in cancer occurrence and third in cancer
mortality. The annual incidence of HCC raised about 80% during the last two decades
(Starley, Calcagno, & Harrison, 2010). NASH has been proposed as a significant risk
factor for primary liver cancer. Cohort studies have demonstrated that patients diagnosed
with fatty liver have an increased risk of liver cancer (Sorensen et al., 2003). My
dissertation work focuses on how oncogenic signaling, specifically PI3K/AKT pathway,
controls mitochondrial function during cancer development using liver as a model. Even
15
though dys-regulated growth factor signaling that facilitates tumor cell growth has been
linked to altered mitochondrial function and ROS production, the intricate molecular
mechanisms still remain unknown. To study these questions, I adopted the mouse model,
whose PI3K pathway activity in the liver is genetically induced by hepatic Pten deletion.
Our laboratory utilized the Cre-loxP system to generate the liver-specific Pten
knockout mice model. By breeding the Pten
loxp/loxp
mice with mice expressing Cre driven
by albumin promoter, we were able to introduce conditional Pten deletion in hepatocytes.
Further analysis using this model revealed that Pten deletion in liver resulted in lipid
accumulation and steatosis, followed by tumor development, which closely resembles
progression of liver diseases in human. PTEN mutation is frequently associated with liver
cancer. Moreover, preliminary results indicated that Pten loss is associated with a highly
condensed mitochondrial distribution and increased mitochondrial number, which is
further confirmed by specific mitochondria staining in hepatocytes. Therefore, the hepatic
Pten deletion mouse model provides a valuable tool to study my central hypothesis:
PTEN and PI3K/AKT pathway regulate metabolism by modulating mitochondrial
biogenesis and respiration.
In Chapter II, I investigated the roles of PI3K signaling in the regulation of
mitochondrial function and provided the molecular mechanisms for the Pten-regulated
mitochondrial alteration. Chapter II also demonstrated that the altered mitochondrial
function is important for maintaining cell proliferation and tumorigenecity. In Chapter III,
I developed therapeutics targeting the molecular signals that I discovered in chapter I and
tested its effects on tumor and fatty liver in the Pten-null model. In Chapter IV, I
16
investigated the distinct roles of different AKT isoforms in the regulation of Pten-
mediated metabolic changes.
17
Chapter II: PTEN signaling regulates mitochondrial biogenesis and
respiration via estrogen-related receptor alpha (ERRα)
II-1: Introduction
A hallmark of tumor cell metabolism is the elevation of glycolysis rate even in the
presence of oxygen (Warburg, 1956). While this increase is not always coupled with a
decrease in mitochondrial respiratory function, clinical studies have reported altered
mitochondrial function and copy numbers in human cancers (Carew & Huang, 2002).
Indeed, mitochondrial function is crucial for the transformation of tumor cells (Jones &
Thompson, 2009). Most tumor cells maintain functional mitochondria that are able to
process oxidative phosphorylation (Ward & Thompson, 2012). Enhanced mitochondrial
function is also not uncommon in tumors (Fogal et al., 2010; Funes et al., 2007;
Weinberg et al., 2010). Particularly, recent evidence using immunohistochemistry and
laser dissection suggests that tumor cells are highly dependent on mitochondrial
respiration while the surrounding stroma often rely on glycolytic respiration with low
expression of mitochondrial genes (Ertel et al., 2012; Sotgia et al., 2012). One
consequence of the enhanced mitochondrial respiration is build-up of reactive oxygen
species (ROS), the byproduct of mitochondrial respiration and oxidative phosphorylation.
By acting as both mutagens and mitogens, ROS is capable of inducing oncogenic
transformation and promoting carcinogenesis and proposed to play major roles in tumor
cell transformation, progression and metastasis (Zager et al., 2006). Growth factor and
mitogenic signals that promote cancer cell growth and survival have been linked to
altered mitochondrial functions and generation of ROS (Abid et al., 2013; Bellavia et al.,
2012; Ilatovskaya, Pavlov, Levchenko, & Staruschenko, 2013; Martel-Gallegos et al.,
18
2013; Xi, Shen, Wai, & Clemmons, 2013; Zamkova, Khromova, Kopnin, & Kopnin,
2013). However, the molecular mechanism of whether and how the aberrant growth
factor/mitogenic signals in tumor cells may control mitochondrial mass and respiration
still remains unclear.
In this study, we investigated the role of PI3K signals in the regulation of
mitochondrial function. We have previously shown that mice lacking PTEN (phosphatase
and tensin homolog deleted on chromosome 10), the negative regulator of PI3K, develop
liver steatosis and cancer (Galicia et al., 2010; Stiles, Wang, et al., 2004); both of these
diseases have been reported to associate with mitochondrial alterations (Watanabe,
Yaginuma, Ikejima, & Miyazaki, 2008). High oxidative stress conditions also
accompanied the steatosis and cancer phenotypes in this model. Here, we investigated
whether mitochondrial functional regulation by PI3K/PTEN signal contributed to the
oxidative stress conditions and tumor transformation phenotypes. We also investigated
the molecular mechanism for the PTEN-regulated mitochondrial functional alteration.
Together, our study established a novel PTEN/PI3K/AKT-pCREB-PGC-
1alpha/ERRalpha signal node for the regulation of mitochondrial mass and function, and
revealed ERRalpha as a regulator of ROS production in Pten-null hepatocytes.
II-2: Results
II-2-1: Levels of reactive oxygen species in hepatocytes are associated with PTEN
status
Reactive oxygen species (ROS) are produced during various cellular processes
and serve distinct biological functions (Thannickal & Fanburg, 2000). Depending on the
levels, ROS has been proposed to promote cell growth and induce cell death (Simon, Haj-
19
Yehia, & Levi-Schaffer, 2000). In mouse models where Pten is specifically deleted in the
liver, we observed increased oxidative stress conditions, indicated as elevated H
2
O
2
level
and trans-4-hydroxy-2-nonenal (4-HNE) production (Galicia et al., 2010). The expression
patterns of GPx and GST differed likely due to the presence of different oxidative stress
triggers at each stage of the phenotype development (Scandalios, 2005). Levels of the
oxidize form of glutathione (GSSG) in the Pten null livers (0.52+0.02 millimolar/g) are
two fold higher than that observed in the control livers (0.26+0.008 millimolar/g) at 18
weeks, further confirming the presence of oxidative stress conditions. In addition,
activity of thioredoxin reductase is also increased (123.4+10.3 vs. 91.8+11.2
millimolar/min/mg, n=4) in the Pten null livers, further indicate higher oxidative stress
conditions. To determine whether the production of ROS is a direct consequence of
PTEN loss or secondary due to other cellular process changes occurring in the liver, we
down-regulated PTEN in immortalized hepatocytes (Zeng et al., 2011) established from
the control mice. Inhibition of PTEN with two independent shRNAs led to increased
ROS production in the hepatocytes (Figure II-1 C), indicating that PTEN status is a
determining factor for ROS levels.
20
Figure II-1. Pten deletion leads to increased reactive oxygen species (ROS)
production and higher oxidative stress in murine liver and hepatocytes.
A and B, Total RNA was extracted from Pten-null (Pm) and control (Con) mice livers.
Quantitative PCR (QPCR) analysis reveals higher expression of glutathione peroxidase
(GPx) and glutathione-S- transferase (GST) in Pm than Con at different ages (6M, 9M,
12M and 15M). Values are expressed as fold change vs. controls (set as 1). *P≤ 0.05,
different from controls (Con) of the same age. n=5. C, PTEN knockdown by shRNA
induces ROS production. Con hepatocytes were transfected with shPTEN or shScramble
and stained with H2DCF for ROS indication. Fluorescence-activated cell sorting (FACS)
was carried out to assess the fluorescence intensity. Left panel: FACS analysis of ROS
production in Con cells with shPTEN or shScramble transfection. Right panel:
Quantification of average fluorescence intensity. * P≤ 0.05, different from shScramble.
n=3.
II-2-2: PTEN and PI3K/AKT signal regulate mitochondrial respiration and
glycolysis in hepatocytes
A. B.
C.
Reactive Oxygen Species (ROS)
Staining
GST
GPx
Fold Change mRNA
0
4
8
12
16
*
*
*
*
6M 9M 12M 15M
Fold Change mRNA
Con
Pm
0
4
8
12
*
*
*
6M 9M 12M 15M
*
0
1000
2000
3000
shScramble shPTEN
Florescence Intensity
(Arbitrary Unit)
shScramble
shPTEN
Florescence Intensity
Events
Con
Pm
21
The major source of ROS in the cells is the mitochondria, which are the primary
site of aerobic respiration. To study whether the functions of mitochondria are affected by
PTEN status, we determined the cellular respiration rate (oxygen consumption rate,
OCR) in primary (Figure II-2 A-B) and immortalized hepatocytes (Figure II-2 C-D) from
livers of control (Con) and Pten-null mice (Pm). Primary hepatocytes from Pten-null
mice exhibited remarkably elevated OCR rates. Both basal and maximal OCRs were
elevated when Pten is deleted. Maximal OCR was determined by the addition of p-
trifluoromethoxy carbonyl cyanide phenyl hydrazone [FCCP], used to uncouple
respiration from ATP production. Approximately 2-fold increase in the basal OCR and a
30% elevation in maximal OCR were observed in Pten-null primary hepatocytes
compared to the controls (Figure II-2 A-B). Similar inductions in immortalized
hepatocytes were observed when Pten is deleted (Figure II-2 C-D), suggesting that the
immortalized hepatocytes can be used to investigate the relationship between PTEN
signal and mitochondrial respiration.
The rate of extracellular acidification (ECAR), an indicator of lactate levels in the
culture produced from glycolysis, was simultaneously determined with OCR. Oligomycin
treatment, which inhibits mitochondrial function, led to increased ECAR in Pten control
and null cells, suggesting that glycolysis and mitochondrial activities remain coupled in
response to metabolic alterations regardless of PTEN status. However, PTEN loss was
able to induce both glycolysis (ECAR) and mitochondrial respiration (OCR)
simultaneously. ECAR rate was 2-3 folds higher when PTEN was deleted in both primary
(Figure II-2 E) and immortalized (Figure II-2 F) hepatocytes at baseline and throughout
the experiment. These observations suggest that PTEN and PTEN-regulated signals may
22
control mitochondrial respiration and glycolysis rate in hepatocytes independent of their
metabolic coupling. This metabolic feature is highly similar to “Warburg effect” where
accelerated glycolysis at the presence of high oxidative phosphorylation confers tumor
cell growth advantage by supplying the necessary macromolecules for biosynthesis
(Vander Heiden, Cantley, & Thompson, 2009).
23
Figure II-2. Increased mitochondrial respiration and glycolysis rate in Pten-deficient
hepatocytes.
A, Basal (first four time points) and maximal (with FCCP addition) oxygen consumptions
(OCR) are higher in Pten-null (Pm) primary hepatocytes vs. controls (Con). OCR was
measured (Seahorse XF24 analyzer) in primary hepatocytes derived from Pten control
(Con) and null (Pm) mice livers. B, Quantification of baseline OCR in Pm and Con
primary hepatocytes. C, Basal and maximal OCR are increased in Pm immortalized
24
hepatocytes. OCR was measured in Pm and Con immortalized hepatocytes. D,
Quantification of baseline OCR in Pm and Con immortalized hepatocytes. E and F,
Extracellular acidification rate (ECAR, indication of glycolysis rate) is constantly higher
in both Pm primary and immortalized hepatocytes. ECAR was simultaneously measured
with OCR. Oligomycin (1μM), FCCP (1μM) and Rotenone (1μM) were added
sequentially after measuring basal OCR to inhibit ATP synthesis capacity, uncouple ATP
synthesis and mitochondrial respiration and completely block the respiration of
mitochondrial, respectively. *P≤ 0.05, different from controls (Con). n=5.
To confirm whether PTEN directly regulates mitochondrial function, we reduced
PTEN expression using sequence-specific shRNA in immortalized control (Con)
hepatocytes. Knockdown of PTEN resulted in increased mitochondrial respiration
compared to scrambled shRNA control (Figure II-3, left panels). Baseline OCR was
approximately 2.5 folds higher when shRNA, against PTEN, was introduced, compared
to the scrambled shRNA group (Figure II-3, right panels). Similarly, maximum OCR was
also higher when PTEN was inhibited, suggesting that PTEN was responsible for
regulating mitochondrial respiration in hepatocytes.
25
Figure II-3. PTEN regulates mitochondrial respiration.
A and B, PTEN knockdown leads to both increased basal and maximal OCR in
immortalized Con hepatocytes. Con hepatocytes were transfected with shPTEN1/2 or
shScramble. After 24 h, transfected and control cells were seeded for OCR measurement.
Left panel, OCR results in shPTEN, shScramble, and naive Con hepatocytes. Right panel
(bottom), quantification of base-line OCR value. Right panel (top), PTEN protein level is
decreased in Con hepatocytes transfected with shPTEN1/2. *, p ≤ 0.05, different from
shScramble. n=3.
To explore the signals downstream of PTEN that may be involved in regulating
mitochondrial function, we evaluated the role of PI3K, which is a major mitogenic kinase
26
mediating essential cell events such as proliferation, growth and survival (Stiles, Groszer,
et al., 2004). Immortalized hepatocytes were treated with Insulin-like growth factor-1
(IGF-1), a growth factor that induces PI3K signaling and LY294002, a PI3K inhibitor
capable of blocking signals downstream of PI3K, prior to OCR measurement. In control
(Con) hepatocytes, a 2-fold induction of baseline OCR was observed with 2 hour IGF-1
pre-treatment (Figure II-4 A); 4-hour pre-treatment further increased OCR (Figure II-4
A). Conversely, pre-treatment of Pten-null cells with LY294002 for 4 hours was
sufficient to reduce the OCR (Figure II-4 B, left panel). Baseline OCR rate was kept at a
constant lower level (30%) in LY294002-treated groups compared to that of vehicle-
treated ones (Figure II-4 B, right panel). Inhibition of PI3K with LY294002 reduced the
maximal OCR by 30% as well. LY294002 treatment also led to a persistent lower ECAR
level in Pten-null hepatocytes compared to vehicle-treated group (Figure II-4 C). A 2.5
folds reduction in baseline ECAR is observed with LY294002 treatment compared with
the vehicle treated cells (Figure II-4 C), confirming that both glycolysis and
mitochondrial oxygen consumption are regulated by PTEN and its downstream PI3K
signaling in the same fashion.
27
Figure II-4. PI3K/AKT signal regulates mitochondrial respiration and glycolysis.
A, IGF-1 treatment leads to increased OCR in Con immortalized hepatocytes. Con
hepatocytes were serum-starved for 24 h followed by IGF-1 treatments for 1, 2, and 4 h.
28
Cells were then subjected to OCR measurement. Left panel, representative OCR results
of Con hepatocytes starved for 24 h and treated by IGF-1 for 4 h. Right panel (top),
quantification of OCR base-line values of 24 h of starvation, 1-, 2-, and 4-h IGF-1
treatments. Right panel (bottom), P-AKT is induced by IGF-1 treatments in Con
hepatocytes. *, p≤0.05, different from time 0. n=5. B, LY294002 treatment leads to
decreased OCR in Pm immortalized hepatocytes. Pm hepatocytes were treated with
vehicle or LY294002 at 1, 2, and 4 h. Cells were then subjected to OCR measurement.
Left panel, representative OCR results of Pm hepatocytes treated with vehicle and
LY294002 for 4 h. Right panel (top), quantification of OCR base-line values of vehicle
and 1-, 2-, and 4-h LY294002 treatments. Right panel (bottom), P-AKT level was
suppressed by LY294002 treatments in Pm hepatocytes. *, p≤0.05, different from time 0.
n=5. C, left panel, LY294002 treatment leads to decreased ECAR in Pm hepatocytes.
ECAR is simultaneously measured with OCR. Right panel, quantification of base-line
ECAR. *, p ≤0.05, different from time 0. n=5.
II-2-3: PTEN/PI3K signal controls mitochondrial mass
To investigate how PTEN and PI3K signal control the mitochondrial function, we
evaluated the morphology of the mitochondria in immortalized hepatocytes.
Immortalized hepatocytes were stained using MitoTracker Red, a fluorescent dye that
specifically accumulates within respiring mitochondria thereby allowing visualization of
mitochondria (Figure II-5 A, right panel). Quantification of the stain intensity showed
that Pten-null hepatocytes exhibited increased mitochondrial intensity compared to
control cells (Figure II-5 A, left panel). The quantified ratio of fluorescent pixels versus
whole cell area pixels was 10% higher in Pten-null hepatocytes. These results suggest
that the mitochondrial mass might increase upon PTEN loss. To further confirm whether
increased MitoTracker staining intensity was due to an increase in mitochondrial mass,
we measured the expression of cytochrome c oxidase subunit II (COX II), a gene
exclusively encoded by mitochondrial genome, using a non-transcribed intron region of
globin as genomic DNA control. The ratio of COX II/Globin (intron) is commonly used
as an indicator of mitochondrial content (Cunningham et al., 2007; Isganaitis et al., 2009;
Schreiber et al., 2004). As expected, the ratio of COX II/Globin is 2-3 folds higher in
29
both Pten-null hepatocytes and liver tissues than that of their control counterparts (Figure
II-5 B), confirming an increased mitochondrial mass under Pten-null condition. To
further demonstrate that this observation occurs in vivo, we performed electron
microscopic analysis of liver sections from 1-month old mice. Mitochondria from control
livers showed a relatively sparse number of cristae whereas those from Pten-null livers
showed densely packed cristae. The mitochondria in the Pten-null liver also seemed to
have occupied a relatively greater fraction of the cytoplasm, thereby supporting the DNA
analysis results, indicating an increase in mitochondrial mass.
To investigate whether PTEN induces mitochondrial biogenesis via PI3K,
immortalized hepatocytes were treated with either LY294002 or IGF-1 followed by
staining of MitoTracker Green, a mitochondria dye of which the accumulation is less
dependent on mitochondrial membrane potential. Thus, the staining intensity reflects
mitochondrial mass. Fluorescence-activated cell sorting (FACS) analysis was employed
to evaluate whether mitochondrial density changes with these treatments. Substantially
higher (>2.5 folds) fluorescent signal was observed in control (Con) cells treated with
IGF-1 (24 hours) vs. vehicle-treated cells, indicating an increase of mitochondrial mass
upon PI3K activation (Figure II-5 C). When the Pten-null cells were treated with
LY294002 (4 hours), the fluorescent peak shifted to the left, suggesting reduced
Mitotracker staining and thus lower mitochondrial density when PI3K signal is inhibited
(Figure II-5 D). Quantification of fluorescent values showed that LY294002 treatment
results in approximately 2 folds decrease of fluorescence intensity in Pten-null vs. control
(Con) hepatocytes. Together, these data suggest that the increased mitochondrial mass
upon PTEN loss is dependent on PI3K activity.
30
Figure II-5. PTEN/PI3K signal controls mitochondrial mass.
0
0.1
0.2
0.3
0.4
Con Pm
Fluorescence Pixels/Whole Cell
Area Pixels
(Relative Mitochondrial Area)
A
*
Con Pm
B
0
1
2
3
4
Con Pm
Ratio of Expression
(COXII/Globin)
Hepatocytes
0
0.5
1
1.5
2
2.5
3
3.5
4
Con
Liver Tissue
Ratio of Expression
(COXII/Globin)
Pm
*
*
Mitochondrial Density by FACS
Con Hepatocytes + IGF-1
Unstained
IGF-Treated
Vehicle
0
1000
2000
3000
Unstained Vehicle IGF-1
Fluorescence Intensity
(Arbitrary Units)
Fluorescence Intensity
Arbitrary Units)
0
200
400
600
Unstained Vehicle LY294002
C
D
Unstained
LY-Treated
Vehicle
Mitochondrial Density by FACS
Pm Hepatocytes + LY294002
Events Events
*
*
Fluorescence Intensity
Fluorescence Intensity
31
A, Immortalized Pten-null (Pm) hepatocytes showed larger relative mitochondrial area
than Control (Con) hepatocytes. Con and Pm hepatocytes were stained by MitoTracker
Red (50 nM, Invitrogen) at 37
o
C for 20 mins and fluorescence signal was analyzed.
Quantification of the ratio, Fluorescence Pixels/Whole Cell Area Pixels was performed
by UN-SCAN-IT software. * P≤ 0.05, different from control (Con). n=3. B, The ratio of
COXII/Globin is higher in both Pten null (Pm) hepatocytes (Left panel) and livers (Right
panel). Genomic DNA was extracted from Con and Pm hepatocytes and livers. QPCR
was performed to analyze COXII and Globin levels. * P≤ 0.05, different from control
(Con). n=5. C, IGF-1 treatment increases mitochondrial mass in Con immortalized
hepatocytes. Con hepatocytes were treated with IGF-1 for 24 hours and stained with
MitoTracker Green (25nM, Invitrogen). FACS was carried out to analyze the
fluorescence intensity. Left panel: FACS results of mitochondrial density in unstained,
vehicle-treated and IGF-1-treated Con hepatocytes. Right panel: Quantification of
average fluorescence intensity. * P≤ 0.05, different from vehicle treated group. n=3. D,
LY294002 decreases mitochondrial mass in Pm immortalized hepatocytes. Pm
hepatocytes were treated with LY294002 for 4 hours and stained with MitoTracker Green
(25nM, Invitrogen). FACS was used to analyze the fluorescence intensity. Left panel:
FACS results of mitochondrial density in unstained, vehicle-treated and LY294002-
treated Pm hepatocytes. Right panel: Quantification of average fluorescence intensity. *
P≤ 0.05, different from Vehicle treated group. n=3.
II-2-4: PTEN regulates mitochondrial respiration partially by reducing the
abundance of transcriptional factor ERRα
Mitochondrial biogenesis is largely dependent on the nuclear transcriptional
machinery, where the nuclear receptor estrogen-related receptor α (ERRα) plays a
predominant role in activating expressions of genes involved in the TCA cycle and
oxidative phosphorylation (Villena et al., 2007). ERRα either directly activates genes of
mitochondrial components or indirectly induces expressions of other transcriptional
factors regulating mitochondrial biogenesis such as nuclear respiratory factor-1 (NRF-1)
and -2 (NRF-2/GABP) (Huss, Torra, Staels, Giguere, & Kelly, 2004; Laganiere et al.,
2004). ERRα is also a central transcription factor that is responsible for adaptive
mitochondrial biogenesis in response to a variety of stimuli, including high fat diet,
thermogenesis, etc. (Scarpulla, 2006, 2008; Villena et al., 2007) The ERRα-null mice
exhibited defective adaptive thermogenesis, a process that relies on induction of
32
mitochondrial mass and energy production under cold exposure. To explore how
PI3K/PTEN signal may control mitochondrial biogenesis, we compared expression levels
of ERRα in control (Con) and Pten-null hepatocytes and livers. The protein level of
ERRα was dramatically up-regulated in Pten-null hepatocytes (Figure II-6 A, left panel)
and the level of ERRα transcript was 5 folds higher when compared to the control (Con)
(Figure II-6 A, right panel). Similar induction of ERRα protein expression was observed
in Pten-null liver while ERRα was undetectable in control (Con) liver lysates (Figure II-
6B).
To confirm that the function of ERRα is induced with the elevated protein and
mRNA levels, we evaluated the transcriptional function of ERRα using two luciferase
constructs: pCytc/-686Luc and pATPsynβ/-385Luc. The pCytc/-686Luc consists of
sequence from codon -686 to +55 of cytochrome c. The pATPsynβ/-385Luc consists of
sequence from codon -385 to +90 of ATP synthase β. Both cytochrome c (cyst c) and
ATP synthase β (ATP5b) were found to be under the transcriptional regulation of ERRα
(Schreiber et al., 2004). In comparison with control (Con) hepatocytes, luciferase activity
of pCytc/-686Luc was approximately 2 folds higher (approximately 800 vs. 400 units) in
Pten-null cells (Figure II-6 C, left panel), suggesting enhanced promoter activities when
Pten is deleted. Similarly, more than 2 folds induction of pATPsynβ/-385Luc luciferase
activity was observed in the Pten-null cells (Figure II-6 C, right panel), indicating that
ERRα transcriptional activity is higher in Pten-null cells compared to controls. We
further evaluated the mRNA levels of cytochrome c and ATP synthase β to verify the
enhanced ERRα transcriptional activity on endogenous ERRα target genes. We show that
mRNA expression of cyt c is 2 folds higher in the Pten-null cells vs. controls (Figure II-6
33
D), similar to what is observed with pCytc/-686Luc activity. The mRNA level of ATP5b
were also higher in the Pten-null cells vs. controls (Figure II-6 D). We also evaluated
another endogenous gene, Medium Chain acyl CoA Dehydrogenase (MCAD), a fatty
acid oxidation enzyme transcriptionally controlled by ERRα. We found that MCAD
mRNA level was 5 folds higher in Pten-null hepatocytes than the controls (Figure II-6
D). Thus, ERRα levels and activities were both higher when Pten is deleted in liver
hepatocytes.
These analyses suggest that PTEN and PTEN-regulated signals may control
ERRα functions. To investigate whether the increased ERRα levels and activity may be
responsible for the higher mitochondrial respiratory function in the Pten-null hepatocytes,
we used two independent siRNAs to knockdown ERRα in Pten-null hepatocytes and
evaluated the respiration rate of these cells. Both siRNAs targeted at ERRα decreased its
protein expression (Figure II-6 E&F, top right panel). Concomitantly, siERRα-transfected
Pten-null hepatocytes displayed significantly decreased basal and maximal OCRs
compared to scrambled siRNA-transfected ones, indicating that ERRα is crucial for
mitochondrial function and may be responsible for the enhanced mitochondrial
respiratory function observed in Pten-null hepatocytes (Figure II-6 E&F, left, bottom
right panels).
34
Figure II-6. PTEN signal regulates mitochondrial function via ERRα.
A
B
D
Fold Change
0
2
4
6
Con Pm
P-AKT
PTEN
ERRα
ACTIN
Con Pm
Immortalized hepatocytes
Con Pm
ERRα
PTEN
ACTIN
Mouse Liver
0
200
400
600
800
1000
Con Pm
0
50
100
150
200
250
Con Pm
C
Fold Change
Luciferase
(pCytc/-686Luc)
Luciferase
(pATPsynβ/-385Luc )
Luciferase activity
(arbitrary units)
0
2
4
Con
Pm
Cyt C ATP5b MCAD
ERRα mRNA
*
*
*
*
*
QPCR
ERRα
ACTIN
si ERRα1 si Scramble Oxygen Consumption Rate (OCR)
(Pm Hepatocyte+siERRα1)
OCR (pMoles/min)
siScramble
siERRα1
FCCP Baseline Oligomycin Rotenone
0
50
100
150
200
250
300
1 23 46 68 91
Time (min)
E
0
50
100
150
200
250
Baseline Maximal
siScramble
siERRα1
OCR (pMoles/min)
*
*
siERRα2 siScramble
ERRα
ACTIN
F
OCR (Pm Hepatocytes + siERRα2)
0
40
80
120
160
1 23 46 68 91
Baseline FCCP Rotenone Oligomycin
siERRα2
siScramble
35
A, PTEN loss increases ERR expression. Left panel, total cell lysate was extracted from
Con and Pm hepatocytes and subjected to Western analysis using antibody against PTEN,
P-AKT, and ERRα. Right panel, total RNA was extracted from Pm and Con hepatocytes.
qPCR was performed using ERRα and GAPDH primers. GAPDH is detected as
housekeeping control. n=5. B, ERRα expression is higher in Pm mouse livers than that in
Con ones. Total protein was extracted from Con and Pm mouse livers and subjected to
Western analysis with primary antibody against PTEN, p-AKT, and ERRα. C, promoter
activities of ATP syn β and cyt c are enhanced in Pm hepatocytes. Con and Pm
hepatocytes were transfected with luciferase reporters, pATPsynβ/-385Luc (right panel)
and pCyt c/-686Luc (left panel). The luciferase activities were measured. *, p≤0.05,
different from Con.n=3. D, PTEN loss induces mRNA levels of cyt c, ATP synthase β
(ATP5b), and medium chain acyl-CoA dehydrogenase (MCAD). Total RNAwas
extracted from Con and Pm immortalized hepatocytes. qPCR was used to analyze the
mRNA abundance of cytochrome c, ATP synthase β, and medium chain acyl-CoA
dehydrogenase using corresponding primers. GAPDH serves as housekeeping control. *,
p ≤ 0.05, different from Con. n=5. E, ERRα knockdown suppresses mitochondrial
respiration in Pm hepatocytes. Immortalized Pm hepatocytes were transfected with
siERRα1 and subjected to OCR measurement. Left panel, OCR results of Pm hepatocytes
transfected with siERRα and siScramble. Right panel (top), ERRα level is down-
regulated by siERRα1 transfection. Right panel (bottom), quantifications of base line and
maximal OCR values. *, p≤0.05, different from siScrambled controls. n=5. F, same
experiments were carried out as E using siERRα2.
II-2-5: PTEN regulates ERRα expression through PI3K/AKT signaling pathway
To elucidate the underlying mechanisms for ERRα up-regulation upon PTEN
loss, we manipulated PTEN and its downstream signaling molecules and examined the
corresponding change in the expression of ERRα. Compared to scramble shRNA-
transfected cells, shPTEN transfection in control (Con) hepatocytes led to increased
ERRα protein expression (Figure II-7 A, left panel) whereas introduction of wild type
PTEN into Pten null (Pm) hepatocytes down-regulated ERRα (Figure II-7 A, right panel),
reconstructing the expression pattern observed in the two genetically distinct cell lines.
Having shown that PTEN manipulations alter ERRα expression and that PI3K/AKT
signal, the primary PTEN target, controls mitochondrial biogenesis and respiration
(Figure 4 and Figure 5), we next asked whether ERRα regulation by PTEN is also
PI3K/AKT-dependent. To test this, IGF-1 and LY294002 were used to treat Pten control
36
and null cells to activate and inhibit PI3K signal, respectively. After a 4-hour treatment of
LY294002 to inhibit PI3K in Pten null hepatocytes, we observed a dramatic reduction of
ERRα expression together with a robust inhibition of p-AKT (Figure II-7 B, left panel).
Conversely, inductions of ERRα and p-AKT were observed in control hepatocytes treated
with IGF-1 (Figure II-7 B, right panel). These data suggested that activation of PI3K
signal leads to increased ERRα expression. To confirm the involvement of AKT in this
regulation, constitutively active myristylated (CA) AKT and dominant negative (DN)
AKT construct were introduced to the Con and Pten-null hepatocytes respectively to
induce and inhibit total AKT activities (Figure II-7 C). WT AKT construct and empty
vectors were also used as controls. In control hepatocytes, overexpression of CA AKT
induced ERRα expression whereas the ERRα protein expression is inhibited by DN AKT
transfection in Pten-null cells. WT AKT also increased the expression of ERRα when
introduced to the control hepatocytes. To further investigate the role of ERRα as a
mediator for AKT-controlled mitochondrial function, we manipulated ERRα with siRNA
in cells expressing constitutively active (CA)-AKT. Introduction of CA-AKT alone led to
robust induction of OCR in control hepatocytes as predicted. ERRα knockdown in these
cells attenuated the induction and brought the OCR level back to a level comparable to
that of the controls (Figure II-7 D). Together, these data suggest that the expression of
ERRα relies on AKT activity downstream of PI3K and ERRα functions as a prominent
PI3K/AKT target that regulates mitochondrial function.
37
Figure II-7. PTEN regulates ERRα expression through PI3K/AKT signaling
pathway.
A, PTEN knockdown in Con hepatocytes results in elevated ERRα levels (left panel), and
PTEN overexpression in Pm levels leads to decreased ERRα expression (right panel).
shPTEN and WT PTEN constructs were introduced into Con and Pm hepatocytes,
respectively. Total cell lysate was extracted, and Western blot was carried out using
primary antibodies against PTEN, p-AKT, ERRα, and actin. N/A, naive cells. B,
shPTEN shScramble
PTEN
P-AKT
ERRα
ACTIN
Con Hepatocytes
ERRα
ACTIN
P-AKT
24hrs Starvation Standard Condition IGF-1
Con Hepatocytes
ERRα
ACTIN
CA AKT WT AKT Vector
Con Hepatocytes
ERRα
P-AKT
PTEN
ACTIN
PTEN+Vector Vector N/A
Pm Hepatocytes
ERRα
P-AKT
ACTIN
Vehicle LY294002
Pm Hepatocytes
Vector
r
P-AKT
WT AKT DN AKT
ERRα
ACTIN
Pm Hepatocytes
A
B
C
ERRα/ACTIN 0.3 0.3 0.4 0.2 0.2 0.1 ERRα/ACTIN
0.7 0.9 0.7 1.2 1.2 1.1 1.2 1.0 1.3
ERRα/ACTIN
0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.3 0.3
ERRα/ACTIN
ERRα/ACTIN
0.6 0.6 0.7 0.5 0.5 0.4 0.1 0.2 0.2 ERRα/ACTIN
0.7 0.6 0.6 0.5 0.5 0.5 0.5 0.4 0.7
P-AKT
0.9 0.8 2.2 0.5 0.1 0.2
D
P-AKT
CA
AKT
CA AKT+
siERRα
Vec+
siScramble
ACTIN
ERRα
Baseline FCCP Rotenone Oligomycin
Con Hepatocytes
0
50
100
150
200
250
300
350
400
CA AKT
CA AKT+siERRa
Control
1 23 46 68 91
Con Hepatocytes
0
50
100
150
200
250
300
CA AKT CA AKT
+siERRα
Control
OCR (pMoles/min)
Baseline
*
38
LY294002 treatment suppressed ERRα in Pm hepatocytes (left panel), and IGF-1
treatment leads to increased ERRα levels in Con hepatocytes (right panel). IGF-1 and
LY294002 treatments were performed in Con and Pm hepatocytes, respectively. Western
blot was employed to assess the protein levels of p-AKT, ERRα, and actin. C, AKT is
involved in mediating ERRα abundance. Constitutively active AKT (CA-AKT) and
dominant negative AKT (DN AKT) constructs were introduced into Con and Pm
hepatocytes, respectively. Wild type AKT (WT AKT) and empty vector were also used as
transfection controls. Western blot was employed to assess the protein levels of p-AKT,
ERRα, and actin. The signal density ratio of ERRα/actin for each Western blot result is
provided at the bottom. D, ERRα knockdown together with CA-AKT overexpression
rescues the OCR induction by CA-AKT-only transfection. Con hepatocytes were
transfected with CA-AKT or CA-AKT and siERRα or empty and siScramble as control.
After 24 h, transfected and control cells were seeded for OCR measurement. Left panel,
siERRα leads to decrease of OCR level from CA-AKT-transfected cells. Right panel
(top), Western blotting results of ERRα, p-AKT, and actin to confirm the plasmid/siRNA
transfections. Right panel (bottom), quantification of base-line OCR.
II-2-6: CREB mediates ERRα regulation downstream of PI3K/AKT independent of
PKA
In order to understand the molecular mechanisms leading to increased expression
of ERRα, we examined the expression levels of PGC-1α, a nuclear co-activator of ERRα
that also transcriptionally regulates ERRα expression (Schreiber, Knutti, Brogli,
Uhlmann, & Kralli, 2003). We found that both mRNA and protein levels of PGC-1α were
induced in Pten-null hepatocytes vs. controls (Figure II-8 A). PGC-1α serves as a co-
activator for a number of nuclear transcriptional factors and is proposed to be a major
moderator for metabolic signals (Knutti & Kralli, 2001). We show here that siRNA-
mediated PGC-1α knockdown led to reduced ERRα expression in the Pten-null
hepatocytes (Figure II-8 B), suggesting that PGC-1α-mediated induction of ERRα
transcription may play a role in the accumulation of ERRα when PTEN is lost.
CREB is proposed to directly induce PGC-1α expression upon phosphorylation at
Ser133 (S. Herzig et al., 2001). To explore the potential PI3K/AKT-downstream
candidates mediating ERRα regulation in the nucleus, we evaluated CREB signals in
39
hepatocytes. A remarkably higher p-CREB was observed in Pten-null hepatocytes vs.
controls (Figure II-8 C), suggesting that Pten deletion might result in elevated
phosphorylation signal that activates CREB. A dominant negative CREB construct
CREB133 (Nishihara, Hwang, Kizaka-Kondoh, Eckmann, & Insel, 2004), which has a
S133A mutation, was used to abolish the phosphorylation event of CREB at Ser133. We
found that overexpression of wild type (WT) CREB, but not CREB133, was able to
induce the expression of PGC-1α in Con hepatocytes (Figure II-8 D). This observation
corroborates with previous studies showing that CREB transcriptionally induces
expression level of PGC-1α (S. Herzig et al., 2001). Concomitantly, expression of
CREB133 dramatically repressed ERRα expression in Pten-null hepatocytes (Figure II-8
E). These data suggested that CREB mediates the increased expression of ERRα that we
observed in Pten-null hepatocytes.
To determine the dependency of CREB phosphorylation on PI3K/AKT signaling,
Con hepatocytes were treated with IGF-1 in order to activate PI3K signaling (Figure II-8
F). Treatment with IGF-1 for 1 hour was sufficient to induce p-AKT and this induction
was sustained throughout the treatment. Meanwhile, a time-dependent induction of p-
CREB by IGF-1 was also observed. ERRα protein starts to be detectable 6 hours after
IGF-1 treatment and sustained at 24 hours concurrent with sustained AKT
phosphorylation. In cells treated with IGF-1 for 24-hour when AKT and CREB were
maximally phosphorylated, significantly higher ERRα levels are also detected compared
to other parallel treatment groups (Figure II-8 F). Together, this data indicated that
sustained p-AKT may be capable of transmitting activation signal towards CREB in a
phosphorylation-dependent manner. Conversely, applying LY294002 treatment in Pten-
40
null hepatocytes showed a robust inhibition of p-CREB at 4 hours accompanied by
suppression of p-AKT (Figure II-8 G). At 8-hours post treatment, p-AKT recovered back
to normal level. Interestingly, p-CREB followed the same trend (Figure II-8 G). Thus,
CREB phosphorylation in the Pten-null hepatocytes appears to depend on PI3K activity.
41
Figure II-8. CREB mediates ERRα regulation downstream of PI3K/AKT signaling.
A, QPCR (Left panel) and western blot (Right panel) analyses of PGC-1α mRNA and
protein levels in control (Con) and Pten-null (Pm) immortalized hepatocytes. *P≤ 0.05,
42
different from controls (Con). n=5. B, Inhibition of PGC-1α with shRNA leads to
reduced ERRα expression. Actin is used as loading control. C. Western blot analysis for
p-CREB, CREB and Actin in Con and Pm immortalized hepatocytes. D, CREB induces
PGC-1α expression. Wild type CREB construct (WT CREB), CREBS133A construct
(CREB133) and empty pCMV vector were transfected into Con hepatocytes. Western
blot shows levels of PGC-1α, p-CREB and Actin. E, CREB133 diminishes ERRα level in
Pm hepatocytes. CREB133 and pCMV vector were transfected into Pm hepatocytes.
Protein levels of p-CREB and ERRα were analyzed by western blot. F, IGF-1 treatment
stimulates p-CREB and ERRα levels. Con hepatocytes were serum-starved for 24 hours
and then treated with IGF-1 for 1 hour, 6 hours and 24 hours. Protein levels of ERRα, p-
CREB, p-AKT and Actin were assessed by western blot. G, LY294002 treatment
suppresses p-CREB. Pm hepatocytes were treated with LY294002 for 4 hours and 8
hours. Protein levels of p-AKT and p-CREB were analyzed by western blot.
Phosphorylation of CREB has been shown to be a consequence of protein kinase
A (PKA) activation upon buildup of intracellular cyclic AMP (cAMP) (Altarejos &
Montminy, 2011). We therefore investigated whether PKA was involved in the
PI3K/AKT-induced CREB phosphorylation. Con and Pten-null hepatocytes were
infected with lentivirus expressing shRNA that targets the catalytic subunit of PKA (PKA
Cα). Our data showed the lentivirus infection in control cells completely abolished PKA
Cα expression, leading to a significant reduction of p-CREB without affecting the levels
of p-AKT (Figure II-9 A, left panel). On the other hand, PKA knockdown in Pten-null
hepatocytes where p-AKT is constantly activated was unable to change p-CREB level
(Figure II-9 A, right panel). In addition, consistent with our earlier data showing p-CREB
mediates ERRα expression (Figure II-8), lentivirus-shPKA infection diminished ERRα
expression in Pten-control hepatocytes but not in Pten-null ones (Figure II-9 A).
Together, these findings indicate that hyper-activated PI3K/AKT signal is sufficient to
induce phosphorylation of CREB independent of PKA.
To further interrogate this signaling relationship, we performed IGF-1 treatment
in Con hepatocytes infected with lentivirus that express either shRNA targeting PKA Cα
43
or LacZ as control. In shLacZ-infected cells, IGF-1 was able to activate p-AKT and p-
CREB (Figure II-9 B, left panel). As anticipated, similar CREB phosphorylation response
was also observed in IGF-1-treated hepatocytes when PKA Cα level is dramatically
decreased by shRNA (Figure II-9 B, right panel). Therefore, the activation of PI3K/AKT
signal was capable of inducing CREB phosphorylation independent of PKA.
Screening of CREB sequence revealed an imperfect AKT substrate consensus
sequence: “R-X-X-S/T”. It has been reported that chronic activation of AKT may lead to
phosphorylation of “secondary substrates with imperfect consensus sequences” (Manning
& Cantley, 2007). We tested this potential kinase-substrate relationship by performing
immunoprecipitation analysis using a p-AKT substrate antibody against a specific AKT
phosphorylation motif. Immunoblot with CREB antibody revealed a positive binding
signal within the pull down sample (Figure II-9 C), implicating that CREB might be a
putative substrate for AKT kinase activity.
44
Figure II-9. CREB serves as a putative AKT substrate and regulates ERRα level
independent of protein kinase A (PKA).
A, Activated AKT signaling due to PTEN loss maintains p-CREB level independent of
PKA. Control (Con) and Pten null (Pm) immortalized hepatocytes were infected with
lentivirus expressing shPKA (against catalytic α domain of PKA) or shLacZ (serves as
control). Protein expression levels of PKA Cα, p-CREB, p-AKT, ERRα and Actin in Con
and Pm cells were assessed by western blot. B, IGF-1 is able to induce p-CREB when
PKA Cα protein level is significantly reduced by lentivirus shRNA in Con hepatocytes.
Con immortalized hepatocytes were infected with shPKA/LacZ lentivirus and then
subjected to 24-hour serum starvation followed by IGF-1 treatment. P-AKT, p-CREB and
Actin levels were assessed by western blot. C, AKT and CREB physically interact.
Potential AKT substrates were pulled down from Pm hepatocytes lysate by the antibody
that specifically recognize AKT substrate motif. Total protein lysate (Input) and pull
down lysate were subjected to western blot analysis using antibody against CREB.
II-2-7: Reactive oxygen species (ROS) level is regulated by ERRα
To clarify the role of ERRα in the elevated ROS production upon PTEN loss, we
either overexpressed or knocked down ERRα in control and Pten-null hepatocytes
Starve IGF-1
P-AKT
P-CREB
Actin
shPKA
shPKA shLacZ
Pm
p-AKT
PKA Cα
p-CREB
Actin
ERRα
p-AKT
PKA Cα
p-CREB
Actin
ERRα
shPKA shLacZ
Con
Starve IGF-1
shLacZ
A
B
CREB
Input P-AKT Substrate Ab
CREB
IP: P-AKT Substrates
IB: CREB
lgG
C
Con Hepatocytes
45
respectively and examined the corresponding ROS levels. H
2
DCFDA staining and FACS
analysis revealed that knocking down ERRα in Pten-null hepatocytes significantly
reduced ROS production by 2 folds compared to the siScramble-transfected cells (Figure
II-10 A&B). Conversely, overexpression of ERRα led to a 30% increase in ROS
production in Con hepatocytes (Figure II-10 C). Thus, ERRα plays a role in regulating
ROS production downstream of PTEN signaling.
46
Figure II-10. ERRα regulates ROS production.
Unstained Pm Hepatocytes
Stained Pm Hepatocytes+siScramble
Stained Pm Hepatocytes+siERRα1
ROS Production
siERRα1 siScramble
ERRα
ACTIN
A
*
0
500
1000
1500
Fluorescent intensity
(Arbitrary Units)
Unstain siERRα1 siScr
Events
Unstained Pm Hepatocytes
Stained Pm Hepatocytes+siScramble
Stained Pm Hepatocytes+siERRα2
ROS Production
0
2000
4000
6000
8000
10000
12000
14000
16000
Unstain siERRα2 siScr
siERRα2 siScramble
ERRα
ACTIN
Fluorescence Intensity
Fluorescence Intensity
Unstained Con Hepatocytes
Stained Con Hepatocytes+Vector
Stained Con Hepatocytes+ERRα
ROS Production
*
0
200
400
600
800
Unstain ERRα vector
B
Events
C
Fluorescence Intensity
*
Fluorescence Intensity
(Arbitrary units)
Fluorescence Intensity
(Arbitrary units)
ERRα
ACTIN
ERRα Vec Naive
47
A and B, ERRα knockdown decreases ROS production in Pm immortalized hepatocytes.
siERRα 1/2 and siScramble were delivered into Pm hepatocytes, which were then stained
with H2DCFDA and subjected to FACS analysis. Left panels, FACS results of ROS
production in siERRα-transfected, siScramble-transfected, and unstained Pm hepatocytes.
Right panels (top), quantification of average fluorescence intensity. Right panels
(bottom), ERRα level is diminished by siERRα in Pm hepatocytes. *, p ≤ 0.05, different
from siScramble (siScr) control. n=3. C, ERRα overexpression in Con hepatocytes
increases ROS production. WTERRα was overexpressed in Con hepatocytes.H2DCFDA
staining and FACS were performed to assess ROS levels (left panel). Right panel (top),
quantification of average fluorescence intensity. Right panel (bottom), ERRα level after
transfection in Con hepatocytes. *, p≤ 0.05, different from vector control. n=3.
II-2-8: Knockdown of ERRα attenuates colony forming potential in Pten-null
hepatocytes and PTEN level is negatively correlated with ERRα level in liver cancer
patients’ samples
ROS production has been shown to correlate with tumorigenesis and tumor cell
transformation (Cui, 2012). We evaluated the role of ERRα in the tumorigenic potential
of the Pten-null hepatocytes. We show here that siRNA inhibition of ERRα expression
led to decreased cell growth compared to scramble siRNA or naive cell controls (Figure
II-11 A). We have shown previously that the Pten-null hepatocytes are capable of
forming colonies on soft agar (Zeng et al., 2011). Here, we found that inhibition of ERRα
with siRNA significantly inhibits the ability of Pten-null hepatocytes to form colonies
(Figure II-11 B). Thus, ERRα induction in Pten-null hepatocytes may support their ROS
production and transformation potential.
To explore the physiological significance of PTEN-ERRα signaling cascade, we
analyzed gene expression data from 5 cohorts of liver cancer patient samples
(www.oncomine.org) (Chiang et al., 2008; Hoshida et al., 2009; Roessler et al., 2010;
Woo et al., 2010) (TCGA: http://tcga-data.nci.nih.gov/tcga/) and assessed the
coexpression pattern of PTEN and ERRα. Significantly negative correlation between
48
expressions of PTEN and ERRα is observed, suggesting that the novel link between
PTEN/AKT/CREB/ERRα may be present in human samples and this signaling pathway
maybe relevant to liver cancer development (Figure II-11 C). Together these results
support the crucial role of ERRα during the transformation of liver cancer cells.
Moreover, the negative correlation between PTEN and ERRα revealed by the cohort
analyses of liver cancer patient datasets confirmed the clinical relevance of the PTEN-
controlled signaling pathway that regulates ERRα expression.
49
Figure II-11. ERRα knockdown attenuates colony forming potential in Pten-null
hepatocytes, and PTEN level is negatively correlated with ERRα level in samples
from patients with liver cancer.
50
A, ERRα knockdown decreases proliferation rate of Pm hepatocytes. Pm hepatocytes
were transfected with siScramble or siERRα1/2. Cells were then seeded, and cell
numbers were counted each day for 5 days. B, ERRα knockdown decreases colony
forming potential of Pm hepatocytes. Pm hepatocytes were transfected with siScramble
or siERRα1/2 and then seeded in a 6-well plate. Colonies were stained after 10 days. Top
left panel, representative results of colony staining in wild type (Con), Pm, siScramble-
transfected, and siERRα1-transfected Pm hepatocytes. Bottom left panel, representative
results of colony staining using siERRα2. Right panel, quantifications of colony numbers
in Con, naive, siScramble, and siERRα1/siERRα2 groups.*, p≤0.05, different from
controls; **, p≤0.05, different from siScrambled (siScr) groups. n=3. C, expressions of
PTEN and ERRα are negatively correlated in patients with liver cancer. Coexpression
analysis was performed on three clinical datasets of patients with liver cancer. Prism
Graphpad is used to analyze the data. Analysis Results: Woo. etal: Pearson ratio, -0.5425;
95% confidence interval, -0.6739 to -0.3779; p value (two-tailed), ≤0.0001. Is this
correlation significant? Yes. R square, 0.2944; TCGA Database: Pearson ratio, -0.2148;
95% confidence interval, -0.3973 to -0.01601; p value (two-tailed), 0.0346. Is this
correlation significant? Yes, R square, 0.04615; Chiang et al., Pearson ratio, -0.5009;
95% confidence interval, -0.5988 to -0.3883; p value (two-tailed),
≤0.0001. Is this correlation significant? Yes, R square, 0.2509.
II-3: Discussion
In this study, we investigated the roles of PTEN, a tumor suppressor and the
mitogenic PI3K/AKT signaling negatively regulated by PTEN in the regulation of
mitochondrial function. Broadly, the results suggest that activation of PI3K/AKT signal
and loss of PTEN function lead to enhanced mitochondrial respiratory capacity and
mitochondrial volume. PI3K/AKT/PTEN signaling was found to control the expression
of ERRα, a critical nuclear transcriptional factor involved in adaptive mitochondrial
biogenesis. Delineation of the signaling events leading to induction of ERRα by PI3K
showed that CREB, a putative AKT substrate, mediates the ERRα up-regulation in
response to PI3K/AKT/PTEN signaling. ERRα induction in Pten-null hepatocytes
promotes the transformation and growth potential of these cells. The clinical cohort
analyses revealed that in tumor samples of liver cancer patients, PTEN expression is
51
negatively correlated with ERRα level. These analyses further demonstrated the
physiological relevance of the PTEN-ERRα signaling pathway established by this study.
Together with PTEN mutation, PI3K amplification and AKT over-activation
occurs frequently in human malignancies (Vivanco & Sawyers, 2002). Studies in recent
years have highlighted the metabolic regulatory function of PI3K/AKT/PTEN signal in
malignancy transformation in addition to its role in cell growth and survival (Shaywitz,
Courtney, Patnaik, & Cantley, 2008). AKT, the prominent downstream function unit of
PTEN/PI3K regulation is a major metabolic enzyme besides being a cell survival kinase.
AKT has been shown to regulate both glycolysis and oxidative phosphorylation
(Gonzalez & McGraw, 2009a; Shaik, Fifer, & Nowak, 2008). Our data here confirm this
observation and demonstrate functionally that the rate of glycolysis (measured as
extracellular acidification rate, ECAR) is induced when AKT activity is high and
inhibited when AKT activation is blocked. Similarly, oxidative phosphorylation
determined as oxygen consumption followed the same correlation with AKT. This
observation suggests that AKT activation alters the cellular metabolic profile in a manner
similar to that exhibited by tumor cells. In non-tumor cells, oxidative phosphorylation is
coupled with glycolysis. When oxidative phosphorylation is inhibited by the addition of
oligomycin, glycolysis increases to maintain the supply of ATP production. In these cells,
the two rates are only simultaneously increased when the two processes are uncoupled by
the addition of an uncoupler to cause proton leakage (Yao et al., 2012). Tumor cells,
however, were shown to metabolize large quantities of glucose via glycolysis and
generate excessive lactate while maintaining the high oxygen consumption through
52
mitochondrial respiration (Warburg, 1956). Our data show that the oncoprotein AKT is
capable of producing such an effect.
Our data indicate that AKT is capable of regulating oxidative phosphorylation and
glycolysis independent of each other. The effects of AKT do not depend on the coupling
of the two processes as both processes increase simultaneously when AKT is activated.
Activation of AKT has been previously shown to accelerate glycolysis by directly
regulating molecules involved in glycolytic pathways (Gottlob et al., 2001; Robey &
Hay, 2006). AKT phosphorylates and promotes the translocation of hexokinase onto
mitochondria where it binds to VDAC (voltage dependent anion channel) (Gottlob et al.,
2001). This is proposed to allow hexokinase to gain direct access to high concentration of
ATP, thus facilitate accelerated glycolysis (Robey & Hay, 2006). These functions of
AKT lead to increased aerobic glycolysis that supply both ATP and probably more
importantly, increased fluxes of substrates used in biosynthetic pathways. We show here
that AKT activation also induces oxidative phosphorylation by inducing mitochondrial
biogenesis and increasing the mass of the mitochondria. This process is mediated by a
key transcriptional factor, ERRα that orchestrates the mitochondrial bioenergetic
response.
ERRα controls expressions of both nuclear and mitochondrial transcriptional
factors involved in mitochondrial biogenesis (Villena & Kralli, 2008). Expressions of
other nuclear transcription factors necessary for mitochondrial biogenesis, including
NRF-1 and -2 are also under the regulation of ERRα (Mootha et al., 2004). In mouse
models lacking ERRα, the ability to maintain the body temperature, when exposed to
cold is attenuated and mice are unable to cope with cold environment. This is primarily
53
due to the inability of the brown adipose tissue to induce mitochondrial biogenesis and
energy production when ERRα is absent (Villena et al., 2007). Thus, ERRα plays crucial
roles in the transcriptional paradigms for mitochondrial biogenesis.
The expression of ERRα has been shown previously to correlate with that of
ErB/Her2/EGFR, which signals through PI3K/AKT signaling pathway (Ariazi, Kraus,
Farrell, Jordan, & Mertz, 2007). In tumor cells, particularly ER negative breast cancer
cells, ERRα level correlates with expression of EGF receptor including ErB2, 3 and
EGFR (Chang et al., 2011). In in vitro kinase assays, incubation of ERRα with MAPK or
AKT both led to gel mobility shifts, indicating a potential kinase-substrate relationship
between the molecules (Ariazi et al., 2007). Phosphorylation status of ERRα is generally
correlated with its transcriptional activity. However, phosphorylation of serine 19 is
found to precondition ERRα for sumoylation, which suppresses its transcriptional activity
(Tremblay, Wilson, Yang, & Giguere, 2008). Major breakthroughs in ERRα regulation
studies came from the realization that PGC-1α, the co-activator for a number of nuclear
receptors has structural motifs specific for binding to ERRα. It was later discovered that
PGC-1α/ERRα complex also acts as a transcriptional activator for ERRα itself, turning
ERRα from a weak to a strong transcriptional activator (Schreiber et al., 2003). We found
that Pten-deficient hepatocytes exhibited elevated levels of both ERRα and PGC-1α. The
inductions of these two factors when PI3K/AKT is activated confirmed that ERRα
transcriptional activity remains high in cells where their signal is induced. The high level
of ERRα transcriptional activity is necessary to execute the mitochondrial biogenic
program to increase mitochondrial mass in Pten-null hepatocytes.
54
Our data indicate that ERRα expression is robustly up-regulated upon Pten
deletion, and this process clearly depends on the activity of PI3K/AKT. We have
identified CREB as a potential AKT substrate that mediates this induction of ERRα
expression. Phosphorylation of CREB on serine 133 is a common consequence of PKA
activation in response to cellular accumulation of cyclic AMP. In cells where AKT is
overexpressed, phosphorylation of serine133 has been observed (Du & Montminy, 1998).
Whether this increased CREB phosphorylation in cultured cells depends on PKA activity
was not clarified. Our data in hepatocytes indicates that PKA is dispensable in the
phosphorylation of CREB regulated by PI3K/AKT signal. Recombinant AKT is shown to
induce phosphorylation of CREB polypeptides containing Ser133 residue in in vitro
kinase assay, suggesting a potential direct kinase-substrate relationship (Du & Montminy,
1998). CREB contains a motif of R-X-X-S/T similar to but different from the typical
AKT recognition site, R-X-R-X-X-S/T (S. Herzig et al., 2001). However, it was
suggested that chronic activation of AKT is capable of phosphorylating the imperfect R-
X-X-S/T sites (Manning & Cantley, 2007). Our immunoprecipitation study using a p-
AKT substrate antibody that recognizes potential AKT substrates shows that CREB can
be effectively pulled down, suggesting that the two proteins do physically interact with
each other in vivo. Therefore, it is likely that AKT may directly phosphorylate CREB,
particularly during a chronic adaptation to constitutively active AKT signals, like those in
cancer cells.
In hepatocytes, a major role of CREB is to regulate hepatic glucose output during
starvation. During fasting, CREB phosphorylation is induced in response to
glucocorticoid induction and buildup of cAMP. The presence of CREB is necessary for
55
inducing expression of gluconeogenic programs (Altarejos & Montminy, 2011). This
process relies on the ability of CREB to induce the transcriptional co-activator PGC-1α.
Chromatin immunoprecipitation analysis shows specific binding of CREB to the
consensus CREB binding element on the PGC-1α promoter (S. Herzig et al., 2001). Thus,
the phosphorylation of CREB induced by AKT activation may directly result in the
induction of PGC-1α and the subsequent expression of ERRα observed in hepatocytes
where PI3K/AKT signals are induced.
Mitochondrial function is crucial for the transformation of tumor cells. When
transformed, human mesenchymal stem cells were found to exhibit increased oxidative
phosphorylation rate (Funes et al., 2007). In several breast cancer cell lines, reducing
oxidative phosphorylation by knocking down a key mitochondrial protein p32 led to
reduced transformation (Fogal et al., 2010). In vivo studies using oncogenic Kras-driven
mouse model of lung cancer showed that mitochondrial transcription factor A (TFAM) is
essential for tumorigenesis (Weinberg et al., 2010). Thus, the AKT-pCREB-ERRα
signal-driven mitochondrial mass increase is likely important for tumor cell
transformation. Clinically, studies have reported increased mitochondrial DNA in liver
cancer specimens. However, a decrease of mitochondrial DNA content has also been
reported (H. C. Lee et al., 2004; Vivekanandan et al., 2010; Yin et al., 2004). As tumor
samples in these clinical studies are primarily from patients with late stage liver cancer, it
is still premature to determine cause and consequence relationship between mitochondrial
mass and tumor development based on the clinical findings. Nonetheless, these studies do
support the notion that mitochondrial abnormalities are associated with tumor phenotypes
clinically.
56
A primary link between mitochondrial function and cell transformation is
proposed to involve the generation of ROS (Ralph, Rodriguez-Enriquez, Neuzil,
Saavedra, & Moreno-Sanchez, 2010). By reacting with the cysteinyl thiols, ROS modify
a variety of proteins involved in multiple cellular processes. In some cases, the thio acts
as a redox-sensitive switch to modulate protein activity. By modulating these proteins,
ROS may control cell growth, survival and tumorigenesis. At low levels, ROS promote
cell proliferation and survival. Higher levels of ROS can lead to mutagenesis and
senescence/cell death (Fleury, Mignotte, & Vayssiere, 2002; Fruehauf & Meyskens,
2007). Mitochondria are the major source of ROS due to the generation of free electrons
during the process of oxidative phosphorylation (Turrens, 2003). The high metabolic rate
(ECAR, glycolysis) and accelerated oxidative phosphorylation (OCR, oxygen
consumption) induced by activation of AKT have the potential to lead to high levels of
free electrons and thus ROS generation. In the Pten-null mouse model, high levels of
ROS (H
2
O
2
) accompanied tumorigenesis (Galicia et al., 2010). Similar results are
observed when PTEN is manipulated in vitro in this study using isolated hepatocytes.
When we manipulate mitochondrial functions with ERRα, we observed reduction in ROS
and also significant attenuation of cell proliferation and transformation ability. Thus, the
ability of PTEN/PI3K/AKT signal to control cell proliferation may be partially regulated
by increased ROS production resulting from mitochondrial biogenesis.
In summary, our study demonstrates a novel molecular interaction between
PI3K/AKT signaling and mitochondrial bioenergetics and establishes the PI3K/AKT-
pCREB-PGC-1α/ERRα signaling relationship. Our study provides for the first time the
molecular mechanisms by which PI3K/AKT/PTEN signaling may control the function of
57
mitochondria concurrent with its role in glycolysis. We show that PTEN/PI3K/AKT
signal contributes to the enhanced mitochondrial biogenesis and respiration as well as
ROS production.
II-4 Materials and Methods
Animals –Pten
loxP/loxP
; Alb-Cre
+
(Pten-null, Pm) and Pten
loxP/loxP
; Alb-Cre
-
(Control, Con)
mice were reported previously (Stiles, Wang, et al., 2004). All experimental procedures
were conducted according to Institutional Animal Care and Use Committee guidelines of
University of Southern California.
Cell culture –Primary hepatocytes were isolated from livers of Control (Con) and Pten-
null (Pm) mice. Immortalized hepatocytes were previously described (Zeng et al., 2011).
Hepatocytes were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM;
Mediatech) supplemented with 10% FBS (Atlas Biologicals), 5ug/ml insulin (Sigma),
and 10ng/ml epidermal growth factor (Invitrogen).
Reagents, plasmids and shPKA-expressing lentivirus –LY294002 and IGF-1 were
purchased from cell signaling Technology. P-IRES CA-AKT, WT-AKT, DN-AKT,
pSG5-wt PTEN and control vectors, p-IRES and pSG5, were previously described. For
shPTEN plasmids, PTEN-targeting sequence, 5-AGAGATCGTTAGCAGAAAC-3 was
inserted into vector pSilencer 3.1 Neo U6 (Invitrogen) and transformed with DH5α
competent cells (Invitrogen). Positive plasmids were amplified by plasmid midi kit
(QIAGEN). Luciferase reporter constructs, pATPsynbeta/-385Luc and pCyt c/-686Luc,
were generous gifts from Dr. Kralli (Schreiber et al., 2004). The shPKA Cα-expressing
lentivirus was generous gifts from Dr. Wei Li (Keck School of Medicine, University of
Southern California).
58
Mitochondrial staining, ROS staining and flow cytometry –Cells were grown on plates to
80% confluence and stained with 50nM MitoTracker Red (Invitrogen, Molecular Probes)
or 25nM MitoTracker Green (Invitrogen, Molecular Probes) or 1μM H2DCFDA (5-(and-
6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester, Invitrogen,
Molecular Probes) for 20 minutes at 37
o
C. For MitoTracker Red staining, cells were
analyzed under fluorescent microscope and the relative mitochondrial area was measured
using UN-SCAN-IT software. For MitoTracker Green and H2DCFDA staining, cells
were then washed in PBS, trypsinized and analyzed with a flow cytometer (BD
Bioscience). Data were collected from 10000 cells from each sample and analyzed with
the software WinMDI.
Plasmids and siRNA transfection –Plasmids and siRNA transfections were conducted
with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. 1×10
5
cells were plated in each well of 6-well plates 24 hours before transfection. 4 µ g DNA
and 10 µ l lipofectamine were delivered into cells. The combination of 100 pmole siRNA
and 5 µ l lipofectamine was used for siRNA transfection.
Luciferase reporter assay –1× 10
5
cells were plated in each well of 6-well plates 24 hours
before transfection. Luciferase constructs containing Firefly luciferase driven by
cytochrome c or ATP synthase β promoter and luciferase construct containing Renilla
luciferase driven by thymidine kinase promoter (internal control) were co-transfected into
the cells. The cell lysates preparation and luciferase activity measurement were done
according to manufacturer’s instructions (Promega, E1910).
Western blot and immunoprecipitation –Cell lysates preparation and immunoblot analysis
were previously described (Zeng et al., 2011). Antibodies against PTEN, p-AKT, CREB
59
and p-CREB were purchased from Cell Signaling Technology. Antibodies against ERRα,
PGC-1α were obtained from Abcam. Anti-actin was purchased from Sigma. P-AKT
substrate antibody is from Cell Signaling Technology. For immunoprecipitation assay, P-
AKT substrate antibody was incubated with cell extracts overnight. 30 μl Protein A beads
(GE Healthcare) was then used to pull down p-AKT substrate antibody complexes (4
o
C
for 3 hours). The lysates were spin down and washed 5 times with cell lysis buffer.
Western immunoblotting was then performed.
Seahorse XF-24 metabolic flux analysis –Primary or immortalized hepatocytes from
Control (Con) and Pten-null (Pm) mice were cultured on XF-24 plates. The medium used
for experiments was DMEM supplemented with 25mM glucose, 1mM pyruvate and
2mM Glutamine. The mitochondrial respiration inhibitors, oligomycin (1µ M), FCCP
(1µ M) and Rotenone (1µ M) were added after 4 measurements of basal OCR and ECAR.
OCR and ECAR were simultaneously measured and recorded. Protein concentration was
measured after the assay and served as normalization values for each well. DMEM is
obtained from Sigma. Pyruvate and Glutamine are from Invitrogen. Chemicals used in
Seahorse experiment are purchased from VWR.
RNA Isolation, reverse transcription and quantitative real-time PCR –Total RNA was
isolated using TRIzol (Invitrogen). Reverse transcription was performed using M-MLV
reverse transcriptase system (Promega). Quantitative real-time PCR was carried out with
SYBR Green qPCR Master Mix (Fermentas) and 7900 HT Fast Real-Time PCR system
(Applied Biosystems). Gene-specific primers are: ERRα: forward 5-
CAAGAGCATCCCAGGCTT-3, reverse 5-GCACTTCCATCCACACACTC-3; PGC-
1α: forward 5-AATCAGACCTGACACAACGC-3, reverse 5-
60
GCATTCCTCAATTTCACCAA-3. ATP synthase β: forward 5-
GCAAGGCAGGGAGACCAGA-3, reverse 5- CCCAAAGTCTCAGGACCAACA -3;
Cytochrome c: forward 5- CCAGTGCCACACCGTTGAA-3, reverse 5-
TCCCCAGATGATGCCTTTGTT-3; COX-2: forward 5-
GCCGACTAAATCAAGCAACA-3, reverse 5-CAATGGGCATAAAGCTATGG-3;
GAPDH: forward 5-GCACAGTCAAGGCCGAGAAT-3, reverse 5-
GCCTTCTCCATGGTGGTGAA-3.
Statistical analysis –Data in this study are presented as mean ± SEM. Differences between
individual groups were analyzed by student t-test, with 2-tailed P value and less than 0.05
was considered statistically significant.
61
Chapter III: Targeting estrogen-related receptor alpha (ERRα) with
pyrrole-imidazole polyamide in PTEN loss-induced fatty liver and
tumorigenesis
III-1: Introduction
Estrogen-related receptor α (ERRα), an orphan member of the nuclear hormone
receptor superfamily, has recently been implicated to play important roles in lipid
metabolism, mitochondrial oxidative phosphorylation (OXPHOS) and cancer (Chang et
al., 2011; Huss et al., 2004; Schreiber et al., 2004). ERRα was first identified using the
DNA-binding domain (DBD) of estrogen receptor α (ERα) as a screen probe (Giguere et
al., 1988). The subsequent sequence analysis showed that ERRα shares about 68%
homology in DBD and 33% homology in ligand-binding domain (LBD) with estrogen
receptor α (ERα), which might explain the divergence in ligand binding nature between
these two receptors (Stein & McDonnell, 2006). Nucleotide sequence binding analysis
revealed ERRα preferentially binds to the consensus DNA sequence 5’-TCAAGGTCA-
3’, termed estrogen-related response element (ERRE) (Sladek et al., 1997). The
homozygous ERRα-null mice are found to be fertile and viable with reduced fat mass in
peripheral organs and resistance to high-fat diet-induced obesity (Luo et al., 2003). Under
cold exposure, ERRα knockouts failed to initiate the adaptive thermogenesis, a process
required to cope with cold environment, because of reduced mitochondrial mass together
with decreased expression of respiratory chain components and reduced mitochondrial
DNA copy number (Villena et al., 2007). Recently, several clinical oncology studies
showed that the elevated ERRα expression was significantly associated with unfavorable
clinical outcome, hormonal insensitivity and increased risk of recurrence in human breast
62
cancer, and ERRα might serve as a potent predictive biomarker of cancer therapy (Ariazi
et al., 2002; T. Suzuki et al., 2004).
We have previously reported liver-conditional deletion of tumor suppressor Pten
(phosphatase and tensin homolog deleted on chromosome 10), the negative regulator of
phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) pathway, led to an early
fatty liver phenotype followed by liver carcinogenesis (L. He et al., 2010). Moreover,
PTEN loss resulted in a dramatic induction of ERRα, which further contributes to
elevated mitochondrial bioenergetics, reactive oxygen species (ROS) production and
hepatocytes tumorigenicity (Figure 1) (Y. Li et al., 2013). Thus, ERRα might play
important roles in the regulation of hepatic lipid deposition and tumorigenesis.
In this study, we investigated the PTEN-ERRα signaling pathway in liver and
prostate cancer cell lines and evaluated the efficacy of targeting ERRα both in vitro and
in vivo using synthetic polyamides. Polyamides are DNA minor groove binders that are
consisting of pyrrole (Py) and imidazole (Im) linked in an anti-parallel fashion (Dervan,
2001). Sequence specificity is achieved by programing the side-by-side pairings of Py
and Im to recognize the different hydrogen bonding patterns of nucleotides. Im-py
recognizes G:C, Py-Im recognizes C:G whereas Py-Py is indifferent for A:T or T:A
(Kielkopf et al., 1998). Previously, specific polyamides have been developed to interfere
expression of genes induced by hypoxia-inducible factor-1, estrogen receptor α and
androgen receptor (Nickols et al., 2013; Olenyuk et al., 2004; Yang et al., 2013).
Therefore, ERRα’s activity could be modulated by polyamide specifically designed to
target the ERRα’s consensus binding site, ERRE.
63
Here, we confirmed that ERRα knockdown resulted in decreased mitochondrial
function and reduced cell proliferation in human liver and prostate cancer cell lines with
diminished PTEN expression, SNU398 and C4-2b (Thalmann et al., 1994; J. B. Wu et al.,
2014). . Using polyamide, 5’-WGGWCW-3’ (W, A or T) against the ERRE core
sequence, we showed that inhibiting ERRα’s transcriptional activity suppressed xenograft
formation and strikingly, completely blocked the endogenous fatty liver development
induced by Pten deletion. Together, our study reinforced the functional importance of
PTEN-ERRα signaling axis in tumor growth and lipid metabolism, and demonstrated for
the first time the potential of targeting ERRα in fatty liver and cancer.
III-2: Results
III-2-1: Pten deletion is associated with elevated ERRα expression in liver and
prostate cancer cell lines
We have shown that hepatic Pten deletion leads to up-regulation of estrogen-
related receptor alpha (ERRα) via activation of PI3K/AKT signaling pathway (Y. Li et
al., 2013). To further examine the PTEN-AKT-ERRα signaling axis in cancer cells, we
analyzed expression levels of p-AKT and ERRα in two Pten-null cancer cell lines, C4-2b,
a sub-line of LNCaP derivative C4-2 and SNU398, a liver cancer cell line. Expression
profiles of these proteins were also assessed in immortalized Pten-wt (wt) and Pten-null
(Pm) hepatocytes derived from Pten-wt/null mice (Figure III-1 A). As expected, Pm
hepatocytes express significantly higher p-AKT and ERRα than wt (Figure III-1 A). C4-
2b and SNU398 also exhibited substantial levels of p-AKT and ERRα. Transient wild
type PTEN (wtPTEN) overexpression in SNU398 cells resulted in decreased p-AKT and
ERRα in comparison with vector (vec)-transfected and naï ve cells, confirming that
64
PTEN/AKT signaling is responsible for controlling the ERRα expression in these human
cancer cell lines (Figure III-1 B). The PTEN-ERRα signaling axis might be important for
maintaining the mitochondrial function in order to meet the cellular energy demand as
ERRα plays a main role in nuclear-controlled mitochondrial biogenesis.
Figure III-1. Pten deletion is associated with elevated ERRα expression in liver and
prostate cancer cell lines.
A. Pten loss increases ERRα expression. Protein expression profiles of PTEN, pAKT,
ERRα and Actin in wt and Pten-null immortalized hepatocytes, as well as Pten-null
prostate (C4-2b) and liver (snu398) cancer cell lines. B. wtPTEN overexpression
suppresses ERRα expression. The wtPTEN plasmid and empty vector were transfected
into snu398 cells using lipofectamine 2000. Protein extract was collected 48 hours after
transfection and subjected to western blot analysis using antibodies against PTEN,
pAKT, ERRα and Actin.
III-2-2: ERRα knockdown reduces cytochrome c expression and impairs
mitochondrial function
ERRα regulates the expression of genes involved in mitochondrial OXPHOS and
the tricarboxylic acid cycle (Scarpulla, 2012). To study the role of ERRα in
mitochondrial respiration in our cell lines, we used siRNA to specifically knockdown
ERRα and assessed the subsequent effect on mitochondrial function. Western blotting
confirmed that protein levels of ERRα were effectively down regulated by siRNA in all
cell lines (Figure III-2 A). Cytochrome c (Cyt c) harbors ERRE in its promoter sequence
PTEN
Actin
pAKT
ERRα
wt Pten-null C4-2b snu398
Immortalized
Hepatocytes
Cancer cell line
Prostate Liver
PTEN
Actin
wtPTEN
pAKT
ERRα
Vec
Naive
SNU398
A B
65
and represents one of the well-established ERRα-downstream targets (Schreiber et al.,
2004). To confirm that the function of ERRα is inhibited in accordance with the siRNA-
mediated protein knockdown, we evaluated ERRα’s transcriptional function by
examining the promoter activity and mRNA level of cytochrome c. To do so, we stably
expressed a luciferase construct encoding cyt c’s promoter sequence of -686 to +55
followed by firefly luciferase in Pm hepatocytes. ERRα knockdown in these hepatocytes
resulted in a 25% reduction of luciferase activity, indicating ERRα’s transcriptional
activity is repressed (Figure III-2 B). The endogenous mRNA level of cyt c was also
quantified in Pm, C4-2b and SNU398 cells transfected with either siScramble or siERRα.
ERRα knockdown led to a 50% reduction of cyt c transcripts in Pm hepatocytes and C4-
2b cells, and approximately 20% decrease in SNU398 (Figure III-2 C).
Figure III-2. ERRα knockdown reduces cytochrome c expression.
A. ERRα protein levels were reduced by siERRα transfection in Pten-null hepatocytes,
C4-2b and snu-398. SiRNA against ERRα was transfected into Pten-null hepatocytes,
C4-2b and snu-398 using lipofectamine 2000. Cell protein lysates were collected 48
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Fold Change
pCytc-Luc Activity
RLU (Relative Light Units)
X 10
6
0
0.2
0.4
0.6
0.8
1
1.2
C4-2b
siScr
C4-2b
siERRα
Cytochrome c expression
ERRα
Actin
siCon siERRα
siERRα siCon
siERRα siCon
Pm hepatocytes
C4-2b
SNU398
0
0.2
0.4
0.6
0.8
1
1.2
SNU398
siScr
SNU398
siERRα
Cytochrome c promoter activity
0
2
4
6
8
10
12
Vehicle siERRα
*** *** **
*
**
A
B C
66
hours after transfection and subjected to western blot analysis using antibodies against
ERRα and Actin. B. Promoter activity of cytochrome c is reduced by siERRα. Pten-null
hepatocytes stably expressing luciferase construct containing cytochrome c’s promoter
sequence -686~+55 were generated. Luciferase activity was measured 48 hours after
siERRα transfection. C. mRNA level of cytochrome c was decreased by siERRα. Total
RNA was extracted from Pten-null hepatocytes, C4-2b and snu398, which were
transfected by siERRα. Reverse transcription and qPCR analysis were then carried out to
measure level of cytochrome c transcripts.
Having observed compromised ERRα transcriptional activity, we moved on to
study the effect of ERRα knockdown on mitochondrial function. Oxygen consumption
rate (OCR) was assessed as an indication of mitochondrial respiration using Seahorse
XF24 extracellular flux analyzer. The methodology was described previously (Y. Li et
al., 2013). SiERRα-transfected cells exhibited significant reductions of both basal and
maximal OCR compared to siScramble-transfected controls (Figure III-3 A, B, C).
Quantification of baseline OCR showed that siERRα resulted in a 50% reduction in all
cell lines (Figure III-3 D). Together, our analysis indicate that knockdown of ERRα
attenuates its transcriptional activity and further resulted in diminished mitochondrial
function.
67
Figure III-3. ERRα knockdown decreases mitochondrial function.
A, B, C. ERRα knockdown leads to decreased oxygen consumption rate (OCR) in Pten-
null hepatocytes, C4-2b and snu398 cells. Cellular OCR was measured using Seahorse
Extracellular Analyzer 48 hours after siERRα transfection in three cell lines. D. siERRα
knockdown leads to 50% decrease of baseline OCR.
III-2-3: ERRα knockdown impedes tumor cell proliferation
Although evidence show cancer cells favor glycolytic pathway instead of
mitochondrial oxidative phosphorylation (OXPHOS) to metabolize glucose, recent
studies suggested that tumor cells also depend on mitochondrial OXPHOS for energy
production and cell proliferation (Tan, Baty, & Berridge, 2014). Having shown ERRα is
responsible for maintaining the cellular respiration, we next investigated the effect of
siERRα on the cell growth. By monitoring the cell number for consecutive five days after
siERRα transfection, we observed that ERRα inhibition leads to a significant decrease of
Oxygen Consumption Rate (OCR)
Pten-null hepatocytes
O
2
pmole/min
O
2
pmole/min
Oxygen Consumption Rate (OCR)
C4-2b Prostate line
Oligomycin
FCCP
Rotenone
Oligomycin
FCCP
Rotenone
-50
0
50
100
150
200
250
300
350
1 8 16 23 31 39 46 54 62 69 77 85 92
siScr
siERR α
O
2
pmole/min
Time Time
Time
Oxygen Consumption Rate (OCR)
SNU398
Oligomycin
FCCP
Rotenone
Baseline OCR Quantification
O
2
pmole/min
-100
0
100
200
300
400
500
600
700
800
900
1000
1 8 16 23 31 39 46 54 62 69 77 85 92
siScr
siERR α
0
50
100
150
200
250
300
350
Pm C4-2b SNU398
siScr
siERRα
0
100
200
300
400
500
600
1 8 16 23 31 39 46 54 62 69 77 85 92
siScr
siERR α
***
***
***
A
B
C
D
68
cell growth rate starting from day 3 in SNU398 and C4-2b, and from day 4 in pm
hepatocytes. At the end of the growth curve measurement, the cell number of siScramble-
transfected groups is approximately two folds higher than the siERRα-transfected ones in
all cell lines (Figure III-4). These data offer robust evidence proving that ERRα inhibition
is capable of suppressing the cell proliferation likely due to the compromised
mitochondrial function. Our data suggest that ERRα plays an important role in regulating
mitochondrial function and reducing ERRα’s expression was able to not only inhibit
cellular respiration but also significantly decrease the cell growth rate. Therefore, ERRα
might be a potential therapeutic target for tumors bearing abnormal PTEN-ERRα
signaling.
Figure III-4. ERRα knockdown reduces tumor cell proliferation.
ERRα decreases proliferation rate of Pten-null hepatocytes, C4-2b and snu398 cells.
Three cell lines were transfected with siERRα for 48 hours. Equal numbers of siScramble
and siERRα-transfected cells were then placed in culture dish. Cell numbers were
monitored for five days.
III-2-4: Polyamide targeted to ERRE inhibits ERRα’s transcriptional activity and
mitochondrial function
To explore strategies targeting ERRα, we developed pyrrole-imidazole (Py-Im)
polyamides (PA), a type of synthetic amino acid oligomers linked in an antiparallel
fashion with the capability of binding to DNA minor groove with sequence specificity
Cell Number X 10
4
Day
Pten-null hepatocytes SNU398 C4-2b
0
50
100
150
200
250
300
350
400
1 2 3 4 5
siScr
siERRα
0
1
2
3
4
5
6
1 2 3 4 5
siScr
siERRα
0
5
10
15
20
25
30
1 2 3 4 5
siScr
siERRα
***
*
**
69
(Nickols et al., 2013). By employing PA targeted to ERRE, we could modulate the
ERRα-ERRE binding and thus, interfere ERRα’s transcriptional activity (Figure III-5 A).
We first verified the inhibitory effect of PA on the binding between ERRα and ERRE
using gel-shift assay (Figure III-5 B). Biotin-labeled DNA fragment containing ERRE
was incubated alone (lane 1), with nuclear extract (lane 2), with nuclear extract and cold
probe (lane 3) or with nuclear extract and PA supplied at two different concentrations
(lane 4 and 5). The “shift” was observed when nuclear extract was incubated with free
probe (lane 2). Cold probe diminished the observed “shift” (lane 3). As expected,
incubation with PA at both concentrations attenuated the “shift” to the same extent as
cold probe (lane 4, 5). Therefore, this result confirmed the ability of PA to disrupt the
ERRα-ERRE complex in vitro. We next employed PA in the cultured cells and assessed
its effect on ERRα’s function. Pm hepatocytes stably expressing luciferase construct
containing cyt c’s promoter were treated with PA at 0.2μM or 1μM followed by
luciferase activity measurement. 0.2μM PA was sufficient to reduce 30% of luciferase
activity compared to vehicle whereas 1μM PA further suppressed the activity by 50%
(Figure III-5 C). Same PA treatments were performed in Pm, C4-2b and SNU398 naï ve
cells followed by qPCR analysis designed to measure cyt c mRNA level. Results showed
a 60% reduction in Pm and C4-2b, and 25% reduction in SNU398 by 0.2μM PA
treatment. 1μM PA treatment resulted in further inhibition of cyt c transcripts in all the
cell lines (Figure III-5 D). Since reducing ERRα’s expression decreased mitochondrial
respiration, we next sought to study whether modulating ERRα’s activity by PA might
affect mitochondrial respiratory function. We measured OCRs of Pm, C4-2b and
SNU398 cells treated with vehicle, 0.2μM PA and 1μM PA. 0.2μM PA was capable of
70
reducing both baseline and maximal OCR levels in these cell lines compared to vehicle-
treated and naïve cells whereas a higher 1μM PA dose led to a more dramatic OCR
reduction (Figure III-5 E). The effect of PA on cell proliferation rate was measured by
cell number count for consecutive 5 days with constant PA treatment (Figure III-5 F).
Starting from day 3, PA-treated C4-2b and SNU398 cells showed slower cell growth
rates than vehicle-treated ones and the trend was sustained throughout the analysis time
range. In Pm cells, the growth suppression by PA was observed at Day 4 and became
magnified at Day 5 (Figure III-5 F). Among PA treated groups in all cell lines, 1μM PA
produced a more remarkable cell growth inhibition than 0.2μM PA. Taken together, these
analyses confirmed the inhibitory effect of PA on ERRα–ERRE binding and ERRα’s
transcriptional activity. Moreover, our data showed PA treatments led to significant
inhibitions on mitochondrial function and cell growth.
71
Free Probe
Nuclear Extract
Cold Probe
Polyamide 0.2μM
Polyamide 1μM
+ + + + +
+ + + +
+
+
+
Gel-Shift Assay
Shift
Lane
1 2 3 4
5
RLU (Relative Light Units)
X 10
6
Fold Change
Cytochrome c expression Cytochrome c promoter activity
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
0.2
0.4
0.6
0.8
1
1.2
0
0.2
0.4
0.6
0.8
1
1.2
Pten-null
hepatocytes
C4-2b
Prostate cancer line
SNU398
Liver cancer line
Pten-null
hepatocytes
0
2
4
6
8
10
12
Vehicle PA
0.2μM
PA 1μM
***
***
*
** **
**
n.s
*
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5
Mean Grey Value
*
**
**
Densitometry
B
C
D
pyrrole
imidazole
Polyamide targeting ERRE
GGWC
5’-A A G G T C A-3’
+ PA
NH
3
+
5’-T T C C A G T-3’
A
72
Figure III-5. Polyamide targeted to ERRE inhibits ERRα’s transcriptional activity,
mitochondrial function and cell proliferation.
A. Schematic structure illustration of polyamide (GGWC) targeted to ERRE. B.
Polyamide disrupts binding between ERRα and ERRE. Gel-shift assay was conducted
using labeled ds DNA probe containing ERRE, protein extract from cells overexpressing
ERRα, cold probe and polyamide supplied at two concentrations, 0.2μM and 1μM. Lane
1, labeled probe alone. Lane 2, labeled probe incubated with nuclear protein extract. Lane
3, cold probe is added into the reaction in lane 2. Lane 4 and 5, polyamide was incubated
with the components in lane 2. C. Polyamide treatment decreases cytochrome c’s
promoter activity. Pten-null hepatocytes stably expressing luciferase construct driven by
cytochrome c’s promoter was treated with polyamide for 48 h with two concentrations.
Cell lysates were collected and subjected to luciferase activity measurement. D.
Polyamide treatment decreases cytochrome c’s mRNA level. Pten-null hepatocytes, C4-
2b and snu-398 cells were pre-treated with polyamide for 48 h. Total RNA was extracted
from the treated cells and subjected to qPCR analysis for cytochrome c’s mRNA level. E.
Polyamide treatment decreases cellular OCR. Pten-null hepatocytes, C4-2b and snu-398
cells were pre-treated with polyamide for 48 h. OCR measurements were then conducted
using the treated cells. F. Cell proliferation was decreased by polyamide treatment. Pten-
null hepatocytes, C4-2b and snu-398 cells were pre-treated with polyamide for 48 h.
Equal numbers of different treatment group of cells were placed into the culture dish.
Cell number was monitored for five days.
Oxygen Consumption Rate (OCR)-PA treatment
Pten-null hepatocytes C4-2b
O
2
pmole/min
Time
Mins
Oligomycin
FCCP
Rotenone
Oligomycin
FCCP
Rotenone
SNU398
Oligomycin
FCCP
Rotenone
-100
0
100
200
300
400
500
600
1 8 16 23 31 39 46 54 62 69 77 85 92
-50
0
50
100
150
200
250
300
350
1 8 16 23 31 39 46 54 62 69 77 85 92
0
100
200
300
400
500
600
700
800
1 8 16 23 31 39 46 54 62 69 77 85 92
Vehicle
PA 0.2 μM
PA 1 μM
Cell Number X 10
4
Day
Pten-null hepatocytes
SNU398
C4-2b
0
50
100
150
200
250
300
350
400
1 2 3 4 5
Vehicle
PA 0.2 μM
PA 1 μM
0
1
2
3
4
5
6
1 2 3 4 5
Veh
PA 0.2 μM
PA 1 μM
0
5
10
15
20
25
30
1 2 3 4 5
Veh
PA 0.2 μM
PA 1 μM
Cell Population Doubling Assay
***
***
**
***
**
*
E
F
73
III-2-5: Inhibition of ERRα’s transcriptional activity by PA blocks fatty liver
development induced by Pten loss
ERRα functions in lipogenesis by transcriptionally regulating either enzymes
involved in lipid metabolism such as MCAD (medium-chain acyl-coenzyme A
dehydrogenase) (Huss & Kelly, 2004) or lipogenic transcriptional factors such as
SREBP-1, whose targets include FAS (fatty acid synthase), ACC (acetyl-CoA
carboxylase), etc (Ju, He, Zhao, Zheng, & Yang, 2012). ERRα knockout mice exhibited
significant reduction of fat deposition in peripheral organs and resistance to high-fat diet-
induced obesity (Luo et al., 2003). Thus, ERRα is strongly associated with lipid
homeostasis. We have reported the development of fatty liver phenotype at 1-3 month old
liver-conditional Pten-deleted mice in accordance with a robust up-regulation of ERRα
protein expression (Y. Li et al., 2013; Stiles, Wang, et al., 2004). To test whether ERRα
inhibition imposes effect on the fatty liver development upon Pten deletion, we treated
1.5-month old Pten-null mice with 25 nmole PA every four days for 1 month. The body
weight of PA-treated pm mice did not significantly differ from the non-treated ones,
suggesting a limited animal toxicity of PA treatment (Figure III-6 B). H&E staining
showed massive lipid depositions in the Pm liver section whereas wt liver maintained
normal hepatic morphology without visible lipid droplets (Figure III-6 A). Oil Red O
staining also showed increased lipid formation in Pm liver compared to wt. Strikingly,
the PA treatment on Pm mice completely inhibited the fatty liver phenotype evidenced by
minimal fat deposition observed in H&E section as well as the significantly reduced Oil
Red O staining signal (Figure III-6 A). In agreement with the tissue section results,
triglyceride (TG) quantification showed that PA treatment remarkably reduced the
74
elevated TG content in Pm livers, even to the level that is lower than wt (Figure III-6 C).
ERRα has been shown to act as a transcriptional activator for itself (Knutti & Kralli,
2001). Western analysis using liver extracts from wt, non-treated Pm and PA-treated Pm
mice revealed that the level of ERRα protein is decreased upon PA treatment (Figure III-
6 D). Further qPCR analysis showed mRNA reductions of two ERRα target genes,
MCAD and cyt c, after PA treatments in both hepatocytes and Pm mice, indicating PA is
able to suppress ERRα’s activity both in vitro and in vivo (Figure III-6 E, F). We
previously showed PTEN deletion in the liver resulted in up-regulation of lipogenic
enzyme expressions such as FAS and ACC, enzymes that catalyzed two initial processes
in lipid synthesis (L. He et al., 2010). Here, the elevated FAS and ACC mRNA levels
were significantly reduced by PA treatment in Pm mice, consistant with remissive fatty
liver phenotype observed in Pm mice treated with PA (Figure III-6 F). Together, the
results above demonstrated that in vivo polyamide administration in Pm mice inhibits
ERRα’s transcriptional activity and prevents the fatty liver development.
75
Figure III-6. Inhibition of ERRα’ activity by PA prevents fatty liver development
induced by Pten loss.
A. ERRα inhibition by PA prevents fatty liver development in Pten-null mice. 1.5-month-
old Pten-null mice were treated with PA for 1 month. At the end of the treatment, liver
slides were developed from wt, Pten-null non-treated and Pten-null PA-treated mice
livers. H&E and oil red o staining were conducted. B. Body weight of wt, Pten-null non-
treated and Pten-null PA-treated mice at the end of treatment. C. PA decreases
triglyceride level. The amount of triglyceride was measured in wt, Pten-null non-treated
and Pten-null PA-treated mice livers. D. PA treatment decreases expression of ERRα in
the liver. Liver lysates were extracted and subjected to western blot analysis using
antibodies against PTEN, pAKT, ERRα and Actin. E. PA treatment reduces MCAD
mRNA level in Pten-null hepatocytes. Pten-null hepatocytes were treated with PA at 0.2
and 1μM concentration. Total RNA was extracted and subjected to qPCR analysis for
MCAD mRNA level. F. PA treatment decreases expressions of Fas, Acc, MCAD and Cyt
C in vivo. Total RNA was extracted from wt, Pten-null non-treated and Pten-null PA-
Pten-wt
Pten-null (pm)
Non-Treated
Pten-null (pm)
PA-Treated
H&E Oil Red O
0
20
40
60
80
100
120
wt pm pm+pa
Triglyceride Quantification
Body Weight
g
0
5
10
15
20
25
30
wt pm pm+pa
mg/ g Liver
* *
A
B
C
PTEN
pAKT
ERRα
Actin
wt pm pm+PA
Fold Change
mRNA Expression
Liver Tissue
0
0.5
1
1.5
2
2.5
3
3.5
4
FAS ACC MCAD Cyt C
wt
pm
pm+pa
Fold Change
MCAD mRNA
Pm Hepatocytes+PA
0
0.2
0.4
0.6
0.8
1
1.2
Key enzymes
In Lipogenesis
ERRα downstream
targets
***
n.s
*** *
** *
** **
* ***
D E
F
76
treated mice livers. Qpcr analysis was used to measure the mRNA levels of Fas, Acc,
MCAD and cyt c.
III-2-6: Inhibiting ERRα’s transcriptional activity by PA impedes tumor growth in
xenograft
Following fatty liver at early age, the Pm mice develop liver tumor at 9-12 month
(Galicia et al., 2010). We next asked whether ERRα inhibition is capable of affecting
hepatocytes’ tumorigenecity. To answer this question, we subcutaneously implanted the
immortalized Pm hepatocytes derived from Pm mice into nude mice. When the engrafted
tumor volume reaches 50-100 mm
3
, mice were treated with PA for 1 month with
continuous tumor volume measurement. After the treatment was initiated, PA-treated
cells showed slower tumor growth rate than Vehicle-treated controls and the trend
sustained throughout the experiment (Figure III-7 A). The average weight of vehicle-
treated tumors is approximately two times higher than the weight of PA-treated tumors
(517± 100.2mg vs. 277± 24.8mg) (Figure III-7 B). Moreover, the PA treatment showed
limited toxicity indicated by the comparable body weight between two treatment groups
(Figure III-7 C). Representative engrafted tumor pictures were presented (Figure III-7 D).
77
Figure III-7. ERRα inhibition by PA exhibits anti-tumor activity in xenograft.
A. PA impedes engrafted tumor growth in xenograft model. 1x10
6
Pten-null
immortalized hepatocytes were subcutaneously injected into nude mice. 1-month PA
treatment was initiated when the engrafted tumor reaches to approximately 100mm
3
. The
treatment regime was 25 nmole per mice per injection, two injections per week. Tumor
size was constantly monitored during the treatment. B. The average weight of engrafted
tumor treated with PA was significantly lower than the one treated with vehicle. C.
Representative tumors from nude mice treated with PA or vehicle. D. Body weight of
treated nude mice.
III-2-7: Expressions of PTEN and ERRα is negatively correlated in cancer patients
and ERRα level is higher in cancer tissue versus normal counterparts
To further research the physiological relevance of PTEN-ERRα signaling cascade
and the therapeutic significance of targeting ERRα, we performed correlation analysis
between expressions of PTEN and ERRα in clinical datasets obtained from prostate
cancer patients (Oncomine.org). Similar to what we observed from liver cancer patients’
cohorts, a significantly negative correlation between PTEN and ERRα was shown in
mm
3
Days after treatment
mg
g
PA Vehicle PA Vehicle PA Vehicle
Tumors Taken From Nude Mice
Tumor Volume
Tumor Weight
Body Weight
*
0
5
10
15
20
25
30
35
0 15 18 22 25 29 32
PA
Vehicle
0
100
200
300
400
500
600
700
PA Vehicle
A
B
C
D
0
100
200
300
400
500
600
700
800
900
1 2 3 4 5 6 7
PA-Treated
Vehicle-Treated
**
*
*
78
seven independent cohorts, indicating the PTEN-ERRα signaling pathway exists in
human samples and might be associated with prostate cancer development (Figure III-8
A, three representatives were shown). Moreover, we also analyzed ERRα expression
levels in normal tissue versus liver/prostate cancer tissues and discovered that the ERRα
expression in liver and prostate tumors is higher compared to normal liver tissue and
prostate gland (Figure III-8 B). Therefore, theses analyses demonstrate the clinical
relevance of PTEN-ERRα signaling pathway and the association between ERRα and
cancer.
79
Figure III-8. Expressions of PTEN and ERRα is negatively correlated in cancer
patients and ERRα level is higher in cancer tissue versus normal counterparts.
A. The expressions of PTEN and ERRα are negatively correlated in prostate cancer
patients. Coexpression analysis was performed on clinical datasets of prostate cancer
patients. Prism Graphpad is used to analyze the data. B. The expression of ERRα in
higher in HCC and prostate carcinoma patients compared to normal counterpart tissues.
The ERRα expression level was analyzed using Prism in HCC versus Normal liver
samples, as well as Prostate Carcinoma versus Normal Prostate Gland. The datasets were
obtained from Oncomine.
PTEN
ERRα
Grasso, et al. Liu, et al.
ERRα
PTEN
TCGA
ERRα
PTEN
ERRα versus PTEN correlation analysis
Prostate Cancer Datasets
Log2 median-centered Intensity
R Squared: 0.2207 R Squared: 0.1285 R Squared: 0.08593
Cancer Vs. Normal Analysis
Chen, et al.
Normal
Liver
HCC
Log
2
Median-centered Intensity
P=0.0167
Normal Prostate
Gland
Prostate
Carcinoma
P=0.0052
Taylor, et al.
Log
2
Median-centered Intensity
Min: -1.14
Max: 1.813
Median: 0.8675
Min: -0.936
Max: 2.683
Median: 1.0405
Min: 0.05975
Max: 0.58197
Median: 0.226
Min: -0.0519
Max: 1.25047
Median: 0.33274
A
B
80
III-3: Discussion
By investigating the metabolic phenotypes of liver-specific Pten-null mice and
delineating the Pten-downstream signaling cascade, we previously reported that PTEN
loss and the subsequent activation of AKT led to enhanced mitochondrial mass and
function, accompanied by up-regulation of ERRα, the predominant mitochondrial
biogenesis regulator. In this study, we confirmed the ERRα induction by PTEN loss
signal in liver and prostate cancer cell lines, SNU398 and C4-2b. We also showed that
ERRα knockdown inhibits mitochondrial function and reduces tumor cell growth. To
modulate ERRα’s activity, the polyamide targeted to its consensus DNA binding site
ERRE was developed and showed desired efficacy against mitochondrial function, cancer
cell proliferation and xenograft tumor formation. Moreover, polyamide exhibits
promising potency in preventing endogenous fatty liver progression in Pten-null mice
due to its role in regulation of the lipogenic pathway. Similar to cohort analysis derived
from clinical datasets of liver cancer patients (Y. Li et al., 2013), negative correlation
between PTEN and ERRα expressions were observed from cohorts of prostate cancer
patients. Furthermore, gene expression analysis in cancer versus normal tissues revealed
increased ERRα expression in clinical liver and prostate carcinomas compared to normal
counterparts. These analyses demonstrated the influential roles and physiological
relevance of ERRα in Pten loss-induced tumorigenesis and lipid metabolic disorder.
ERRα controls nuclear transcriptional network for mitochondrial biogenesis via
mediating both mitochondrial components’ genes and transcriptional factors that
facilitate mitochondrial biogenesis (Villena & Kralli, 2008). ERRα itself shows limited
transcriptional activity. However, co-expression of its nuclear co-activator PGC-1α not
only potentiates its transcriptional activity but also induces its mRNA and protein level
81
(Laganiere et al., 2004; Schreiber et al., 2003). Following studies indicated other PGC-1
family members such as PGC-1 and PGC-1-related co-activator (PRC) also belong to
the nuclear co-activator family that interacts with ERRα (Kressler, Schreiber, Knutti, &
Kralli, 2002; Shao et al., 2010). Our previous reports indicate that PGC-1α’s expression
is induced by the p-AKT/p-CREB signaling axis, which is hyper-activated upon Pten
deletion. Thus, the PTEN/AKT/CREB/PGC-1α signaling pathway might underlie the
ERRα induction observed in our Pten-null model. Two other crucial nuclear
transcriptional factors in the mitochondrial regulatory machinery, nuclear respiratory
factor -1 and -2 (NRF-1, 2), are thought to activate mitochondrial gene expression
downstream of ERRα signal (Dufour et al., 2007). Thus, ERRα serves as a central
regulator to orchestrate the mitochondrial biogenesis, commonly reported in cardiac and
skeletal muscle, as well as in adipose tissue (Huss et al., 2004; Soriano et al., 2006). Our
study showed in hepatocytes and several cancer cell lines where PTEN signal is absent,
ERRα expression is induced and leads to increased mitochondrial mass and cellular
respiration. Knockdown of ERRα effectively impairs the mitochondrial metabolism in
these cell lines and decreases the cell growth rate.
Mitochondrial function has gained extensive attention in cancer research
primarily due to its crucial roles in energy homeostasis and glucose/lipid metabolism.
Even though cancer cells preferentially rely on glycolysis rather than OXPHOS to
catabolize glucose and it is hypothesized that the defects in mitochondrial respiratory
chain are the cause of the glycolytic phenotype, an increasing number of studies showed
mitochondrial function remain intact or even enhanced in many cancer cells, suggesting
the mitochondrial bioenergetics might play supportive roles during tumor transformation
82
and progression. For instance, a radio-resistant cell line derived from a patient with
esophageal adenocarcinoma showed increased numbers of mitochondria, more condensed
inner membrane structure, elevated rates of oxygen consumption, compared to its radio-
sensitive counterpart, revealing an association between mitochondria and cancer cell
radio-resistance (Lynam-Lennon et al., 2014). The cancer stem cell (CSC) population
isolated from epithelial ovarian cancer patient overexpressed genes involved in oxidative
phosphorylation and key enzymes for directing pyruvate to TCA cycle. Interestingly,
inhibitors for electron transport chain led to apoptosis in this CSC population (Pasto et
al., 2014b). Together these studies support the notion that mitochondrial oxidative
phosphorylation is strongly associated with the cancer initiation and progression, as well
as cancer cells’ tumorigenecity and resistance.
Our data clearly indicate that ERRα inhibition by either siRNA knockdown or PA
against ERRE resulted in not only decreased mitochondrial function but also
compromised cell proliferation, supporting the pivotal role of ERRα-controlled
mitochondrial metabolism in cancer cells. Those findings are coincident with previous
reports. Employing the ERRα inverse agonist XCT790 induced apoptosis in HepG2
hepatocarcinoma cell line and its multi-drug resistant sub-line, accompanied by dose-
dependent mitochondrial mass reduction (F. Wu et al., 2009). Moreover, a screening in
800 breast tumor samples revealed a positive correlation between shorter disease-free
survival in patients and higher ERRα activity. In vitro knockdown of ERRα’s co-
activator, PGC-1β, reduced proliferation of breast cancer cells (Chang et al., 2011).
While ERRα’s function is extensively studied in breast tumors due to its shared
homology and functional interference with estrogen signaling (Johnston et al., 1997), our
83
study shows ERRα inhibition has significantly inhibitory effects on mitochondrial
function and tumorigenecity of the prostate cancer cell line C4-2b. Our in vivo study
using xenograft showed the growth of subcutaneously engrafted tumor is dramatically
suppressed by the PA treatment. Thus, both genetic and pharmacological interventions of
ERRα are able to repress cell growth and tumorigenesis in mice. In line with our clinical
cohort analysis revealing the expression level of ERRα is significantly higher in liver and
prostate tumors compared with corresponding benign counterparts, a clinical examination
in prostate cancer patients showed higher ERRα immuno-reactivity (IR) score in
cancerous tissue compared to benign control and a poor survival rate in patients bearing
tumors with high ERRα expression (Fujimura et al., 2007). Therefore, others and we
together demonstrated the significant association between ERRα and tumor phenotypes,
as well as the therapeutic potential of targeting ERRα in cancer.
Besides its role in mitochondrial metabolism, ERRα functions to regulate lipid
homeostasis. The ERRα knockouts exhibit reduced body weight, decreased peripheral fat
deposition, and resistance to high fat diet-induced obesity (Luo et al., 2003). Histological
analysis of ERRα-knockout adipose tissue showed normal adipocyte number but with
decreased size. Concurrent microarray analysis showed significantly reduced expression
of genes involved in lipid synthesis, including fatty acid synthase (Fasn), stearoyl
coenzyme A desaturase 2 (Scd2), elongation of very long chain fatty acids-like 3
(Elovl3), etc (Luo et al., 2003). Moreover, it’s been shown that ERRα induces
adipogenesis via regulating expressions of lipogenic nuclear co-activators and
transcriptional factors such as Srebp-1c and PGC-1β (Ju et al., 2012). In our mice model
that carries liver-conditional PTEN deletion, hepatomegaly and fatty liver phenotype
84
were developed at early age from 1 to 3 month together with a significant induction of
ERRα expression (Galicia et al., 2010; Y. Li et al., 2013; Stiles, Wang, et al., 2004).
Thus, the liver Pten-null mice provide a valuable model to investigate the effect of
endogenous ERRα inhibition on fatty liver development. In this study, we administered
polyamide that targets ERRα in Pten-null mice at the age of 1.5 month when lipid
deposition is initiated. Surprisingly, we observed a noticeable restoration of fatty liver
phenotype together with reduced triglyceride content and lipogenic enzyme expression in
the Pten-null liver after 1-month polyamide treatment. Therefore, it is likely that under
the control of PTEN loss signaling, ERRα represents the main driving factor for the fatty
liver progression and offers promising therapeutic potential.
Given that ERRα plays an essential role in the nuclear regulatory network
governing the metabolic machinery and its prominent associations with fatty liver and
cancer etiology, targeting ERRα offers substantial therapeutic benefit. The strategy we
adopted is polyamide, a class of synthetic DNA-binding amino acid oligomers (Dervan,
2001). Other than small molecules modulating protein-protein interactions, polyamide
offers an alternative approach by interfering the binding between transcriptional factors
and their corresponding DNA sequence owing to its flexibility of Im-Py pairing that can
recognize different DNA base pairs. Recently, the possibilities of targeting nuclear
transcription factors have been widely explored. Polyamide targeted to hypoxia response
element (HRE), estrogen response element (ERE) and androgen response element (ARE)
were developed and demonstrated to possess distinct activity against their specific targets
(Nickols & Dervan, 2007; Nickols et al., 2013; Olenyuk et al., 2004). In this study, we
first showed polyamide, specifically designed against ERRE, is capable of disrupting the
85
binding between ERRα and ERRE in the gel shift assay. Further evaluations in both cell
and xenograft models revealed polyamide’s efficacy in inhibiting ERRα’s activity,
evidenced by markedly reduced mRNA transcripts of ERRα target genes and suppressed
engrafted tumor growth. Moreover, polyamide greatly rescued the endogenous fatty liver
progression and decreased the high triglyceride deposition in Pten-null mice without
causing detectable toxicity. Further biochemical analysis using liver extracts showed
decreased expressions of ERRα’s transcriptional targets involved in lipogenesis and
ERRα itself. Therefore, the polyamide targeted to ERRE showed phenomenal efficacy
against ERRα’s activity both in vitro and in vivo.
In summary, our study demonstrates that ERRα, controlled by PTEN/AKT
oncogenic signal, functions to regulate mitochondrial bioenergetics and lipid metabolism.
Reducing ERRα’s expression impedes cancer cells’ mitochondrial respiration and
proliferation. Polyamide designed to target ERRα-ERRE binding, as an ERRα inhibitor,
exhibits remarkable potency against ERRα’s activity in cell, fatty liver and xenograft
model.
III-4 Methods and Materials
Animals
Pten
loxP/loxP
; Alb-Cre+ (Pten-null Pm) and Pten
loxP/loxP
; Alb-Cre- (Pten-wt wt) mice were
reported previously (Stiles, Wang, et al., 2004). 4-6 weeks old nude mice used for
xenograft were ordered from The Jackson’s Laboratory. All experimental procedures
were conducted according to the Institutional Animal Care and Use Committee
guidelines of the University of Southern California.
Cell Culture
86
Wt and Pm hepatocytes were isolated from livers of wild type and Pten-null mice as
previously described (Zeng et al., 2011). Hepatocytes were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Mediatech) supplemented with 10% FBS (Atlas
Biologicals), 5μg/ml insulin (Sigma), and 10ng/ml epidermal growth factor (Invitrogen).
C4-2b prostate cancer cell line was cultured in RPMI-1640 medium supplemented with
10% FBS (Atlas Biologicals). SNU398 liver cancer cell line was cultured in DMEM
supplemented with 10% FBS. Pm hepatocytes were transfected with pGL4 luciferase
construct (Promega) expressing cytochrome c’s promoter followed firefly luciferase and
puromycin-resistant selection marker. Pm hepatocytes stably expressing luciferase
construct were subsequently selected by puromycin.
Reagents, Plasmids and siRNAs
Oligomycin, FCCP, and Rotenone were purchased from VWR. Wild type PTEN-
expressing plasmids pSG5-wtPTEN and pSG5 empty vector were previously described
(Zeng et al., 2011). Two siERRα targeting sequences are 5’-
ATCGAGAGATAGTGGTCACCATCAG-3’ and 5’-
ATCGAGAGATAGTGGTCACCATCAG-3’. Cytochrome c’s luciferase reporter
construct was generated by cloning the cytochrome c’s promoter sequence (-686 - +55)
into the luciferase construct pGL4 (Promega).
Transfection
Plasmids and siRNAs were transfected using lipofectamine 2000 (Invitrogen) according
to the manufacturer’s instructions. 4μg of DNA and 10μl of lipofectamine were delivered
into cells growing at 50% confluency in 60mm dishes.
Luciferase Reporter Assay
87
1x10
5
Pm hepatocytes stably expressing pCytc/-686Luc luciferase construct were plated
in each well of 6-well plates. The cell lysate preparation and luciferase activity
measurement were conducted according to the manufacturer’s instructions (Promega,
E1910)
Western Blot
Cell lysate preparation and immunoblot analysis were described previously (Zeng et al.,
2011). Antibodies against PTEN and p-AKT were purchased from Cell Signaling
Technology. Antibody against ERRα was purchased from Abcam. Anti-actin was
obtained from Sigma.
Seahorse XF24 Metabolic Flux Analysis
Cells were cultured on XF24 plates during experiment. The mitochondrial inhibitors,
oligomycin (1μM), FCCP (0.1μM) and Rotenone (1μM) were added after four
measurements of basal OCR. Details were described previously (Y. Li et al., 2013).
RNA Isolation, Reverse Transcription and Quantitative PCR
Total RNA was isolated using Trizol reagent from Invitrogen. Reverse transcription was
conducted using the reverse transcription system from Promga. Quantitative PCR was
performed using SYBR green qPCR mix (Thermo) and 7900 HT fast real-time PCR
system (Applied Biosystems). Gene-specific primers are as follows, cytochrome c
forward 5’-CCAGTGCCACACCGTTGAA-3’ and reverse 5’-
TCCCCAGATGATGCCTTTGTT-3’; Fas forward 5’-AGCGGCCATTTCCATTGCCC-
3’ and reverse 5’-CCATGCCCAGAGGGTGGTTG-3’; ACC forward 5’-
ACAGTGGAGCTAGAATTGGAC-3’ and reverse 5’-
ACTTCCCGACCAAGGACTTTG-3’; MCAD forward 5’-
88
TGTGGAGGTCTTGGACTTGGA-3’ and reverse 5’-
TCCTCAGTCATTCTCCCCAAA-3’; GAPDH forward 5’-
GCACAGTCAAGGCCGAGAAT-3’ and reverse 5’-GCCTTCTCCATGGTGGTGAA-
3’.
Xenograft
2x10
6
Pm cells suspended in 100μl PBS were injected subcutaneously into the right
flank area of every nude mouse (4-6 weeks, JAX). Polyamide treatment started when the
engrafted tumor reaches approximately to 50-150 mm
3
.
Tumor volume was measured
every three days after the polyamide treatment started.
Polyamide Treatment
For Pten-null mice and xenograft nude mice, 25nmole polyamide dissolved in 100μl PBS
supplemented with 5% DMSO was given per mouse every three days. Control group
received 5% DMSO in 100μl PBS per mouse. Treatments for both Pten-null mice and
xenograft sustained for one month.
H&E, Oil Red O staining and Triglyceride Content Determination
The methodologies were previously described (L. He et al., 2010).
Statistical Analysis
Data in this study were presented as mean ± SEM. Differences between individual groups
were analyzed by Student’s t-test, with 2-tailed p value, and p≤0.05 was considered
statistically significant.
89
Chapter IV: Distinct roles of AKT1 and AKT2 in the regulation of
hepatic mitochondrial metabolism.
IV-1: Introduction
In response to ligand binding such as hormones and growth factors, the receptor
tyrosine kinase (RTK) is phosphorylated at its cytoplasmic domain and recruits PI3Ks to
the plasma membrane via interaction of phospho-tyrosine residues on the receptor and
SRC-homology 2 (SH2) domain on the PI3K’s p85 regulatory subunit. Under some
circumstances, activated RTK phosphorylates the adaptor protein insulin receptor
substrate 1 or 2 (IRS1/2), which provides docking sites for p85’s SH2 domain and
mediates the interaction between PI3K and RTK. Following PI3K activation, the second
messenger PIP3 is generated and is responsible for recruiting pleckstrin-homology (PH)-
domain-containing proteins, such as AKT, a serine/threonine kinase, to the cell
membrane where it is phosphorylated and activated. AKT acts downstream of PI3K and
controls a variety of essential cellular processes, such as proliferation, growth, apoptosis
and metabolism. Three AKT isoforms, AKT-1, -2, -3, have been identified. AKT1 is
ubiquitously expressed and its homozygous deletion resulted in growth retardation and
high rate of apoptosis (Chen et al., 2001). AKT3 is mainly enriched in neuronal tissues
(Chin & Toker, 2009). AKT2 is also universally expressed but accounts for 84% of total
AKT protein in the liver and thus, represents a major hepatic AKT isoform (Lu et al.,
2012). To date, the insulin-mediated glucose homeostasis is the best-characterized
process with AKT isoform specificity. AKT2 knockout mice exhibit a type 2 diabetes-
like phenotype evidenced by fasting hyperglycemia, glucose intolerance and impaired
glucose uptake, suggesting a role of AKT2 in controlling glucose metabolism (Cho et al.,
90
2001). In vitro studies showed that AKT2 knockdown in adipocytes resulted in decreased
insulin-mediated GLUT4 translocation to plasma membrane (Jiang et al., 2003).
Interestingly, overexpression of AKT1 failed to rescue the impaired glucose uptake and
GLUT4 plasma membrane translocation in AKT2-deficient adipocytes, suggesting
divergent roles of AKT1 and AKT2 in regulating such events (Gonzalez & McGraw,
2009b).
Our laboratory generated a liver tumor mouse model that carries deletion of
PTEN, a prominent tumor suppressor, specifically in the liver. As a major effector
molecule downstream of the PTEN pathway, AKT2, which represents the dominant
hepatic AKT isoform, is highly up-regulated in liver cancer. We have shown that liver-
conditional Pten deletion resulted in enhanced glucose utilization via glycolysis and
mitochondrial OXPHOS, accompanying increased expressions of nuclear metabolic
regulators such as ERRα and PGC-1α. Eventually Pten-null mice experienced liver
inflammation followed by tumor development. Furthermore, the simultaneous deletion of
AKT2 largely attenuated the liver injury and inhibits liver cancer onset in Pten-null mice.
How AKT2 controls hepatic glucose metabolism however is not clear. In the present
study, we show that AKT2 regulates hepatic expressions of ERRα and PGC-1α, the
major nuclear transcription factor and co-activator for mitochondrial function. AKT2 and
Pten double deletion restores the induced mitochondrial content and respiration observed
with Pten deletion alone. Knockdown of AKT1 in Pten-deficient hepatocytes does not
cause significant change in mitochondrial function. Together, our findings suggest that as
the major AKT isoform that is hyper-activated upon Pten deletion, AKT2 predominantly
91
regulates mitochondrial function, as well as expressions of nuclear factors governing
mitochondrial biogenesis such as ERRα and PGC-1α.
IV-2: Results
IV-2-1: AKT2 is responsible for up-regulations of ERRα and PGC-1α upon Pten
loss
Our previous study using wild type and hepatic Pten-null mice showed that the
master regulator for mitochondrial transcription, ERRα and its co-activator PGC-1α, are
dramatically up-regulated by PTEN loss and contribute to elevated glucose metabolism
via mitochondrial OXPHOS. To elucidate the role of AKT2 in this process, we generated
the AKT2 knockout (A2) and PTEN/AKT2 double knockout (DMA2) mice and isolated
the primary hepatocytes from the mice strains, respectively. Protein expression analysis
using liver extracts shows that ERRα level is induced in Pm liver, and this induction is
significantly rescued in PTEN/AKT2 double knockout mouse liver (Figure IV-1 A).
ERRα’s expression level in DMA2 mice is also lower compared to Wt and Pm. Western
blot analysis using cultured primary hepatocytes revealed similar induction of ERRα and
its main co-activator, PGC-1α in Pm hepatocytes. However, this induction is remarkably
restored to the level comparable with Wt, suggesting that under the PTEN signaling
pathway, AKT2 plays a pivotal role in the regulation of nuclear factors controlling
mitochondrial function (Figure IV-1 B).
92
Figure IV-1. AKT2 is responsible for up-regulations of ERRα and PGC-1α upon
Pten loss.
A. Liver extracts were obtained from WT (wild type), PM (Pten-null), A2 (AKT2
knockout) and DMA2 (PTEN/AKT2 double knockout) mice. Western blot analysis was
employed to analyze protein expression levels of p-AKT, AKT2, p-CREB, ERRα and
PGC-1α. B. Cell lysates were obtained from the primary hepatocytes isolated from the
corresponding mice stains. Western blot analysis was employed to analyze expression
profiles of proteins in A.
IV-2-2: Increased mtDNA content and oxygen consumption in Pten-deficient
hepatocytes are rescued by AKT2 deletion
Since we established that ERRα and PGC-1α are subjected to AKT2 regulation
and given that ERRα and PGC-1α are major nuclear factors that control mitochondrial
homeostasis, we next sought to assess the impact of AKT2 deletion on mitochondrial
number and function. The ratio of COX II/Globin, an indication of mtDNA content, is
significantly higher in Pm cells. Deleting AKT2 together with PTEN reduces the
mitochondrial content evidenced by decreased COX II/Globin ratio (Figure IV-2 A). The
ratio in A2 hepatocytes is comparable to wt. Oxygen consumption rate (OCR) is
measured as a readout of mitochondrial function. Consistent with our previous results,
loss of PTEN by itself markedly induces OCR in immortalized hepatocytes. Interestingly,
Actin
AKT1
AKT2
PTEN
WT PM DMA2 A2
PGC-1α
ERRα
AKT1
PTEN
Primary Hepatocytes
P-AKT
AKT2
P-CREB
ERRα
Actin
WT PM A2 DMA2
Liver Extracts
A
B
93
double deletion of AKT2 and PTEN significantly restored the Pten loss-induced OCR
increase (Figure IV-2 B). Together these findings indicate that under the hepatic PTEN
loss stimuli, AKT2 plays an important role to regulate the mitochondrial bioenergetics.
Figure IV-2. Increased mtDNA content and oxygen consumption in Pten-deficient
hepatocytes are rescued by AKT2 deletion.
A. Genomic DNA was isolated from WT, PM, DMA2 and A2 primary hepatocytes. The
expression ratio of COX II/Globin was measured by qPCR. B. WT, PM and DMA2
primary hepatocytes were subjected to OCR measurement.
IV-2-3: Hepatic mitochondrial function did not significantly change by AKT1
deletion
To further explore the functional discrepancy in the metabolic regulation between
AKT1 and AKT2, we generated shAKT1/2-expressing GFP-positive lentivirus to stably
reduce the expressions of AKT1 or AKT2 in Pten-null hepatocytes. Western blotting
analysis confirmed that lentivirus-infected hepatocytes showed desired knockdown
efficiency against AKT1 or AKT2 (Figure IV-3 A). We then subjected the shAKT1
virus-infected hepatocytes to flow cytometry cell sorting in order to establish the GFP-
positive population, which represents the cell population that harbors AKT1 knockdown.
Following western blotting analysis showed high AKT1 knockdown efficiency in this
population (Figure IV-3 B). Interestingly, the expression level of ERRα and the
0
2
4
6
8
10
12
WT PM DMA2 A2
COX II/Globin
mtDNA
Fold Change
0
100
200
300
400
500
600
700
800
1 2 3 4 5 6 7 8 9 10 11 12 13
WT
PM
PTEN/AKT2 DM
OCR
94
mitochondrial OCR did not significantly differ between shScramble and shAKT1 groups
(Figure IV-3 C). The results suggest that hepatic AKT1 isoform may be dispensable for
mitochondrial function.
Figure IV-3. Hepatic mitochondrial function did not significantly change by AKT1
deletion.
A. Lentivirus expressing shRNA against mouse AKT1 and AKT2 and GFP were
constructed. Pten-null hepatocytes were infected with shAKT1/2 lentivirus. Cell lysates
from infected cells were subjected to western blot analysis for examination of AKT1 and
AKT2 expression. B. shLaZ and shAKT1-infected hepatocytes were subjected to flow
cytometry sorting for GFP-positive population after 5 days infection. Western blot
analysis was used to examine AKT1 and ERRα levels in sorted cell population. C.
FACS-sorted hepatocytes stably expressing either shLaZ or shAKT1 were subjected to
OCR measurement.
AKT2
Actin
shLacZ shAKT2
AKT1
Actin
shLacZ shAKT1
Lentivirus-infected Hepatocytes
AKT1
Actin
ERR
shLacZ shAKT1
FACS-Sorted Hepatocytes (GFP)
A
B
C
95
IV-3 Discussion
PTEN is a lipid phosphatase that dephosphorylates PIP3 and antagonizes the
PI3K/AKT pathway. Loss of PTEN results in hyper-activated AKT signaling and
tumorigenesis in multiple tissues (Vivanco & Sawyers, 2002). We have demonstrated
that hepatic PTEN loss led to liver cancer development, as well as increased
mitochondrial biogenesis and enhanced mitochondrial function in hepatocytes (Galicia et
al., 2010; Y. Li et al., 2013). The roles of two hepatic AKT isoforms, AKT1 and AKT2,
in this metabolic regulation still require further elaboration. Our previous study indicated
that liver-specific PTEN loss induces expressions of ERRα and PGC-1α, major nuclear
receptor and co-activator for regulating transcription of genes involved in mitochondria
assembly. In this study, in addition to wild type and liver-conditional Pten-null mice, we
generated unique mice models that carry PTEN/AKT1, or PTEN/AKT2 double deletion
and evaluated the functions of AKT1 and AKT2 in liver mitochondrial metabolism. Our
results show that AKT2 loss together with PTEN can rescue the up-regulations of ERRα
and PGC-1α induced by PTEN deletion. Expectably, AKT2 deletion in Pten-null
hepatocytes resulted in significant reductions of mitochondrial content and OCR.
Moreover, AKT1 knockdown in Pten-null hepatocytes showed marginal impacts on
ERRα’s expression and mitochondrial OCR. Together, those observations support a
crucial role for AKT2 in hepatic mitochondrial metabolism.
Acting downstream of growth factors and PI3K, AKT protein kinases are critical
regulators that control diverse cellular functions, from cell proliferation and growth to
survival and apoptosis. In mammals, AKT exists as three closely related isoforms, AKT1,
2, and 3. While AKT isoforms share a high degree of sequence similarity, they possess
distinct functions, suggested by distinct phenotypes of AKT isoforms knockouts. In
96
agreement with AKT’s canonical roles such as pro-survival and pro-growth, AKT1
knockout mice experience growth retardation and AKT1-null cells show high rate of
apoptosis (Chen et al., 2001). Interestingly, genetic AKT2 deletion did not affect cell
growth and survival. Instead, mice lacking AKT2 exhibited a type 2 diabetes-like
phenotype accompanied by impaired cellular glucose utilization, suggesting AKT2’s
dominant role lies in metabolic regulation (Garofalo et al., 2003). Recent studies
investigated the roles and mechanisms of AKT2 in the regulation of hepatic glucose
metabolism. In response to insulin signaling, AKT2 is the primary effector that blocks the
gluconeogenesis and directs the newly synthesized glucose-6-phosphate to the glycogen
synthesis. Mice lacking haptic AKT2 displayed elevated hepatic glucose output and
reduced glycogen accumulation, as well as failure to suppress glycogenolysis in response
to feeding (Wan et al., 2013). Therefore, AKT2 is required to maintain hepatic glucose
homeostasis in response to insulin signaling. A subcellular location study revealed that
AKT2 is co-localized with mitochondria (Santi & Lee, 2010). Given that AKT is found to
translocate onto mitochondria and enhance hexokinase II’s activity, AKT2, in the liver,
might couple the mitogenic signaling to glucose metabolism (Miyamoto, Murphy, &
Brown, 2008; Pelicano et al., 2006). In our study, we showed that AKT2 deletion in
hepatocytes led to decreased mitochondrial function likely due to the reduced
mitochondrial content. However, AKT1 deletion failed to produce the similar effect.
Therefore, AKT2 not only facilitates glycolysis by regulating hexokinase II, but also
promotes mitochondrial function via up-regulating nuclear factors that control
mitochondrial transcription, such as ERRα and PGC-1α. In line with previous reports,
others and we have demonstrated that the major role of hepatic AKT2 is to regulate
97
hepatic lipid and carbohydrate metabolism (L. He et al., 2010; X. Li, Monks, Ge, &
Birnbaum, 2007).
IV-4 Methods and Materials
Animals
Pten
loxP/loxP
; Alb-Cre+ (Pten-null Pm) and Pten
loxP/loxP
; Alb-Cre- (Pten-wt wt) mice were
reported previously (Stiles, Wang, et al., 2004). Pten
loxP/loxP
; Alb-Cre+, AKT1-/- and
Pten
loxP/loxP
; Alb-Cre+, AKT2-/- were generated. All experimental procedures were
conducted according to the Institutional Animal Care and Use Committee guidelines of
the University of Southern California.
Western Blot
Cell and liver lysate preparation and immunoblot analysis were described previously (Y.
Li et al., 2013; Zeng et al., 2011). Antibodies against PTEN, p-AKT, AKT1, AKT2 and
p-CREB were purchased from Cell Signaling Technology. Antibody against ERRα and
PGC-1α was purchased from Abcam. Anti-actin was obtained from Sigma.
MtDNA copy number measurement and oxygen consumption measurement
Refer to methods and materials section in chapter I.
Lentivirus generation, Hepatocytes infection and Flow cytometry cell sorting
Lentivirus expressing shRNA sequences was constructed according to the published
protocols (Tsen et al., 2013). The selected sense sequence for mouse shAKT1 is 5′-
TGAGGTTGACAGAGGAACA -3′, the anti-sense shAKT1 sequence is 5′-
TGTTCCTCTGTCAACCTCA -3′; The selected sense sequence for mouse shAKT2 is 5′-
CCAACCTTGGCTGTTACAC -3′, the anti-sense shAKT2 sequence is 5′-
GTGTAACAGCCAAGGTTGG -3′.
98
Pten-null hepatocytes were infected with shAKT1/AKT2 lentivirus and then cultured for
five consecutive days. Fluorescence for GFP was checked to examine the overall
efficiency of infection. Flow cytometry was employed to sort out the GFP-positive cells
from shAKT1 and shAKT2-infected hepatocytes. Stable GFP-positive lines were
established.
99
Chapter V: Overall Discussion
Ever since the “Warburg effect” illustrating tumor cell’s glycolytic dependency
was established, mitochondrial alterations have long been suspected to contribute to
cancer development and progression. In my study, I utilized the mouse model that carries
hepatic Pten deletion and investigated the molecular basis of how mitochondrial function
is regulated by PI3K/AKT pathway, which is the major mitogenic signal targeted by
PTEN and is hyper-activated in various cancer types. Having discovered that ERRα
functions to mediate mitochondrial biogenesis under Pten signal, my study adopted the
polyamide strategy to modulate ERRα’s activity and evaluated polyamide’s efficacy both
in vitro and in vivo. Moreover, I also investigated the distinct roles of AKT isoforms in
the regulation of these Pten loss-induced metabolic phenotypes.
Altered mitochondrial function has been intensively reported in clinical cancer
cases. Even though dys-regulated growth factor and mitogenic signaling have been linked
to altered mitochondrial function in cancer cells, molecular mechanisms of how these
signals control mitochondrial bioenergetics remain elusive. Using hepatic Pten deletion
as a molecular strategy, my results showed that the hyper-activation of PI3K/AKT signal
by PTEN loss led to increased mitochondrial mass and enhanced mitochondrial
respiratory capacity, accompanied by elevated glycolysis. Further analysis revealed that
PTEN/PI3K/AKT pathway regulates expression of ERRα, a major nuclear receptor
mediating mitochondrial transcription. My results also suggested that CREB, as a
putative AKT substrate, mediates the ERRα induction in response to PTEN loss.
Increased ERRα promotes hepatocyte’s proliferation, tumorigenecity and ROS
100
production. This part of my research provides for the first time the molecular evidence
linking aberrant mitogenic signaling and altered mitochondrial function.
As a major downstream effector of PI3K and a critical transducer of growth factor
signaling, AKT is traditionally recognized as a pro-survival kinase since its pivotal roles
in proliferation, apoptosis and growth. Recent studies have also underscored its functions
in the metabolic regulation. AKT has been shown to regulate glycolysis via multiple
mechanisms. First, AKT increases expressions or in some circumstances, plasma
membrane translocation of glucose transporters, and thus increases cellular glucose
uptake (Zaid, Antonescu, Randhawa, & Klip, 2008). Second, AKT phosphorylates and
activates phosphofructokinase-2 (PFK-2), whose product, fructose-2, 6-biphosphate acts
as an allosteric activator of the rate-limiting glycolytic enzyme, phosphofructokinase-1
(PFK-1) (Deprez, Vertommen, Alessi, Hue, & Rider, 1997). Third, the phosphorylation
and inactivation of FoxO by AKT is able to restore the suppression of glycolytic gene
expression (Zhang et al., 2006). Finally, AKT is found to promote the association of
hexokinase II with mitochondrial outer membrane, and therefore, primes hexokinase II to
immediate ATP availability for glucose phosphorylation and enhances glycolysis
(Gottlob et al., 2001). However, AKT has been shown to not only regulate glycolysis but
also oxidative phosphorylation. ATP-citrate lyase (ACL), the enzyme utilizing citrate
exported to cytoplasm to generate acetyl-CoA, is directly phosphorylated and activated
by AKT, so that AKT might function to drive the TCA cycle flux via increasing citrate
utilization (Berwick, Hers, Heesom, Moule, & Tavare, 2002). AKT, when
phosphorylated and activated, is found to translocate to mitochondria and promotes ATP
synthase activity, complex I expression and mitochondrial substrate availability (C. Li et
101
al., 2013). As discussed above, the mitochondria-hexokinase II association promoted by
AKT also increases the coupling efficiency between glycolysis and mitochondrial
oxidative phosphorylation. In the present study, my results demonstrated that Pten loss
enhances both glycolysis and respiration measured as ECAR and OCR, respectively.
Moreover, ECAR and OCR are positively correlated with AKT’s activity, suggesting that
AKT activation is able to produce a metabolic profile highly similar to that displayed by
tumor cells. In addition, my data suggested that AKT independently promotes both
oxidative phosphorylation and glycolysis, evidenced by simultaneous increase of both
ECAR and OCR when Pten is deleted or PI3K/AKT signaling is activated. To summarize,
in addition to being recognized as “pro-survival kinase” or “Warburg kinase” due to its
well-known roles in cellular proliferation and glycolysis, AKT also functions to facilitate
the mitochondrial bioenergetics.
ROS is produced as a byproduct of mitochondrial OXPHOS during the oxidation
of energy storage molecules. By reacting with catalytic cysteine residues to modulate
protein activity, ROS controls a variety of physiological processes such as cell
proliferation, growth and oncogenic transformation. At low levels, ROS is found to
promote cell growth and survival. However, if produced excessively, ROS might also be
harmful, leading to mutagenesis, cell death and cancer formation (Fruehauf & Meyskens,
2007). The accelerated ROS accumulation could be the leading cause for oxidative stress,
a condition defined as an imbalance state favoring the ROS production over anti-oxidant
defense. Cells within the highly metabolic organ are more likely to suffer from ROS-
induced damage due to the high rate of oxidation. Given that mitochondria process
oxidative phosphorylation and represent the major source for ROS, AKT-stimulated
102
increase in mitochondrial respiration can potentially lead to ROS accumulation. In
addition, studies discovered that AKT alternatively contributes to high level of ROS via
inactivating the scavenger enzymes (Nogueira et al., 2008). By investigating the liver
cancer mouse model where PI3K/AKT signaling is hyper-activated due to hepatic Pten
loss, we discovered high levels of ROS (H
2
O
2
) and oxidative stress condition,
accompanying fatty liver and tumor developments. In vitro Pten knockdown in isolated
wild type hepatocytes showed similar induction of cellular ROS level. The activated
AKT-ERRα signaling pathway governing mitochondrial biogenesis could be responsible
for this highly oxidative phenotype since when we manipulated mitochondrial function
by reducing ERRα expression, we observed significantly attenuated ROS production and
cell’s transforming ability together with decreased mitochondrial mass and function.
Thus, the oncogenic PI3K/AKT pathway might contribute to transformation and
tumorigenesis partially through elevating the mitochondrial bioenergetics and ROS
production.
Identified as the first orphan nuclear receptor, ERRα is recognized as a key
regulator of mitochondrial biogenesis and lipid metabolism. Disruption of these two
metabolic processes could lead to increased free radical production, lipid peroxidation
and oxidative stress, all of which are extensively implicated in the pathogenesis of
hepatic diseases such as fatty liver, nonalcoholic steatohepatitis (NASH) and
hepatocellular carcinoma (HCC) (Pagano et al., 2014; Sanyal et al., 2001; Y. Suzuki et al.,
2013; Tanaka et al., 2013; Vivekanandan et al., 2010). There are also strong indications
suggesting the involvement of ERRα in cancer and lipid disorders. Genetic deletion of
ERRα in mice resulted in reduced lipogenesis and resistance to high-fat diet induced
103
obesity. An allelic variant of ERRα’s promoter sequence, which is believed to sensitize
ERRα to PGC-1α activation, is found to be associated with significantly higher BMI and
might be a genetic factor in human obesity (Kamei et al., 2005). Moreover, high levels of
ERRα’s activity and expression are positively correlated with poor prognosis and low
survival rate in several cancer occurrences. Ever since the discovery of oncogenes and
tumor suppressors, inhibition of the oncogenic proteins and reactivation of tumor
suppressors has become the ultimate goal for cancer therapy. Although efforts have been
dedicated to develop strategies to restore silenced tumor suppressor functions, it remains
challenging to employ the tumor suppressor reactivation as a cancer therapy. Therefore,
inhibition of oncogenic proteins by small molecules has become a more feasible
pharmacological objective. Even though the oncogenic pathways involve numerous
proteins, they eventually converge upon nuclear transcriptional factors and change the
transcription pattern within the cell. Therefore, targeting this relatively small set of
oncogenic nuclear factors may offer a direct and effective way for cancer therapy. ERRα,
as a nuclear receptor, resides at the central signaling node to regulate cellular energy
metabolism that is frequently dys-regulated in metabolic disorders. My data suggest that
up-regulation of ERRα not only induces mitochondrial OCR but also leads to increased
ECAR, implying that excess citrate generated during glycolysis and TCA cycle might
travel back into cytosol and participate in fatty acid synthesis, an assumption which might
explain the Pten loss-associated lipogenic liver phenotype. The results of my study using
hepatic Pten deletion animal model highlighted the importance of ERRα in fatty liver and
cancer development. Together with previous reports, ERRα may proved to be an
104
attractive therapeutic target for pharmacological intervention in treating cancer and lipid
disorders.
The first x-ray crystallographic analysis of ERRα’s structure showed that ERRα’s
ligand binding domain (LBD) complexed with PGC-1α is able to adopt a
transcriptionally active conformation in the absence of a ligand in the ligand binding
pocket (LBP), providing strong evidence of ERRα’s ligand-independent activation
(Kallen et al., 2004). Furthermore, the estimated volume of LBP is only 100 Å and can
accommodate the binding of agonist containing only 4 or 5 carbons, which might explain
that the majority of pharmacologically active ERRα ligands identified via screening act
as antagonists. However, many of the identified antagonists exhibit cross-reactivity with
other ERR isoforms and nuclear receptors. As an alternative approach to specifically
target ERRα, we employed pyrrole-imidazole polyamide, which is able to bind to the
DNA minor groove and interfere with protein-DNA interactions. By programing the side-
by-side pairings of pyrrole and imidazole rings, we generated the polyamide with
sequence specificity against ERRE so that ERRα’s transcriptional activity could be
modulated. To our surprise, polyamide targeted to ERRE not only exhibits potency
towards ERRα in vitro, but also remarkably inhibits fatty liver phenotype and reduces
engrafted tumor growth in vivo. Thus, polyamide represents a novel targeting strategy
and offers attractive therapeutic potential.
Lipogenesis and fatty acid β-oxidation coordinately regulate lipid homeostasis.
Disruptions of both processes could contribute to abnormal fatty acid metabolism and
lipid disorders. When there is excessive carbohydrate intake, the surplus glucose is
converted to fatty acids and stored in the form of triglyceride (TG) preferentially in
105
adipose tissue. Liver is an important organ that metabolizes glucose and fatty acids, with
the ability to store a significant amount of TG as well in the condition of sustained excess
energy intake or impaired lipid homeostasis. In this study, mice with hepatic Pten
deletion develop severe fatty liver and hepatic steatosis phenotypes with increased fatty
acid synthesis and hepatic TG content. Potential reasons for the lipogenic phenotype
include: 1) excess dietary intake; 2) increased de novo synthesis of fatty acids, which
further form hepatic TG; 3) diminished oxidation of fatty acids. We reported that two key
enzymes involved in de novo fatty acid synthesis, fatty acid synthase (FASN) and acetyl
CoA carboxylase (ACC) are induced by Pten loss. Meanwhile, the expression of a fatty
acid oxidation enzyme, medium-chain acyl-coenzyme A dehydrogenase (MCAD) is
significantly increased both in vitro and in vivo in response to Pten loss, suggesting β-
oxidation might also be increased. Therefore, even though PTEN signaling is able to
affect both synthesis and oxidation of fatty acid, the perturbation caused by Pten loss
might favor lipogenic pathway and lead to development of fatty liver and hepatic
steatosis. Defective mitochondrial β-oxidation has long been recognized as a contributing
factor of increased lipid deposits and the development of non-alcoholic fatty liver
diseases. Impaired fatty acid oxidation has also been found in many patients (Pessayre,
Fromenty, & Mansouri, 2004). In the present study we discovered that Pten deletion
caused increased expression of ERRα, as well as enhanced mitochondrial biogenesis and
function, which might conflict with the established understanding of mitochondrial
dysfunction in the pathophysiology of fatty liver diseases. However, another important
function of ERRα is the regulation of lipogenesis pathway, supported by the ERRα
knockout study and the discovery that ERRα induces SREBP-1, the central lipogenic
106
transcription factor. Therefore, it is likely that the effect of ERRα on the mitochondrial
function is overshadowed by its impact on the lipogenic program, leading to the
development of fatty liver diseases. Therefore, when polyamide treatment was adopted,
the de novo lipid synthesis is affected to a relatively higher extent compared to the
mitochondrial function, leading to the ultimate prevention of fatty liver phenotype in
Pten-null mice. However, there are solid in vitro evidences showing that polyamide
affects mitochondrial function. In the future, it will be interesting to study 1) how PTEN
globally regulates hepatic lipid metabolism and mitochondrial β-oxidation by using
deuterium labeling and seahorse methods; 2) whether ERRα ablation suppresses Pten
loss-induced tumorigenesis by in vivo employing polyamide or the generation of
ERRα/PTEN knockout mice; 3) a microarray analysis using polyamide treatment to gain
a comprehensive view of how ERRα-targeting polyamide affects the overall
transcriptional network.
107
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Abstract (if available)
Abstract
Mitochondrial abnormalities are associated with cancer development, yet how oncogenic signals affect mitochondrial function has not been fully understood. The purpose of the current study is to investigate whether and how the oncogenic signal, PTEN (Phosphatase and tensin homolog deleted on chromosome 10)/PI3K (phosphoinositide 3-kinase)/AKT (Protein kinase B) pathway, might control mitochondrial biogenesis and function. ❧ Using a isogenic cell system established from mice carrying liver-specific deletion of Pten, which leads to activation of PI3K/AKT, We showed that PTEN loss leads to elevated oxidative stress, increased mitochondrial mass and augmented respiration, accompanied by up-regulation of estrogen-related receptor α (ERRα), an orphan nuclear receptor known for its role in mitochondrial biogenesis. Our pharmacological and genetic studies further illustrated that the PI3K/AKT/CREB/PGC-1α signal axis acts downstream of PTEN to regulate ERRα’s expression. Preliminary analysis suggests that AKT may directly phosphorylate CREB and controls its activity in a manner that is independent of PKA. Our data further demonstrated that AKT2, but not AKT1, is responsible for regulating the expressions of ERRα and PGC-1α, establishing isoform specific functions for AKT2. ERRα knockdown significantly attenuated proliferation and colony forming potential in Pten-null hepatocytes as well as human tumor cell lines where PTEN expression is dimished. Moreover, analysis of datasets from clinical samples showed a negative correlation between expressions of ERRα and PTEN in multiple tumor types. Together, our data established a previously unrecognized link between a growth signal and mitochondrial metabolism. ❧ To explore the effect of inhibiting ERRα in vivo, a class of synthetic amino acid oligomers capable of binding to DNA minor groove with sequence specificity, polyamide, was developed to specifically bind to the DNA sequence that ERRα needs to bind for its function. This binding thus blocks the binding of ERRα and its transactivation activity. We show here that inhibiting ERRα by polyamide hampered the growth of grafted tumors. Furthermore, In vivo polyamide administration in 1.5-month-old Pten-null mice completely blocked the fatty liver development and led to decreased expression of key lipogenic enzymes such as Fas (fatty acid synthase) and Acc (acety-CoA carboxylase). ❧ In summary, data from these studies established for the first time the PI3K/AKT/CREB/PGC-1α signaling pathway for the regulation of ERRα and mitochondrial biogenesis. The ERRα up-regulation upon PTEN loss is important for maintaining the mitochondrial function, ROS production and cell proliferation. As a major hepatic AKT isoform, AKT2 is found to be responsible for the regulation of the observed metabolic events induced by Pten loss. By adopting the novel polyamide strategy to modulate ERRα’s transcriptional activity, we demonstrated the potential of ERRα inhibition in targeting fatty liver and tumorigenesis.
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Li, Yang
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Core Title
Regulation of mitochondrial bioenergetics via PTEN (phosphatase and tensin homolog deleted on chromosome 10)/estrogen-related receptor alpha (ERRα) signaling
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School of Pharmacy
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Doctor of Philosophy
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Molecular Pharmacology and Toxicology
Publication Date
10/24/2014
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10/24/2014
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