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Maf1 is a novel target of the tumor suppressor PTEN and a negative regulator of lipid metabolism

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Maf1 is a novel target of the tumor suppressor PTEN and a negative regulator of lipid metabolism

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MAF1 IS A NOVEL TARGET OF THE TUMOR SUPPRESSOR PTEN AND A
NEGATIVE REGULATOR OF LIPID METABOLISM

by

Beth Marie Palian

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 PHILOSPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)

August 2012

Copyright 2012 Beth Marie Palian
ii

Dedication
To my mother and daughter.

iii

Acknowledgements
I would like to thank my mentor, Debbie Johnson, for her guidance and support in my
professional and personal life, and for providing a s e c o n d h o m e f o r m y f i r s t b a by … my
dog, Bella. I would also like to thank my committee members Dr. Bangyan Stiles and Dr.
Louis Dubeau for helpful suggestions and collaboration. My research would not have
been possible without the many reagents generously contributed by Dr. Stiles and Dr.
Dubeau provided valuable assistance with our human cancer studies. I am grateful to all
the members of the Johnson lab, past and present, for their emotional and technical
support. Special thanks go to Aarti Rohira for her friendship and for assistance with
chromatin immunoprecipitaion experiments, and to Dr. Sandra Johnson for her work on
the human prostate cancer immunohistochemistry and for her assistance with generating
stable cell lines. I would also like to thank the Stiles Lab, especially Lina He and Ni Zeng
for generating lysates from all the mouse liver tissue that was used in this work.

T h e s e l a s t 7 y e a r s w i t h o ut m y m o t h e r h a v e n’ t been easy and I want to thank my family
and friends that have supported me during this time. Their love and humor has helped me
deal with the stresses of graduate school and I know they are as excited as I am for me to
graduate. I especially want to thank my husband, Zack, for always being my biggest
cheerleader. I would not have made it this far without his unwavering love and support.
And finally, I want to thank my daughter, Summer, for being the biggest and best
motivation to finish this dissertation.
iv

Table of Contents
Dedication ................................................................................................................................. ii
Acknowledgements ................................................................................................................. iii
List of Figures.......................................................................................................................... vi
Abstract ................................................................................................................................... vii
CHAPTER 1: Introduction ...................................................................................................... 1
1.1 The Warburg Effect. ...................................................................................................... 1
1.2 De Novo Lipogenesis ..................................................................................................... 2
1.3 PTEN and PI3K Signaling ............................................................................................. 3
1.4 Maf1 ................................................................................................................................ 7
CHAPTER 2: PTEN Regulates Maf1 ................................................................................... 11
2.1 PTEN Positively Regulates Maf1 Expression in Various Cell Lines and Tissues. . 11
2.2 Maf1 Expression Is Decreased in Human Cancers. ................................................... 15
2.3 Summary ....................................................................................................................... 17
CHAPTER 3: Maf1 Is Regulated through the Cell Cycle ................................................... 18
3.1 PTEN Induction Increases Maf1 Protein and Requires Its Phosphatase Activity. .. 18
3.2 PTEN-Mediated Induction of Maf1 Requires Changes in Cell Cycle Regulators. . 20
3.3 Maf1 Is Regulated through the Cell Cycle. ................................................................ 21
3.4 Summary ....................................................................................................................... 23
CHAPTER 4: PI3K Signaling Negatively Regulates Maf1 ................................................ 24
4.1 Inhibition of PI3K Signaling Induces Maf1 Expression............................................ 24
4.2 AKT2 Negatively Regulates Maf1 Expression .......................................................... 25
4.3 mTOR Negatively Regulates Maf1 Activity but Has No Effect on Its Expression. 27
4.4 FoxO1 Positively Regulates Maf1 Expression........................................................... 29
4.5 Maf1 Expression Is Regulated by PI3K Signaling In Vivo. ...................................... 31
4.6 Summary ....................................................................................................................... 32
v

CHAPTER 5: Maf1 Regulates Genes Involved in De Novo Lipogenesis .......................... 33
5.1 Maf1 Regulates FASN and ACC1 Transcription....................................................... 33
5.2 Maf1 Regulates FAS and ACC1 Protein Levels. ....................................................... 35
5.3 Maf1 Directly Represses FASN at the Promoter. ...................................................... 36
5.4 Maf1 Represses Intracellular Lipid Accumulation. ................................................... 38
5.5 Summary ....................................................................................................................... 39
CHAPTER 6: Discussion ...................................................................................................... 40
6.1 PTEN Regulates Maf1 Expression ............................................................................. 40
6.2 A Novel Mechanism of Maf1 Regulation .................................................................. 41
6.4 Maf1 Represses These Genes at the Promoter ........................................................... 43
6.5 PI3K Coordinately Regulates Maf1 and SREBP1c to Control Lipogenesis. ........... 44
6.6 Potential Role of Maf1 in Human Disease ................................................................. 46
CHAPTER 7: Materials and Methods .................................................................................. 48
References............................................................................................................................... 57

vi

List of Figures
Figure 1. Maf1 expression is decreased in cells lacking Pten. 12
Figure 2. PTEN regulates Maf1 expression in mouse livers 13
Figure 3. PTEN regulates Maf1 expression in mouse prostates 14
Figure 4. Maf1 expression is altered in human cancers 16
Figure 5. Induction of wild-type PTEN but not a phosphatase defective mutant form
induces Maf1 protein expression in cells lacking endogenous PTEN.

19
Figure 6. Overexpression of cyclin D1 prevents PTEN-mediated increases in Maf1
expression.

20
Figure 7. Maf1 expression is lowest in S phase. 22
Figure 8. Pharmacologic inhibition of PI3K signaling increases Maf1 expression in
various cell lines.

24
Figure 9. Akt2 negatively regulates Maf1 expression in vitro and in vivo. 26
Figure 10. Rapamycin treatment does not regulate Maf1 expression, but does
enhance Maf1 repression of target genes.

28
Figure 11. FoxO1 positively regulates Maf1 expression and negatively regulates
Maf1 target genes.

30
Figure 12. Mice fed a high carbohydrate diet display a reduction in Maf1 protein
levels in the liver.

31
Figure 13. Maf1 negatively regulates fatty acid synthase (FASN) and acetyl-coA
carboxylase (ACC1) mRNA expression

34
Figure 14. Maf1 negatively regulates cellular FASN and ACC1 protein expression. 35
Figure 15. Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the
FASN promoter.

37
Figure 16. Maf1 represses intracellular lipid accumulation. 38
Figure 17. Schematic representing the regulation and function of Maf1. 45
vii

Abstract
Although Maf1 has been identified as a central negative regulator of transcription, little is
known about its regulation and it is likely that many of its target genes have yet to be
identified. Our finding that Maf1 can suppress cellular transformation led us to examine
whether Maf1 might be regulated by the tumor suppressor, PTEN. Indeed, Pten-deficient
cell lines and mouse tissues show marked decreases in Maf1 protein expression.
Consistent with these results, induction of PTEN expression in human glioblastoma cells
results in increased Maf1 expression. Pharmacologic inhibitors of PI3K signaling also
induce Maf1 expression, and expression of a phosphatase defective mutant PTEN fails to
alter Maf1 expression. These data suggest that regulation of Maf1 expression by PTEN is
due, at least in part, to its ability to inhibit the PI3K signaling pathway. Loss of Akt2 in
the liver of mice results in increased Maf1 protein expression. Additionally, loss of Akt2
in combination with Pten loss rescues the decreased Maf1 expression that was caused by
Pten loss alone. Ectopic expression of FoxO1, a downstream target of Akt, increases
Maf1 protein expression. Given the established role for the PI3K/AKT/FoxO1 pathway in
regulating lipid biosynthesis, we investigated whether Maf1 could regulate these genes.
We show that enzymes required for lipid biogenesis, including FASN and ACC1, are
repressed by Maf1. Repressing Maf1 expression in Huh7 hepatoma cells resulted in
increased lipid accumulation. A marked decrease in Maf1 staining is observed in Pten-
null mouse prostate and human prostate cancer tissue. Together these studies identify
Maf1 as a downstream target of PTEN and a novel regulator of lipogenic gene
expression.
1

CHAPTER 1: Introduction
1.1 The Warburg Effect.
Nearly a century ago, Otto Warburg observed that cancer cells metabolize glucose
differently than normal cells. While normal cells preferentially metabolize glucose using
mitochondrial oxidative phosphorylation to generate energy, cancer cells rely on
glycolysis even in the presence of sufficient oxygen (Warburg, 1956). This preference
for “ a e r o bi c g ly c o l y s i s ” wa s r e f e r r e d to a s t h e W a r b ur g e f f e c t . It was initially suggested
that the Warburg effect was due to a defect in mitochondrial function and that it may
represent a causal event in cancer progression (Warburg, 1956). However, recent work
has shown that mitochondrial function is not impaired in many cancer cells (Vander
Heiden et al., 2009), and ATP production in cancer cells was not significantly altered
when mitochondrial function was completely blocked (Ramanathan et al., 2005). While
some have suggested that increased rate of glycolysis in cancer cells is merely an
adaptation to a hypoxic tumor environment (Furuta et al., 2010), the idea of the Warburg
effect as a causal event in cancer has returned to favor with the recent findings that both
oncogenes and tumor suppressors may regulate the metabolic switch between aerobic
glycolysis and oxidative phosphorylation (Levine and Puzio-Kuter, 2010). In order for
cancer cells to rapidly proliferate, they must generate a large amount of nucleotides,
amino acids and lipids used for synthesis of DNA, protein and cell membranes.
Accordingly, three hallmarks of the transformed phenotype are increased carbon flux
through glycolysis, increased rates of protein and DNA synthesis, and finally increased
rate of de novo fatty acid synthesis (Menendez and Lupu, 2007). Recently it has been
2

proposed that cancer cells preferentially metabolize glucose through glycolysis because
the requirements to generate this cellular biomass are greater than the need for ATP
(Vander Heiden et al., 2009). Therefore, it has become clear that understanding cancer
metabolism will have an important impact on our treatment of cancer.

1.2 De Novo Lipogenesis
De novo fatty acid synthesis is normally low in most adult tissues, and dietary lipids are
preferentially utilized for the production of new structural lipids. By contrast, cancer
cells undergo a significant induction of endogenous fatty acid synthesis independent of
the amount of circulating lipids (Menendez and Lupu, 2007). In fact, practically all of the
esterified fatty acids in tumor cells are derived from de novo synthesis, and the majority
of these are incorporated into the membranes of proliferating cells (Menendez and Lupu,
2007; Ookhtens et al., 1984). This dramatic induction in de novo fatty acid synthesis is
caused by the increased expression and activity of key lipogenic enzymes including ATP
citrate lyase (ACLY), Acetyl-CoA Carboxylase (ACC), and Fatty acid synthase (FASN).
Induction of these enzymes is largely dependent on the transcription factor Sterol
Regulatory Element- binding Protein (SREBP) (Eberle et al., 2004). Although these
enzymes are abundantly expressed during embryonic development, they are maintained
at low levels in most normal adult tissues. However, lipogenic enzymes are markedly
increased in several human epithelial cancers including breast, prostate, ovary,
endometrial, colon, lung, and hepatocellular carcinoma (Kuhajda, 2000). Overexpression
of FASN is positively correlated with clinical aggressive cancers and poor patient
3

prognosis (Alo et al., 1996; Kuhajda, 2000). Elevated expression of lipogenic enzymes
occurs at an early stage in cancer and is thought to play an important role in tumor
progression (Furuta et al., 2010). Consequently, inhibition of these enzymes has been
shown to preferentially induce growth arrest and apoptosis of tumor cells, making these
enzymes attractive targets for cancer therapy (Menendez and Lupu, 2007; Pizer et al.,
1996).

1.3 PTEN and PI3K Signaling
PTEN, or phosphatase and tensin homolog deleted on chromosome 10, is the second
most frequently inactivated tumor suppressor gene in human cancer behind only p53
(Stiles et al., 2004a; Stokoe, 2001). Germline mutations of PTEN are linked to Cowden
disease and other proliferative syndromes that are classified as PTEN hamartoma tumor
syndromes (Hollander et al., 2011). These patients develop benign tumors in multiple
organs and have increased risk of development of brain, breast and thyroid cancers (Eng,
1998; Liaw et al., 1997). High frequency of PTEN mutation or deletion is also observed
in sporadic cancers of the brain, breast, bladder, prostate and endometrium (Stiles et al.,
2004a). Since Pten null mouse embryos die early in embryogenesis, much of what is
known about PTEN function comes from tissue-specific mouse models. Deletion of Pten
is sufficient to cause tumors in certain tissues, and in other tissues Pten loss enhances
tumorigenesis in combination with other genetic alterations (Hollander et al., 2011).
While it is clear that complete loss of PTEN function contributes to cancer, it has also
been reported that PTEN dose is a critical determinant in cancer progression, and even a
4

slight reduction in PTEN levels can dictate cancer susceptibility (Alimonti et al., 2010;
Trotman et al., 2003). Recent studies demonstrated that systemic elevation of PTEN in
mouse models induces an “ a n t i-Warbur g” metabolic state with decreased glucose uptake
and increased mitochondrial ATP production that renders cells and mice resistant to
transformation and cancer development (Garcia-Cao et al., 2012). These studies point to
the important role of PTEN in maintaining normal cellular homeostasis and indicate that
P T E N’ s e f f e c t s o n m e t a b o l i s m m i g h t b e c r i t i c a l f o r t hi s t u m o r s uppr e s s i v e f u n c t i o n .

PTEN is a dual specificity phosphatase that acts on both protein and lipid substrates, and
its phosphatase domain and lipid binding domain are both required for its tumor
suppressor activity (Hollander et al., 2011). Although PTEN has been shown to also
function in the nucleus, it primarily functions at the cell membrane where it opposes the
action of phosphatidyl- i n o s i t o l 3 ’ kinase (PI3K). In response to growth factors and
insulin, PI3K phosphorylates the 3 ’ position of phosphatidylinositol (4,5) bisphosphate
(PIP
2
) to create phosphatidylinositol (3,4,5) trisphosphate, (PIP
3
). PIP
3
acts as an
important second messenger leading to the activation of several downstream kinases with
roles in cell cycle progression, metabolism, migration, apoptosis, transcription and
translation (Hollander et al., 2011). PTEN directly dephosphorylates PIP
3
thereby
limiting its growth- promoting downstream effects (Maehama and Dixon, 1998;
Stambolic et al., 1998). Several cancer drugs that inhibit PI3K and/or its downstream
effectors are now in clinical trials and have had some success, highlighting the
importa n c e o f P T E N’ s f u n c t i o n in tumor suppression.
5

AKT
The serine/ threonine kinase, AKT, is a major downstream target of PI3K pathway and is
thought to be “one of the most important and versatile protein kinases at the core of
h u m a n p hy s i o l o g y a n d d i s e a s e ” (Manning and Cantley, 2007). By acting on an ever-
increasing list of substrates, AKT has diverse roles in regulating growth, proliferation,
migration, cell survival, angiogenesis, and metabolism (Manning and Cantley, 2007).
Although the three AKT isoforms are structurally homologous, they have different
patterns of expression, and animal models and human cancer studies have shown that
they have distinct functions (Bellacosa et al., 2004). As AKT2 is the predominant
isoform in the liver, it acts as the major mediator of the metabolic actions of insulin (Wan
et al., 2011). AKT2 is required to induce hepatic lipid accumulation through activation of
the transcription factor, SREBP1c (Krycer et al., 2010; Leavens et al., 2009). AKT2 is
overexpressed in breast, prostate, colon, liver, ovarian, and pancreatic cancers, and has
been shown to play a critical role in metastasis (Rychahou et al., 2008). Consequently,
increased expression of AKT2 in tumor tissue is positively correlated with aggressiveness
and poor prognosis (Bellacosa et al., 1995).

mTOR
Mammalian target of rapamycin (mTOR) is a downstream target of AKT and an
important sensor of nutrient, growth factor, and stress signals (Sengupta et al., 2010). It is
a highly conserved serine/threonine kinase that functions in two distinct protein
complexes, mTORC1 and mTORC2, which differ in their regulation and downstream
6

targets (Guertin and Sabatini, 2007). Phosphorylation of the downstream targets of
TORC1 leads to increases in translation, proliferation, and cell growth. While a role for
mTORC2 in lipid biosynthesis is still unclear, mTORC1 has been shown to play an
important role in lipid biogenesis through its regulation of the master lipogenic regulator,
SREBP1 (Laplante and Sabatini, 2009). mTORC1 induces SREBP1 through multiple
mechanisms including transcription, processing, and translocation, and its activity is
required for Akt2-dependent lipogenesis (Lewis et al., 2011; Porstmann et al., 2008). As
mTORC1 is responsible for stimulating protein synthesis and lipogenesis, elevated
mTOR activity is commonly detected in obesity and the majority of human cancers
(Duvel et al., 2010).

FoxO1
The FoxO proteins belong to a larger family of Forkhead box transcription factors that
contain a highly conserved winged helix/forkhead DNA-binding domain. FoxO proteins
are potent transcriptional activators that play an important role in longevity and tumor
suppression through diverse functions on differentiation, proliferation, apoptosis, stress
resistance, and metabolism (Calnan and Brunet, 2008; Zhang et al., 2006). Members of
the FoxO subfamily, particularly FoxO1, have received attention due to discovery that
they are key downstream targets of insulin signaling that regulate metabolism. Through
the activation of the PI3K signaling pathway, insulin stimulates AKT to directly
phosphorylate FoxO1 at multiple residues causing its nuclear export and inactivation
(Biggs et al., 1999; Brunet et al., 1999). Although FoxO1 is clearly regulated by insulin
7

signaling, its role in regulating lipid metabolism has been somewhat controversial (Gross
et al., 2008). Expression of a constitutively active FoxO1 in post-prandial mouse livers
repressed genes involved in de novo lipogenesis, including SREBP-1c, FASN, and
ACLY (Gross et al., 2008; Zhang et al., 2006). However, two other studies observed
enhanced lipogenesis and liver steatosis in response to delivery of an active form of
FoxO1 to the liver (Matsumoto et al., 2006; Qu et al., 2006). At this point, the role of
FoxO1 in regulating lipogenic gene expression is still unclear and seems to be
complicated by nutritional status and metabolic background of the various mouse models.
More work will need to be done to clarify its mechanism of action.

1.4 Maf1
Maf1 was originally identified in Saccharomyces cerevisiae as a global repressor of RNA
polymerase (pol) III-dependent transcription (Pluta et al., 2001). As yeast lack structural
homologs of the tumor suppressors, p53 and pRb, Maf1 is the only negative regulator of
RNA pol III transcription that acts as a downstream effector of multiple stress signaling
pathways including nutrient limitation, DNA damage, and secretory pathway defects
(Boguta and Graczyk, 2011; Desai et al., 2005; Upadhya et al., 2002; Willis and Moir,
2007). In yeast, Maf1 is phosphorylated by Sch9 and PKA which cause it to be mostly
cytoplasmic under favorable growth conditions. It is also phosphorylated by mTOR and
CK2 which negatively regulate its repressive activity without affecting its nuclear
localization (Graczyk et al., 2011). Under stress, Maf1 is rapidly dephosphorylated and
accumulates in the nucleus at RNA pol III-dependent gene promoters. However, recent
8

studies have determined that nuclear localization is not sufficient for Maf1 to repress
RNA pol III transcription as it requires further dephosphorylation once inside the nucleus
in order to function (Oler and Cairns, 2012). Maf1 interacts with RNA pol III and Brf1
and recent structural studies have shown that it represses transcription through direct
interaction with RNA pol III which prevents pol III recruitment to TFIIIB complexes at
tRNA and 5S RNA gene promoters (Vannini et al., 2010).

Mammalian Maf1 also represses RNA pol III transcription and interacts with RNA pol III
itself, and the TFIIIB subunit, Brf1 (Reina et al., 2006). Although Maf1 is highly
conserved, the human protein lacks the specific Sch9 and PKA phosphorylation sites
present in the yeast protein that direct its subcellular localization (Pluta et al., 2001).
Consequently, the regulation of subcellular localization of human Maf1 is still unclear.
One group showed that treatment with an mTOR inhibitor (WYE-132) increases Maf1
accumulation in the nucleus (Shor et al., 2010), whereas another group showed that
rapamycin had no effect on Maf1 subcellular distribution in various cell lines (Kantidakis
et al., 2010). This conflicting data could be due to the use of different cell lines, drug
treatments, or different antibodies recognizing Maf1. Regardless of whether mTORC1
regulates Maf1 subcellular localization, it is clear that mTORC1 is present at RNA pol
III- targeted promoters in mammalian cells where it directly phosphorylates Maf1 on
multiple residues to inhibit its repressive function (Kantidakis et al., 2010; Michels et al.,
2010; Shor et al., 2010).

9

Unlike its yeast homolog, mammalian Maf1 has been shown to repress transcription from
all three nuclear RNA polymerases (Johnson et al., 2007). Maf1 directly represses select
Elk-1-regulated RNA pol II-dependent genes including the central transcription factor,
TATA-binding protein (TBP) (Johnson et al., 2007). Elevated expression of TBP is
sufficient to induce oncogenic transformation (Johnson et al., 2003). Since TBP is a
component of transcription complexes used by all three nuclear RNA polymerases,
increases in TBP expression can induce increases in RNA pol I and pol III transcription
(Wang et al., 1998; Wang et al., 1995). RNA pol I transcribes ribosomal RNAs (rRNAs)
and RNA pol III transcribes transfer RNAs (tRNAs) and 5S rRNA. Together, these
products make up the protein synthetic machinery of the cell and can account for up to
80% of cellular transcription. Elevated RNA pol I and III transcription as observed by
abnormally large nucleoli, is considered a hallmark of cancer. In addition, enhanced RNA
pol III activity is required for oncogenic transformation (Johnson et al., 2008; Marshall et
al., 2008). The ability of mammalian Maf1 to regulate TBP and biosynthetic capacity
through regulation of RNA pols I and III suggest that Maf1 is a novel tumor suppressor.
Consistent with this hypothesis, ectopic expression of Maf1 in PTEN-deficient human
glioblastoma cells inhibits anchorage-independent growth (Johnson et al., 2007).

Much of the current research focuses on the regulation of Maf1 through its modification
by phosphorylation and its consequences on RNA pol III transcription. Here, we begin to
elucidate a mechanism for regulation of Maf1 protein expression through the PTEN/PI3K
10

pathway. In doing so, we identified Maf1 as a novel regulator of lipogenic gene
expression, expanding the list of RNA pol II-dependent targets that are regulated by Maf1
and uncovering a new mechanism by which the PI3K/Akt/FoxO1 pathway regulates lipid
metabolism.

11

CHAPTER 2: PTEN Regulates Maf1
2.1 PTEN Positively Regulates Maf1 Expression in Various Cell Lines and Tissues.
Previous work demonstrated that Maf1 represses the expression of genes that promote
oncogenic transformation, and overexpression of Maf1 can repress cellular
transformation (Johnson et al., 2007). Together these findings support the hypothesis that
Maf1 is a novel tumor suppressor. Since PTEN has been shown to regulate the expression
or function of other well-known tumor suppressors, p53 and Rb (Freeman et al., 2003;
Mayo and Donner, 2002; Paramio et al., 1999; Trotman and Pandolfi, 2003), we asked
whether PTEN might also regulate Maf1. To address this question, we used mouse
embryonic fibroblast (MEF) cells lines that were wild-type or that lacked Pten. Protein
lysates were isolated from these cell lines and analyzed by Western blot. We found that
loss of Pten resulted in a substantial decrease in Maf1 protein levels compared to the
wild-type control cell line (Fig. 1A). Next, we isolated RNA from these cell lines to
determine if Maf1 mRNA levels are also altered by loss of Pten. Using qPCR, we found
that Maf1 mRNA levels were similarly decreased in Pten null cells (Fig. 1B). These
results suggest that changes in PTEN may regulate Maf1 expression at the level of RNA
and/or protein.

12

Figure 1. Maf1 expression is decreased in cell lines lacking PTEN expression. (A) Protein
lysates were isolated from wt and Pten null MEF cells and immunoblot analysis was performed.
A representative blot is shown (left). Densitometry was performed and Maf1 protein expression
w a s no r m a l i z e d t o β -actin and wild-type levels were set to 1. The graph represents quantification
of 3 independent experiments (right). Values shown are the means + S.E. (B) RNA was isolated
from wild-type and Pten null MEFs and qRT-PCR was performed with primers specific for Maf1.
Graph represents fold change in Maf1 mRNA expression normalized to GAPDH where wild-type
was set to 1. Values shown are the means + S.E.

13

In order to determine if PTEN might regulate Maf1 in vivo, we made use of two mouse
models with organ-specific Pten deletions. First we used a mouse model in which Pten is
specifically deleted in the liver. These mice begin to develop fatty liver phenotype by 1
month and liver tumors by 9-12 months (Galicia et al., 2010; Horie et al., 2004; Stiles et
al., 2004b). Protein lysates were harvested from the livers of one month old Pten
loxP/loxP
;
Alb-Cre
-
(control) and Pten
loxP/loxP
; Alb-Cre
+
(Pten null) mice and analyzed by Western
blot. Maf1 protein expression was dramatically decreased in lysates from Pten null mice
compared to controls (Fig. 2A). RNA was also isolated from these livers and qPCR was
performed to analyze Maf1 mRNA levels. Although Maf1 mRNA was decreased in the
livers lacking Pten, the difference was not as dramatic as seen at the protein level (Fig.
2B). This suggests that Maf1 regulation is more complicated and may be regulated at
multiple levels such as transcription, translation, and/or protein stability.

Figure 2. PTEN regulates Maf1 in vivo. (A) Protein was isolated from the livers of 1 month old
Pten
loxP/loxP
; Alb-Cre- (n=4) and Pten
loxP/loxP
; Alb-Cre+ (n=3) mice and lysates were subjected to
immunoblot analysis. A representative blot from 4 mice is shown (left). Densitometry was
performed and Maf1 protein expression was normalized to β-actin. Fold change was calculated
by setting wild type values to 1 (right). (B) RNA was isolated from livers of 1 month old
Pten
loxP/loxP
; Alb-Cre- (n=3) and Pten
loxP/loxP
; Alb-Cre+ (n=3) mice and qRT-PCR was performed.
Maf1 mRNA expression was normalized to GAPDH and fold change was calculated by setting
wild type levels to 1.
14

Next we determined if PTEN also regulates Maf1 in prostate tissue. Here we made use of
a mouse model in which Pten is specifically deleted in the prostate. These mice develop
prostatic intraepithelial neoplasia lesions by 6 weeks, invasive adenocarcinomas by 9
weeks, and metastasis by 12 weeks of age (Wang et al., 2003). Protein lysates were
isolated from individual lobes of the prostate of 5.2 week old mice and analyzed by
Western blot. Similar to our results in mouse livers, we observed a dramatic decrease in
Maf1 protein expression in prostates that were lacking Pten (Fig. 3). In both liver and
prostate tissue, the decrease in Maf1 protein expression occurs before the onset of tumors
suggesting that changes in Maf1 are due to Pten loss rather than being a consequence of
disease state.

Figure 3. PTEN regulates Maf1 expression in mouse prostate tissue. Protein was isolated
from individual lobes of the prostate from 5.2 week old Pten
loxP/loxP
; PB-Cre+ mice and
Pten
loxP/loxP
;PB-Cre- littermate controls and immunoblot analysis was performed. A representative
blot is shown (left). Densitometry w a s pe r f o r m e d a nd Ma f 1 e xpr e ss i on w a s no r m a l i z e d t o β -actin.
Graph (right) represents quantification of Maf1 expression where fold change is calculated by
setting control to 1.
15

2.2 Maf1 Expression Is Decreased in Human Cancers.
Given that Maf1 expression was decreased in livers and prostates of Pten conditional
knockout mice, we investigated whether Maf1 might be deregulated in human liver and
prostate cancers. Immunohistochemistry was used to analyze Maf1 expression in frozen
human liver tissue. We compared three matched normal and hepatocellular carcinomas
(HCC) that were negative for hepatitis B and hepatitis C virus. A representative case is
shown (Figure 2A). Maf1 was predominantly localized in the nuclei of normal liver
tissue. In contrast, matched tumor tissue displayed a significant reduction in nuclear
Maf1 staining. PTEN staining is also decreased in HCC compared to normal adjacent
tissue.

We further examined whether Maf1 expression is deregulated in human prostate cancer.
Tissue specimens from four human prostate cancer cases that did not express PTEN were
examined by immunostaining. A representative case is shown (Fig. 2B). Compared
with the predominant nuclear staining of Maf1 in normal prostate epithelium, the
adjacent cancerous tissue from the same individual exhibited a substantial decrease in
Maf1 expression, correlated with loss of PTEN in the malignant tissue. Together, these
results are consistent with the idea that Maf1 expression is deregulated in both human
prostate and liver cancer and that the loss of PTEN drives the alterations in Maf1
expression.
16

Figure 4. Maf1 expression is altered in human cancers. Immunohistochemistry was performed
on frozen human liver tissue (A) and paraffin-embedded human prostate tissue (B) with
antibodies against Maf1 or PTEN. Photomicrographs show representative staining of cancerous
tissue (right) and adjacent normal tissue (left). Insets represent enlargements of areas highlighted
in red. Scale bars represent 50µm.
17

2.3 Summary
These studies identify Maf1 as a novel downstream target of the tumor suppressor,
PTEN. We demonstrate that loss of Pten in mouse embryonic fibroblasts and mouse liver
and prostate tissue causes a decrease in Maf1 protein expression. We show that this
decrease is also seen at the level of mRNA, although to a lesser extent. More importantly,
we provide the first evidence that Maf1 is altered in human cancers. Maf1 nuclear
staining is decreased in human hepatocellular carcinoma and prostate tumors compared to
normal adjacent tissue.

18

CHAPTER 3: Maf1 Is Regulated through the Cell Cycle
3.1 PTEN Induction Increases Maf1 Protein and Requires Its Phosphatase Activity.
Our results from Chapter 1 indicate that loss of PTEN results in decreased Maf1
expression. We next took the opposite approach and increased PTEN expression to
confirm these findings. Here, we used U87 human glioblastoma cells that have been
engineered to express PTEN under the control of a doxycycline-inducible promoter.
Since these cells lack endogenous PTEN, Maf1 levels are low before treatment. Upon
induction of PTEN expression with doxycycline, we observe a corresponding increase in
Maf1 protein expression (Fig 5A left). This increase in Maf1 expression is dependent on
P T E N’ s ph o s p h a t a s e a c t i vi t y a s i nduc t i o n of the phosphatase defective C124S PTEN
mutant did not yield changes in Maf1 expression (Fig. 5A, right). We next sought to
determine if PTEN-mediated regulation of Maf1 was occurring at the level of RNA.
Induction of PTEN or a phosphatase defective mutant PTEN did not produce changes in
Maf1 mRNA levels (Fig, 5B). These results were surprising since we had previously
observed changes in Maf1 protein and mRNA expression upon loss of PTEN. This
difference could be due to the fact the Pten loss is a chronic state whereas induction of
PTEN was not followed past 48 hours. Alternatively, these data may suggest that loss of
PTEN and overexpression of PTEN can induce different pathways in the cell producing
differing effects on Maf1 expression.

19

Figure 5. Induction of wild-type PTEN but not a phosphatase defective mutant form
induces Maf1 protein expression in cells lacking endogenous PTEN. U87 cells engineered to
stably express inducible PTEN or phosphatase defective PTEN-C124S were used. (A) Protein
lysates were isolated from cells treated with 1µg/ml doxycycline for times indicated.
Immunoblot analysis was performed using antibodies as indicated. Maf1 amounts were
no r m a l i z e d t o β -actin. The graphs represent quantification of 3 independent experiments. Values
shown are the means + S.E. (B) RNA was isolated from cells treated with 1µg/ml doxycycline for
times indicated. RT-qPCR was performed with primers specific for Maf1 and results were
normalized to GAPDH.

20

3.2 PTEN-Mediated Induction of Maf1 Requires Changes in Cell Cycle Regulators.
Overexpression of PTEN has been shown to induce G1 cell cycle arrest in cancer cells
(Li and Sun, 1998). These effects on cell cycle progression a r e due to P T E N’ s a bil i t y t o
inhibit PI3K signaling as phosphatase defective PTEN does not induce cell cycle arrest
(Ramaswamy et al., 1999; Weng et al., 1999). In addition to increasing expression of the
cyclin- dependent kinase inhibitor p27Kip1 (Cheney et al., 1999; Gottschalk et al., 2001),
PTEN has been shown to decrease the expression and nuclear localization of cyclin D1
(Radu et al., 2003). The PTEN inducible U87 cells were transiently transfected with a
mutant cyclin D1 T286A that harbors a mutation rendering it resistant to phosphorylation
and subsequent export from the nucleus (Diehl et al., 1997). Western blot analysis
demonstrated that expression of nuclear-persistent cyclin D1 prevents PTEN-mediated
increases in Maf1 protein expression (Fig. 6). These data suggest that increases in Maf1
expression observed with PTEN induction are due, at least in part, to PTEN-mediated
effects on the cell cycle progression.

Figure 6. Overexpression of cyclin D1 prevents PTEN-mediated increases in Maf1
expression. U87 cells were transiently transfected with a nucleus-persistent form of cyclin D1 or
empty vector control and treated with 1 µg/ml doxycycline to induce PTEN expression. After 48
hours, protein lysates were isolated and subjected to immunoblot analysis. A representative
Western blot is shown (left). Densitometry was performed and Maf1 expression was normalized
t o β -actin.
21

3.3 Maf1 Is Regulated through the Cell Cycle.
Given that our data suggest that PTEN regulation of Maf1 is mediated at least in part
through cell cycle effects, we further examined Maf1 expression through the cell cycle.
MCF10A breast epithelial cells were synchronized in G1/G0 phase by serum starvation.
Serum containing media was added back to cells to release the block and allow them to
reenter cell cycle progression. Protein lysates were collected every four hours after serum
release and analyzed by Western blot. Maf1 protein levels were the lowest at 12 hours
post serum release (Fig. 7A). Cells were simultaneously stained with propidium iodide
and analyzed by flow cytometry to determine DNA content. Although the cells were not
completely synchronized, the 12 hour time point corresponded with the highest
percentage of cells in S phase (Fig. 7B). When Maf1 protein expression and percentage
of cells in S phase are graphed together, it suggests that Maf1 expression is inversely
correlated with the percentage of cells were in S phase (Fig. 7C). More work is required
to further elucidate the mechanism of cell cycle regulation of Maf1.
22

Figure 7. Maf1 expression is lowest in S phase. MCF10A cells were synchronized in G1 by
serum starvation. (A) Protein lysates were isolated and subjected to immunoblot analysis with
antibodies against Maf1. Graph represents quantification of Maf1 protein levels normalized t o β -
actin at various time points after serum release. (B) Cells were stained with propidium iodide and
analyzed by flow cytometry. Graph represents percentage of cells in S phase at time points
indicated. (C) Maf1 protein levels are graphed against the percentage of cells in S phase.
23

3.4 Summary
We demonstrate that PTEN phosphatase activity is required for PTEN-induced increases
in Maf1 expression. In addition, we find that increases in Maf1 expression may be due at
least in part to PTEN-mediated changes in the cell cycle mediator, cyclin D1. Finally, we
demonstrate that Maf1 protein levels are changed through the cell cycle and that Maf1
levels are lowest in S phase.

24

CHAPTER 4: PI3K Signaling Negatively Regulates Maf1
4.1 Inhibition of PI3K Signaling Induces Maf1 Expression.
PTEN opposes PI3K signaling through its lipid phosphatase activity. Since this
phosphatase activity is required for PTEN-mediated regulation of Maf1, we next
examined how PI3K signaling might regulate Maf1 expression. MEF and HepG2 human
hepatoma cell lines were treated with LY294002, a pharmacologic inhibitor of PI3K.
Consistent with our results from the PTEN- inducible cell line, pharmacologic inhibition
of PI3K also resulted in increased Maf1 protein expression (Fig. 8A). Increased Maf1
expression correlated with decreased AKT activation as demonstrated by loss of AKT
phosphorylation (p-AKT). Interestingly, Maf1 mRNA levels were also increased in MEF
cells, indicating that PI3K inhibition may regulate Maf1 at the level of RNA (Fig 8B).

Figure 8. Pharmacologic inhibition of PI3K signaling increases Maf1 expression in various
cell lines. (A) MEF and HepG2 cells were treated with LY294002 or DMSO control for 6 hours.
Protein lysates were subjected to immunoblot analysis. Maf1 lev e l s w e r e no r m a l i z e d t o β - actin
and fold change was calculated normalizing to DMSO controls. (B) MEF cells were treated with
LY294002 or DMSO control for 6 hours. RNA was isolated and qRT-PCR was performed with
primers specific for Maf1 and GAPDH internal control.
25

4.2 AKT2 Negatively Regulates Maf1 Expression
We next sought to determine which downstream effectors of PI3K are responsible for
regulating Maf1expression. As mentioned previously, AKT is the major downstream
target of PI3K that carries out many diverse functions. Since AKT2 is the predominant
form in liver, we investigated whether AKT2 might regulate Maf1 expression. We used
mouse models in which Akt2 is specifically deleted in the liver alone or in combination
with Pten deletion. Loss of Akt2 in mouse livers lacking Pten results in a dramatic
decrease in lipid accumulation and expression of lipogenic enzymes (He et al., 2010).
Consequently, Akt2 loss delays tumor onset by 6 months (Galicia et al., 2010). Here, loss
of Akt2 alone resulted in an increase in Maf1 protein expression compared to wild-type
mice. Compared to Pten-deficient mice, additional loss of Akt2 in the double mutant mice
restored Maf1 levels almost to that of the wild-type mice (Fig. 9A). Consistent with these
results, transient transfection of a constitutively active form of AKT2 resulted in a
reduction in Maf1 expression in Huh7 hepatoma cells (Fig. 9B). Together these results
demonstrate that PTEN-mediated regulation of Maf1 expression is due to the ability of
PTEN to negatively regulate PI3K/AKT signaling.

26

Figure 9. Akt2 negatively regulates Maf1 expression. (Left) Protein lysates from wild-type
(n=4), Pten -/- (n=4), Pten-/-; Akt2-/- (n=3), and Akt2-/- (n=2) mice were subjected to
immunoblot analysis. A representative blot is shown. (Right) Huh7 cells were transiently
transfected with HA-tagged AKT2-Myr or empty vector control. Protein lysates were subjected to
immunoblot analysis with antibodies indicated. For both experiments, Maf1 levels were
no r m a l i z e d t o β - actin and fold change was calculated from the wild-type or vector controls.

27

4.3 mTOR Negatively Regulates Maf1 Activity but Has No Effect on Its Expression.
Mammalian target of rapamycin (mTOR) is a downstream target of AKT. As mentioned
previously, a number of groups are currently investigating the effects of mTOR on Maf1
function. Here, U87 glioblastoma cells were treated with rapamycin, a specific inhibitor
of mTORC1. Western blots demonstrated that Maf1 protein expression was not affected
by rapamycin treatment (Fig 10A). However, known targets of Maf1 repression, tRNA
Leu

and TBP, were repressed under these conditions indicating that Maf1 function is
enhanced with rapamycin treatment (Fig. 10B). It is interesting to note that we did not
observe a shift in the Maf1 band representing change in phosphorylation state as other
have demonstrated upon rapamycin treatment (Reina et al., 2006). To further investigate
the mTOR-mediated regulation of Maf1, U87 cells were transiently transfected with and
HA-tagged Maf1 expression vector to examine the subcellular localization of Maf1 with
and without rapamycin treatment. Previous studies haves shown conflicting results
concerning the effect of mTOR inhibitors on subcellular localization (Kantidakis et al.,
2010; Shor et al., 2010). Here, immunofluorescence staining demonstrates that nuclear
localization of Maf1 may be slightly increased in the presence of rapamycin (Fig. 10C).
However, this very modest effect on subcellular localization does not account for the
dramatic repression of Maf1 target genes. Altogether, these data suggest that mTOR does
regulate Maf1 function but the mechanism is not through altering Maf1 expression. This
suggests that an alternate pathway downstream of AKT2 is responsible for changes in
Maf1 expression.

28

Figure 10. Rapamycin treatment does not regulate Maf1 expression, but does enhance Maf1
repression of target genes. U87 cell stably expressing HA-tagged Maf1 were treated with
DMSO or rapamycin for 16 hours. (A) Protein lysates were isolated and subjected to immunoblot
a n a l y s i s w i t h a n t i b od i e s a g a i n st Ma f 1 a n d β -actin. (B) RNA was isolated and reverse transcribed.
qPCR was performed with primers recognizing Maf1 target genes, tRNAleu and TBP. (C)
Immunoflourescence was performed with antibody against Maf1 and DAPI to stain for nuclei.

29

4.4 FoxO1 Positively Regulates Maf1 Expression.
FoxO1 is a transcription factor that is another downstream target of AKT. Unlike mTOR,
phosphorylation of FoxO1 by AKT results in its inactivation and exclusion from the
nucleus. We examined whether FoxO1 might be the downstream target of AKT that
regulates Maf1 expression. Here, we transiently transfected U87 cells with a mutant form
of FoxO1 that cannot be phosphorylated and inactivated by AKT. Overexpression of this
constitutively active form of FoxO1 which would mimic loss of Akt2, resulted in an
increase in Maf1 protein expression (Fig, 11B, left) and a more modest increase in Maf1
mRNA levels (Fig. 11B, right). Overexpression of FoxO1 also resulted in repression of
Maf1 target genes, tRNA
Leu
and tRNA
i
Met
. Consistent with these results, shRNA-
mediated knockdown of FoxO1 resulted in decreased Maf1 protein and mRNA levels and
a corresponding increase in Maf1 target genes (Fig. 11A). Again, the change in Maf1
mRNA levels was modest compared to the 60% decrease at the protein level, suggesting
that FoxO1 may regulate Maf1 at the level of protein rather than at the level of
transcription. These results were surprising as FoxO1 is a transcription factor. The
mechanism by which FoxO1 regulates Maf1 expression is still unclear at this time and
more work needs to be done to determine if FoxO1 affects Maf1 at the level of protein
stability. Together our results reveal that Maf1 expression is positively regulated by
FoxO1 and negatively regulated through PI3K/AKT inactivation of FoxO1. In addition,
these results identify a new FoxO1-dependent signaling pathway that controls RNA pol
III-dependent transcription.

30

Figure 11. FoxO1 positively regulates Maf1 expression and negatively regulates Maf1
target genes. (A) Left: Protein lysates were isolated from MEF cells stably expressing
nonsilencing nsRNA or FoxO1 shRNA and immunoblots were performed. Right: RNA was
isolated from stable MEF cell lines and qRT-PCR was performed with primers specific for
precursor tRNA
Leu
and tRNA
iMet
. Values shown are the means + S.E (n=3). (B) U87 cells were
transfected with a FLAG-tagged constitutively active FoxO1 mutant or empty vector control.
Protein lysates and RNA were isolated after 48 hrs and subjected to immunoblot analysis and
qRT-PCR. Values shown are the means + S.E (n=3).

31

4.5 Maf1 Expression Is Regulated by PI3K Signaling In Vivo.
In order to determine the biological relevance of Maf1 regulation by PI3K signaling, we
examined how diet-induced activation of this pathway would affect Maf1 expression in
mice livers. Mice were fed a high carbohydrate diet or standard chow diet for 2 days, and
livers were harvested for protein analysis. Feeding of a high carbohydrate diet has been
shown to stimulate insulin- induced activation of PI3K signaling and increased de novo
lipogenesis (Miyazaki et al., 2001). Consistent with these studies, we observed enhanced
phosphorylation of AKT along with induction of lipogenic enzymes, FASN and ACC1
(Fig. 12). In addition, the high carbohydrate diet caused a dramatic reduction in Maf1
expression. Together, these studies demonstrate that Maf1 is negatively regulated by
PI3K signaling in vitro and in vivo.

Figure 12. Mice fed a high carbohydrate diet display a reduction in Maf1 protein levels in
the liver. Groups of 4 mice were fed control or high carbohydrate (CHO) diets for 2 days and
livers lysates were harvested. Immunoblot analysis was performed with antibodies against the
proteins designated. Densitometry was performed and fold change of Maf1 expression was
calculated from the average of 4 mice and normalized to the control diet.
32

4.6 Summary
This chapter identifies the PI3K/Akt/FoxO1 pathway as a novel regulator of Maf1
expression. Pharmacologic inhibition of PI3K increases Maf1 protein and mRNA
expression. Loss of Akt2 in mouse livers increases Maf1 protein levels, whereas ectopic
expression of AKT2 in cell lines represses Maf1 expression. FoxO1, a downstream target
of AKT2, positively regulates Maf1 protein and therefore negatively affects its RNA pol
III target genes. The effects of FoxO1 on Maf1 mRNA are insignificant and the exact
mechanism of its effects on Maf1 is unclear at this time. Finally, we demonstrate that
activation of PI3K signaling in vivo through administration of a high carbohydrate diet in
mice represses Maf1 expression.

33

CHAPTER 5: Maf1 Regulates Genes Involved in De Novo Lipogenesis
5.1 Maf1 Regulates FASN and ACC1 Transcription.
Given the important role of FoxO1 in regulating metabolism (Nakae et al., 2008) and our
results indicating that a reduction of Maf1 expression was correlated with an increase in
lipogenic enzyme expression in mice fed a high carbohydrate diet, we examined whether
changes in Maf1 expression could modulate the expression of lipogenic enzymes. A
murine hepatocyte cell line was transiently transfected with siRNA targeting Maf1 and
we examined expression of genes required for de novo lipogenesis. A reduction in Maf1
levels resulted in an increase in FASN and ACC1 mRNA levels however expression of
SREBP1c, the master lipogenic regulator, remained unchanged (Fig. 13A). Next we used
HepG2 cells that stably express Maf1 under the control of a doxycycline inducible
promoter to examine Maf1 effects on lipogenic genes. Induction of Maf1 expression
resulted in repression of FASN and ACC1, but again had no effect on SREBP1 mRNA
expression (Fig. 13B). In order to examine the effects of Maf1 on FASN promoter
activity, we transiently transfected murine hepatocytes with Maf1 siRNA or a Maf1-HA
expression plasmid along with a FASN promoter-reporter construct containing 178bp
upstream of the transcription start site (Swinnen et al., 1997). Decreased Maf1 expression
resulted in an induction of FASN promoter activity, whereas ectopic expression of Maf1
repressed promoter activity (Fig. 13C). Together, these data demonstrate that Maf1
represses transcription of select genes required for de novo lipogenesis. Furthermore, the
Maf1-responsive region of FASN is contained within the first 178 bp upstream of the
transcription start site.
34

Figure 13. Maf1 negatively regulates fatty acid synthase (FASN) and acetyl-coA carboxylase
(ACC1) mRNA expression. (A) Decreased Maf1 expression increases FASN and ACC1 mRNA.
Murine hepatocytes were transiently transfected with mismatch (mm) RNA or siRNA targeting
Maf1. RNA was isolated and qRT-PCR was performed. mRNA amounts were normalized to
that of GAPDH. (B) Increased Maf1 expression represses FASN and ACC1 expression. HepG2
cells were stably infected with Maf1-HA under the control of a dox-inducible promoter. After
treatment with 250ng/ml dox for 48 hrs, RNA was isolated and qRT-PCR was performed. (C)
Maf1 negatively regulates FASN promoter activity. Murine hepatocytes were transfected with
Maf1 siRNA or Maf1-HA and a FASN promoter-reporter construct. Luciferase activity was
measured from resulting lysates and normalized to protein levels. Values shown are the means +
S.E (n=4).
35

5.2 Maf1 Regulates FAS and ACC1 Protein Levels.
Next we determined the effects of Maf1 alterations on lipogenic protein expression. We
found that down regulation of Maf1 in Huh7 cells resulted in a 3.5 fold increase in FASN
expression and a 4.9 fold increase ACC1 protein expression (Figure 14, left). Consistent
with these results, ectopic expression of Maf1 in HepG2 cells produced a ~50% reduction
in these proteins (Figure 14, right). Maf1 levels may not be limiting in HepG2 cells
which might explain why ectopic expression of Maf1 does not produce as dramatic an
effect as decreasing Maf1 expression. SREBP1c is synthesized as an inactive precursor
protein which is then cleaved to yield the “mature ” active form. We measured levels of
mature SREBP1c protein and found that they were unchanged in both cell lines. These
data suggest that FASN and ACC1 are regulated by Maf1 through a mechanism that does
not require changes in SREBP1c expression.

Figure 14. Maf1 negatively regulates cellular FASN and ACC1 protein expression. Protein
lysates isolated from Huh7 cells stably infected with nsRNA or Maf1 shRNA (left) or dox-
inducible Maf1-HA HepG2 cells (right) were subjected to immunoblot analysis using antibodies
against the designated proteins. Representative blots are shown.

36

5.3 Maf1 Directly Represses FASN at the Promoter.
We next determined whether Maf1 repression of FASN transcription was mediated by the
direct recruitment of Maf1 to the FASN promoter. Analysis of Maf1 occupancy revealed
that Maf1 was bound near the transcription start site on the FASN promoter.
Furthermore, down regulation of Maf1 resulted in diminished recruitment of Maf1 to the
FASN promoter (Fig. 15A, top left), whereas induction of Maf1-HA expression resulted
in the enhanced recruitment of the HA-tagged Maf1 to the FASN promoter (Fig. 15A, top
right). These changes in Maf1 occupancy on the FASN promoter observed when cellular
Maf1 levels are altered further validate the ChIP signals and were similar to those
observed for the Maf1-targeted tRNA
Leu
gene (Fig. 15B). Since Maf1 represses TBP by
displacing transcription factor Elk-1 from the promoter (Johnson et al., 2007), we
hypothesized that Maf1-mediated effects on FASN might occur through regulation of
SREBP1c occupancy on the gene promoter. We further assessed whether changes in
Maf1 expression and promoter occupancy would affect SREBP1c binding. Analysis
revealed that changes in Maf1 occupancy did not alter SREBP1c binding to the FASN
promoter (Fig. 15A, bottom). Together, these results support the idea that Maf1 directly
targets and negatively regulates genes required for de novo lipogenesis and that this effect
does not impair the binding of SREBP1c at the promoter.

37

Figure 15. Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the FASN
promoter. (A) Chromatin immunoprecipitations were performed with Huh7 cells stably
expressing Maf1 shRNA or HepG2 cells engineered to express dox-inducible Maf1-HA. ChIP
analysis was performed with antibodies against Maf1, SREBP1c, and IgG. qPCR was performed
with an upstream primers set (gray bars) and a set encompassing the transcription start site TSS
(black and white bars). Bars represent Maf1 (top) or SREBP1c occupancy relative to input and
IgG. (B) Altering Maf1 levels changes Maf1 occupancy at the tRNA
Leu
gene promoter. ChIP
analysis was performed as in (A) with primers specific for the tRNA
Leu
promoter. Values shown
for all graphs are the means + S.E (n=4).
38

5.4 Maf1 Represses Intracellular Lipid Accumulation.
We further examined the biological consequences of Maf1-mediated repression of
lipogenic gene expression. Since the activity of lipogenic enzymes controls de novo
lipogenesis, we asked whether Maf1-mediated repression of these genes would affect
intracellular lipid accumulation. Huh7 stable cell lines expressing non-silencing RNA or
shRNA targeting Maf1 were stained with Oil Red O to analyze intracellular lipid
accumulation. Compared with control cells, the cells expressing Maf1 shRNA displayed
an increase in the size and number of visible lipid droplets (Fig. 16). Together, these
results support the idea that Maf1 negatively regulates intracellular lipid accumulation at
least in part through its ability to directly repress the transcription of lipogenic enzymes.

Figure 16. Maf1 represses intracellular lipid accumulation. Huh-7 cells infected with nsRNA
(left) or Maf1 shRNA (right) were stained with Oil Red O and Maye r ’ s H e m a t o x y l i n . Intracellular lipid droplets were detected as red spheres and nuclei are shown in purple.
Magnification 40x.

39

5.5 Summary
These studies identify a novel class of genes that are regulated by Maf1 and identify a
role for Maf1 in lipid metabolism. Maf1 represses transcription of FASN and ACC1,
which are enzymes required for lipid biogenesis. Maf1 directly represses FASN by
occupying the promoter although its mechanism of action is still unclear. Regulation of
these genes by Maf1 is independent of the lipogenic transcription factor SREBP1c, as
alterations in Maf1 levels did not affect SREBP1c mRNA and protein expression, or its
occupancy on the FASN promoter. Decreasing Maf1 levels is sufficient to induce
intracellular lipid accumulation presumably through changes in lipogenic gene
expression.

40

CHAPTER 6: Discussion
6.1 PTEN Regulates Maf1 Expression
These studies set out to investigate the potential mechanisms for regulation of the
transcriptional repressor Maf1. Previous work has shown that Maf1 is able to repress the
biosynthetic capacity and the transforming properties of cells suggesting that Maf1 may
be a novel tumor suppressor (Johnson et al., 2007). Identifying how this gene is regulated
will give us important insights into how it might become deregulated in human cancer
and metabolic disease. Our results identify Maf1 as a novel target of the tumor
suppressor, PTEN. We show that loss of PTEN in various cell lines and mouse models
results in decreased Maf1 expression. Although we only assessed changes in Maf1 in a
small subset of PTEN negative cancers, we are the first to demonstrate that Maf1
expression is altered in any human cancer, further supporting the hypothesis that Maf1 is
a novel tumor suppressor. Since deregulation of PTEN is one of the most common
aberrations in many human cancers, a reduction in Maf1 expression is likely to be
observed in many other types of cancers that possess alterations in PTEN function or
mutations that activate PI3K signaling. It will be interesting to examine a larger cohort of
cases from liver and prostate, or other cancers to determine if loss of Maf1 expression is
statistically associated with loss of PTEN or FoxO1 expression and/or inversely
correlated with increased activation of PI3K or AKT. Additional studies could also tell us
if Maf1 loss is associated with particular stages of cancer or if it is particularly associated
with aggressive tumors or metastatic tumors. These findings might give us more insight
into the role of Maf1 in the development and progression of human cancer.
41

6.2 A Novel Mechanism of Maf1 Regulation
Previously, little was known regarding how Maf1 is regulated in mammalian cells. It was
recently discovered that mTORC1 directly phosphorylates Maf1 on multiple residues to
inhibit its repressive activity on RNA pol III-dependent transcription (Michels et al.,
2010). Our studies are the first to investigate a mechanism of Maf1 regulation other than
post- translational modification. We identified a novel mechanism by which PI3K
activation negatively regulates Maf1 through the Akt2-FoxO1 axis. Loss of Pten in MEF
cells and in mouse livers resulted in decreased Maf1 protein and mRNA levels.
Pharmacologic inhibition of PI3K resulted in increased Maf1 protein and mRNA levels
suggesting that Maf1 may be regulated at the level of transcription. Next, we showed that
Maf1 protein expression is negatively regulated by Akt2. Since we know that Maf1
expression is not altered by inhibition of mTOR, we investigated other downstream
targets of Akt2 that might regulate Maf1 transcription. We found that the transcription
factor FoxO1, which is inhibited by AKT, positively regulates Maf1 expression.
However, when FoxO1 levels were altered, the changes in Maf1 mRNA expression were
insignificant compared to the changes in protein expression. These data suggest that
FoxO1 may regulate Maf1 at the level of protein, but not at the level transcription. As
FoxO1 can induce G1 cell cycle arrest through increasing p27Kip1 levels, it may be that
it is regulating Maf1 protein expression in part through its effects on the cell cycle. This
hypothesis would be consistent with our studies that showed Maf1 protein, but not
mRNA was changed when G1 arrest was induced by PTEN expression. Future studies
will investigate how FoxO1 might regulate Maf1 either at the level of protein stability or
42

through its ability to induce cell cycle changes. Additionally, it will be interesting to
investigate other pathways downstream of AKT2, besides FoxO1 or mTOR that might
regulate Maf1. An appealing candidate for investigation would be the serine/threonine
kinase, GSK 3β. L i ke F o x O1, GSK 3 β is directly phosphorylated and inactivated by AKT,
and it has also been shown to regulate metabolism and cell proliferation in part through
its ability to directly phosphorylate SREBP and cyclin D1 (Manning and Cantley, 2007).

6.3 Novel Targets of Maf1 Repression
Previously, the only known targets of Maf1 repression were RNA pol III- dependent
genes and select RNA pol II- dependent genes, including TBP and egr-1. Our studies
identify a novel class of genes that are regulated by Maf1. We found that select genes
required for lipid biogenesis, including FASN and ACC1, are repressed by Maf1 and this
regulation occurs at the level of transcription. It will be interesting to see if Maf1
regulates other genes involved in lipid biosynthesis such as ATP-citrate lyase (ACLY), or
even Elovl6 and SCD-1 which are genes involved in fatty acid elongation and fatty acid
desaturation. As increased de novo lipogenesis and increased RNA pol III-dependent
transcription are both hallmarks of cancer, these studies suggest that Maf1 may act as a
global tumor suppressor by inhibiting multiple pathways that are critical for
transformation. In addition to RNA pol III transcription and de novo lipogenesis, cancer
cells also upregulate other metabolic pathways including glucose intake, glycolysis, de
novo nucleotide synthesis and glutamine metabolism. It will be interesting to see if Maf1
also represses genes involved in these metabolic pathways.
43

6.4 Maf1 Represses These Genes at the Promoter
Much of what is known about Maf1 function comes from studies in yeast where it
selectively represses genes transcribed by RNA polymerase III through its ability to
associate with RNA polymerase III itself. Since the components of the transcriptional
machinery differ between RNA pol III and RNA pol II genes, it is likely that the
mechanism of Maf1 repression may differ as well. Previous studies demonstrated that the
Maf1 responsive region within the TBP promoter is within 100 bp of the transcription
start site, and that Maf1 recruitment results in the dissociation of Elk-1 from the
promoters (Johnson et al., 2007). The Maf1-responsive region within the FASN promoter
is contained within the first 178 base pairs upstream of the transcription start site and
Maf1 is recruited to this region to repress gene activity. This region encompasses a
binding site for the master lipogenic gene inducer, SREBP1c. However, changes in Maf1
occupancy do not affect SREPB1c binding. Since the Maf1 responsive regions within the
TBP and FASN promoters contain distinct transcription factor binding sites, this suggests
that Maf1 may repress RNA pol II-dependent gene expression independent of the
composition of activator elements present. Maf1 does not have an apparent DNA binding
domain, therefore, its recruitment to promoters must occur through its association with
other transcription components. While Maf1 genome targets and the mechanism by
which Maf1 represses gene expression need to be further illuminated, it is possible that
Maf1-mediated repression of RNA pol II-dependent promoters may involve its ability to
repress the function or binding of coactivators or components of the general transcription
machinery. Preliminary data from our laboratory indicates that Maf1 interacts with the
44

CDK8 submodule of the larger mediator protein complex. As mediator is required for
activator- dependent transcription (Myers and Kornberg, 2000), this interaction could
provide a potential mechanism for Maf1 recruitment to various promoters. Future studies
will investigate this hypothesis.

6.5 PI3K Coordinately Regulates Maf1 and SREBP1c to Control Lipogenesis.
The PI3K pathway through activation of its downstream effector, AKT2, plays a critical
role in metabolism, proliferation, and growth control. Activation of AKT2 can stimulate
lipogenic gene expression through mTORC1, which enhances transcription, processing
and activity of SREBP1c (Lewis et al., 2011). In addition, mTORC1 can directly
phosphorylate Maf1 to inhibit its ability to repress RNA pol III-dependent transcription
(Kantidakis et al., 2010; Michels et al., 2010; Shor et al., 2010). Alternatively, FoxO1 can
reduce SREBP1c expression and induce Maf1 function to repress lipogenic gene
expression. These studies support opposing roles for Maf1 and SREBP1c in regulating
lipogenic gene expression. The finding that Maf1 is able to repress transcription even in
the presence of SREBP1c at the FASN promoter suggests that induction of lipogenesis
may require not only stimulation by TORC1 and SREBP1c, but also alleviation from
Maf1 repression. Our studies support the idea that Maf1 is a central node that integrates
multiple signals from the PTEN/PI3K pathway to co-repress genes involved in
proliferative, biosynthetic and metabolic processes that lead to oncogenesis. The findings
from this work and previous work from our lab are summarized in the following
schematic (Fig. 17).
45

Figure 17. Schematic representing the regulation and function of Maf1. Specific findings
from these studies are highlighted in gray.

46

6.6 Potential Role of Maf1 in Human Disease
Recent studies suggest that up to 34% of the general adult population, or 60 million
Americans, may have non-alcoholic fatty liver disease (NAFLD) (Adams and Angulo,
2005). NAFLD encompasses a spectrum of disease states ranging from hepatic steatosis,
steatohepatitis, fibrosis, to cirrhosis, and in some cases may represent a preneoplastic
stage to hepatocellular carcinoma (HCC) (Peyrou et al., 2010). NAFLD is strongly
associated with other features of metabolic syndrome including obesity, type 2 diabetes,
and hypertension (Postic and Girard, 2008). Alterations in PTEN expression and activity
play a critical role in the development of NAFLD and HCC, and excessive accumulation
of triglycerides is a hallmark of NAFLD (Peyrou et al., 2010; Postic and Girard, 2008).
As we have identified Maf1 as a novel downstream target of PTEN that regulates lipid
accumulation, it seems likely that Maf1 might also play a role in development or
pathogenesis of liver metabolic diseases.

In 2010, the CDC estimates that more than one third of adults in the United States are
obese (Ogden et al., 2012), and the World Health Organization estimates that 1.6 billion
adults worldwide are overweight (Roberts et al., 2010). In addition to its role in metabolic
disorders, obesity can account for approximately 20% of cancer cases (Wolin et al., 2010)
and is linked to approximately 14% of cancer deaths in men and 20% in women (Calle et
al., 2003). While epidemiologic studies have established a link between obesity and
cancer, the biological mechanisms connecting the two are still unclear. Many candidate
mechanisms have been explored including insulin and insulin-like growth factors, sex
47

hormones, adipokines, inflammation, and obesity- induced hypoxia (Roberts et al., 2010).
However, due to the complex pathology of obesity, experimental evidence for a direct
connection has remained elusive. From our studies, we might extrapolate that chronic
insulin signaling through the PI3K pathway that occurs in obese individuals with
hyperinsulinaemia would maintain Maf1 expression at low levels leading to higher levels
of de novo lipogenesis as well as increased RNA pol III-dependent transcription. As
increased levels of lipids and RNA pol III products (rRNA and tRNAs) are hallmarks of
cancer, and increased RNA pol III transcription is sufficient to induce transformation
(Marshall et al., 2008), Maf1 could provide a critical mechanistic link between obesity
and cancer. It will be interesting to further explore this possibility in the future. As
obesity is a growing epidemic it will be important to understand the biological link
between obesity and cancer so that we may better develop preventative and therapeutic
strategies.

Future studies will build on this work to further elucidate the role of Maf1 in metabolic
diseases and cancer. A mouse model expressing Maf1 under a liver-specific promoter
will serve as a valuable tool in this effort. Crossing this mouse with the Pten
loxP/loxP
; Alb-
Cre
+
mice (Stiles et al., 2004b) will allow us to determine if increased Maf1 expression in
the liver is able to prevent or delay fatty liver disease and tumor formation that is caused
by Pten loss. Increasing expression of Maf1 in various models of metabolic disease and
cancer will allow us to more precisely determine the role of Maf1 in these diseases and
hopefully contribute to the development of improved therapeutic strategies.
48

CHAPTER 7: Materials and Methods
Cell Lines
Mouse embryonic fibroblast (MEF), U87, Huh7, and HepG2 cell lines were obtained
from American Type Culture Collection and were grown in D u l be c c o ’ s M o di f i c a t i o n o f E a g l e ’ s M e d i u m ( D M E M ) ( M e d i a t e c h ) with 4.5 g/L glucose supplemented with 10%
fetal bovine serum, penicillin (500 units), streptomycin (500 µg), and L-glutamine (500
units). MCF10A cells were obtained from ATCC and grown in DMEM/F12 (USC
Cancer center Core) supplemented with 5% Horse serum, Hepes (2.5mM), L-glutamine
(2mM), Penicillin Streptomycin, (100 untis/ml), EGF (20ng/ml), Cholera toxin (100
ng/ml), bovine Insulin (0.01 mg/ml) and hydrocortisone (500ng/ml). Pten -/- MEF (Sun
et al., 1999) and murine hepatocyte cell lines were obtained from Dr. Bangyan Stiles
(USC School of Pharmacy). Murine hepatocytes were grown in DMEM supplemented
with 4.5 g/L glucose supplemented with 10% fetal bovine serum, penicillin (500 units),
streptomycin (500 µg), L-glutamine (500 units), EGF, and bovine insulin. All cells were
maintained in 5% CO
2
at 37ºC.

PTEN and PTEN C124S-inducible U87 cell lines were obtained from Maria-Magdalena
Georgescu (MD Anderson Cancer Center, Houston, TX). They were grown in DMEM
supplemented with 10% Tet-free fetal calf serum, G418 at 1 mg/ml, and blasticidine at 10
µg/ml. These cells express wild-type or phosphatase defective (C124S) PTEN under the
control of a doxycycline-inducible promoter. Cells were treated with 1 µg/ml
doxycycline in order to induce PTEN expression for the times indicated.
49

Generation of Lentivirus and Stable Cell Lines
Lentiviral particles were produced by transfection of HEK293T using calcium phosphate.
Lentiviral vectors were transfected with pMD2.G (vesicular stomatitis virus envelope
protein (VSV-G) expression vector) and psPAX2 (packaging vector). After 48 h,
conditioned media was collected, sterile filtered and centrifuged at 100,000 x g (90 min,
4°). Pelleted virus was resuspended in media and sterile filtered. Cell lines were
transduced with concentrated virus from conditioned media for 6-16 h. After 3 days, the
infected cells were selected for with 2-4 µg/ml puromycin or GFP expression determined
by fluorescence microscopy.

MEF cells stably expressing FoxO1 shRNA were generated using non-silencing empty
vector control and pLKO.1- mouse FoxO1 shRNAs (clone ID TRCN0000054880 and
clone ID TRCN0000054879) obtained from Thermo Open Biosystems. Three days after
transduction, cells were stably selected with 4 µg/ml puromycin. They were grown in
DMEM with 4.5 g/L glucose supplemented with 10% fetal bovine serum, penicillin (500
units), streptomycin (500 µg), and L-glutamine (500 units). Cells were serum starved
(0.5% FBS) for ~16 hours before isolation for protein or RNA analysis.

Huh7 hepatoma cells stably expressing Maf1 shRNA were generated using non-silencing
GIPZ lentiviral shRNAmir control vector (GIPZ-ns shRNA) and GIPZ lentiviral
shRNAmir expression clones to human Maf1 (GIPZ-Maf1 shRNA) obtained from
T h e r m o Ope n B i o s y s t e m s ( C l o n e I Ds V2L HS_13 8266, 3’ UT R t a r ge t ; V3L HS_380771,
50

cDNA target; V3LHS_380770, cDNA target). Cells were transduced individually with
the 3 Maf1 shRNAs and pooled together. Pooled populations were FACS sorted for GFP
expression by the USC Flow cytometry core.

HepG2 cells stably expressing Maf1-HA under the control of a doxycycline-inducible
promoter were generated. Briefly, Maf1-HA was PCR amplified from pcDNA 3.1( –)
Maf1-HA (Lyu et al., 2009) to a dd B a m HI a n d a K o z a k c o n s e ns us s e que n c e 3 ’ a n d X b a I 5’ o f t h e M a f 1 -HA cDNA. Inducible lentiviral vector pFTREW (Lyu et al., 2009) was
restriction digested with BamHI and XbaI to remove GFP and subclone in Maf1-HA
(pFTREW-Maf1-HA). FTREW and FUIPW-tTA (lentiviral tetracycline transactivator)
vectors were a gift from Wange Lu (USC). Stable cells were selected with 3 µg/ml
puromycin. Cells were grown in DMEM with 4.5 g/L glucose supplemented with 10%
Charcoal-stripped Fetal Calf Serum, penicillin (500 units), streptomycin (500 µg), and L-
glutamine (500 units). Cells were seeded on 60mm (for RNA isolation), 100mm (for
protein isolation), or 150 mm (for chromatin isolation) plates and allowed to grow
overnight. The following day, Maf1-HA expression was induced by replacing media with
fresh media containing 250 ng/ml doxycycline. After 48 hours of induction, cells were
harvested for RNA, protein, or ChIP analysis.

Mouse Tissues
Pten
loxP/loxP
; Alb-Cre
+
, and Pten
loxP/loxP
; Alb-Cre
+
; Akt2
-/-
double mutant mice were
generated as previously described (He et al., 2010; Stiles et al., 2004b). Briefly,
51

Pten
lopP/loxP
mice were crossed with Alb-Cre

mice that express Cre recombinase under the
control of a liver-specific albumin promoter to generate mice with specific deletion of
Pten in hepatocytes. Livers from 1 month old mice were harvested and flash frozen in
liquid nitrogen for protein analysis. Livers were homogenized in PBS (pH 74)/1% NP-
40/0.5% sodium deoxycholate/0.1% SDS containing protease inhibitors and used for
Western blot analysis. Protein concentrations were determined using the Bio-Rad DC
protein assay kit.

16 week old C57Bl/6 mice were fed standard chow diet (5008 Formulab Diet; PMI
Nutrition International) or high carbohydrate diet (TD 99252; Harlan Teklad) ad libitum
for 2 days. High carbohydrate diet contained, by weight, 55% sucrose, 21% casein, 14%
maltodextrin, , 5.2% cellulose, 3.6% mineral mix (AIN-93G-MX), 1.1% vitamin mix
(AIN-93-VX), 0.32% L-Cystine, and 0.26% choine bitartrate. Mice were sacrificed and
livers were harvested for protein analysis as described above.

Pten
loxP/loxP
; PB-Cre
+
mice were generated as previously described (Wang et al., 2003).
Briefly, Pten
loxp/loxp
mice were crossed with the PB-Cre4 line that expresses Cre
recombinase under the control of a prostate-specific probasin promoter, yielding mice
with specific deletion of Pten in prostatic epithelial cells.

52

Plasmids and siRNA
Mouse Maf1 siRNA t a r ge t s e que n c e 5 -TGTGACATCTACAGCTATA- 3 wa s i de n t i f i e d us i n g D h a r m a c o n’ s s i DE S I GN C e n t e r to NC B I a c c e s s i o n n u m be r NM_026859. Maf1-
HA and cyclin D1 T286A plasmids were described previously (Diehl et al., 1997;
Johnson et al., 2007). FoxO1-AAA (He et al., 2010), AKT2-Myr, and FAS luciferase
reporter construct (Swinnen et al., 1997) were gifts from Dr. Bangyan Stiles.

Transient Transfections
HepG2 cells were transiently transfected with F1 transfection reagent (Targeting
Systems). MEF and Huh7 cells were transiently transfected with Lipofectamine 2000
f o l l o w i n g m a n u f a c t ur e r ’ s pr oto c o l (Invitrogen).

Quantitative Real Time PCR
Total RNA was isolated from cells 48 hours post- transfection using RNA-Stat60
(TelTest). RNA was DNase treated with Turbo DNA-free kit (Ambion) and reverse
transcribed to cDNA using SuperScript III first strand synthesis kit (Invitrogen). RT-
qPCR was performed with Brilliant II SYBR Green qPCR Mastermix (Stratagene) on the
MX3000P System (Stratagene). Primer sets for FAS, ACC1, SREBP1c (He et al., 2010),
pre-tRNA
Leu
(Johnson et al., 2007), pre-tRNA
iMet
(Marshall et al., 2008), and GAPDH
(Johnson et al., 2007) have been described previously. Relative amounts of transcripts
we r e qua n t i f i e d by t h e c o m p a r a t i v e t h r e s h o l d c y c le m e t h o d ( Δ Δ C t ) us i n g G A P DH a s t he
53

endogenous reference control. Fold change was calculated from the control or vector cell
lines.

Western Blot Analysis
Cells were plated on 100mm dishes and isolated when they reached 80-90% confluency.
Cells were washed two times with cold PBS and lysed in 150-350 µl of cell lysis buffer
(20mM Tris pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 2.5 mM sodium pyrophosphate,
1 m M β -Glycerolphosphate, 1mM Na
3
VO
4
, 1 mM PMSF, 10 µl/ml protease inhibitor
cocktail III, and 10 µl/ml Halt phosphatase inhibitor). After incubation on ice for 15
minutes, cells were scraped and transferred to microcentrifuge tubes for 15 second
sonication. Lysates were then cleared by centrifugation at 10,000 g for 20 minutes at 4 ºC
and supernatants were collected and stored at -80 ºC. Protein concentrations were
determined by the Bradford method using the Bio-Rad Protein Assay reagent. Protein
lysates (150ug) were subjected to SDS-PAGE and transferred to Hybond ECL
Nitrocellulose membranes. Nonspecific binding was blocked with 5% nonfat milk or 5%
BSA in TBS for at least 45 minutes. Membranes were probed with the following 1º
antibodies: Maf1 (provided by Ian Willis), Maf1 (Santa Cruz), Maf1 (Abcam), PTEN, p-
AKT, AKT2, FoxO1, ACC1 (Cell Signaling), HA (Roche), FLAG (Sigma), and FAS
( B D) . M e m b r a n e s we r e a l s o pr o b e d wi t h a n t i b o d i e s a ga i ns t β -actin and vinculin (Sigma)
as loading controls. Bound primary antibody was visualized using HRP-conjugated
secondary antibodies (Pierce) or biotinylated secondary antibodies complexed with
avidin/peroxidase (Vector Labs) and enhanced chemiluminescence reagents (Pierce or
54

Perkin-Elmer). Densitometry was performed using UN-SCAN-IT software (Silk
Scientific). Fold changes were calculated by setting vector or control cell lines to 1.

Chromatin Immunoprecipitation
HepG2 and Huh7 stable cell lines were used for chromatin immunoprecipitation
experiments. Cells were plated on 150mm plates so they would reach 80-90% confluency
on the day of isolation. The following day, HepG2 inducible cells were treated with 250
ng/ml doxycycline for a total of 48 hours to induce Maf1 expression and were serum
starved (0.5% Tet-free Fetal Calf Serum) for the final 16 hours of that period. Chromatin
was isolated from cells after formaldehyde cross-linking, and then sheared into smaller
fragments (200-700 bp) using the Branson Sonifier 150 at setting 3 with 4, 10 sec pulses.
100 µl of chromatin was then diluted 10 times with dilution buffer and pre-cleared for
one hour with Protein A/G beads. After pre-clearing the samples were incubated
overnight with antibodies against Maf1 (Santa Cruz), Maf1 (Abcam), HA (Abcam),
SREBP-1c (Santa Cruz), and IgG (Bethyl Labs). The next day, samples were
immunoprecipitated using Protein A/G beads. Following IP washes, DNA-protein
crosslinks were reversed and DNA was isolated using the phenol-chloroform protocol
followed by ethanol precipitation. DNA was then amplified by quantitative PCR using
Terra qPCR Direct SYBR Premix (Clontech) or Brilliant II SYBR Green qPCR
Mastermix (Stratagene) on the MX3000P System (Stratagene). P r i m e r s e que n c e s f o r t h e F A S ge n e r e g i o n a r e a s f o l l o w s F A S ups t r e a m f o r wa r d 5 - C T GG T C A C AC T C T GC C C AC A GC- 3 , r e v e r s e 5 - A G C T GC AA A GG T C C C A C G A- 3 ,
55

a n d F A S pr o m o t e r f o r wa r d 5 -CAGCCC C GA C GC T C A T T GG- 3 , r e v e r s e 5 -
GG C T GC T C GT AC C T GG T GA G - 3 . P r i m e r s f o r t h e t R NA
Leu
gene were described
previously (Johnson et al., 2007). Normalized Ct values for antibody pulldowns were
normalized to input using the antibody IP*10/input calculation and immunoglobulin G.

Cell Cycle Synchronization and Propidium Iodide Staining
MCF10A cells were washed 2 times with PBS and serum free media was added to each
plate. Cells were serum starved (0.5% Horse serum) for 24 hours to induce cell cycle
arrest. Serum starve media was removed and replaced with normal growth medium to
release cell cycle block. Cells were harvested at time 0 and every 4 hours after for 24
hours. Protei n wa s i s o l a t e d a s de s c r i b e d u n de r “ W e s t e r n bl o t s ” . C e l l s we r e ha r v e s t e d
from duplicate plates and stained for propidium iodide to determine cell cycle profile.
Briefly, cells were trypsinized and washed 2 times with PBS. They were resuspended in
200 µl PBS and fixed with 2 ml of cold 70% ethanol for 2 hours. Cells were washed
twice with PBS then resuspended in 1 ml of PBS containing DNase free RNase I (40
µg/ml) and propidium iodide for 3 hours at room temperature. Cells were washed 1 time
and resuspended in PBS for flow cytometry analysis.

Oil Red O Staining
Glass coverslips were sterilized with 100 % ethanol then placed in 6 well culture plates.
Coverslips were coated with Poly-D-Lysine (0.5 mg/ml) followed by rat tail collagen I
(20 µg/ml). Approximately 7.5 x 10
4
Huh7 stable cells were seeded in each well and were
56

grown overnight at 37 ºC. The following day, cells were fixed in 10% formalin for 3 to 5
minutes. Cells were then stained with Oil Red O Isopropanol Solution (Electron
Microscopy Sciences #26503-02) for 10 minutes followed by Mayers Hematoxylin for 5
minutes. Coverslips were mounted on glass slides with Aqueous Mounting Media.
Staining was performed by the Cell and Tissue Imaging Core of the USC Research
Center for Liver Diseases and images were visualized with the Nikon 80i light
microscope.

Immunohistochemistry
Human tissues were obtained from the USC/Norris Comprehensive Cancer Center Tissue
Procurement Core. Fresh frozen liver samples were courtesy of Dr. Linda Sher.
Hematoxylin & Eosin staining was performed and slides were analyzed by a pathologist
to identify normal vs. cancerous tissue. Matched normal and tumor samples from the
same patient were compared. Frozen human liver sections were stained with anti-Maf1
(Abcam) at a concentration of 1:80 or anti-PTEN (Cell Signaling) and counterstained
with hematoxylin. Paraffin-embedded human prostate sections were stained with Maf1
(Santa Cruz) and PTEN antibodies. Staining was performed by the USC Department of
Pathology Immunohistochemistry Lab and images were visualized on the Nikon 80i light
microscope.

57

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Abstract (if available)

Abstract Although Maf1 has been identified as a central negative regulator of transcription, little is known about its regulation and it is likely that many of its target genes have yet to be identified. Our finding that Maf1 can suppress cellular transformation led us to examine whether Maf1 might be regulated by the tumor suppressor, PTEN. Indeed, Pten-deficient cell lines and mouse tissues show marked decreases in Maf1 protein expression. Consistent with these results, induction of PTEN expression in human glioblastoma cells results in increased Maf1 expression. Pharmacologic inhibitors of PI3K signaling also induce Maf1 expression, and expression of a phosphatase defective mutant PTEN fails to alter Maf1 expression. These data suggest that regulation of Maf1 expression by PTEN is due, at least in part, to its ability to inhibit the PI3K signaling pathway. Loss of Akt2 in the liver of mice results in increased Maf1 protein expression. Additionally, loss of Akt2 in combination with Pten loss rescues the decreased Maf1 expression that was caused by Pten loss alone. Ectopic expression of FoxO1, a downstream target of Akt, increases Maf1 protein expression. Given the established role for the PI3K/AKT/FoxO1 pathway in regulating lipid biosynthesis, we investigated whether Maf1 could regulate these genes. We show that enzymes required for lipid biogenesis, including FASN and ACC1, are repressed by Maf1. Repressing Maf1 expression in Huh7 hepatoma cells resulted in increased lipid accumulation. A marked decrease in Maf1 staining is observed in Pten-null mouse prostate and human prostate cancer tissue. Together these studies identify Maf1 as a downstream target of PTEN and a novel regulator of lipogenic gene expression.

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Asset Metadata

Creator Palian, Beth Marie (author)

Core Title Maf1 is a novel target of the tumor suppressor PTEN and a negative regulator of lipid metabolism

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 06/22/2012

Defense Date 05/04/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag de novo lipogenesis,FASN,Maf1,OAI-PMH Harvest,PTEN

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Johnson, Deborah L. (committee chair), Dubeau, Louis (committee member), Stiles, Bangyan L. (committee member)

Creator Email beth.palian@gmail.com,palian@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-48553

Unique identifier UC11289525

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Legacy Identifier etd-PalianBeth-900.pdf

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Document Type Dissertation

Rights Palian, Beth Marie

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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