University of Southern California Dissertations and Theses

The noncanonical role of telomerase in prostate cancer cells: exploring a non-telomeric signaling role for telomerase protein (TERT) in a cancer cell line

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The noncanonical role of telomerase in prostate cancer cells: exploring a non-telomeric signaling role for telomerase protein (TERT) in a cancer cell line

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Content THE NONCANONICAL ROLE OF TELOMERASE IN PROSTATE CANCER
CELLS:
EXPLORING A NON-TELOMERIC SIGNALING ROLE FOR TELOMERASE
PROTEIN (TERT) IN A CANCER CELL LINE

by

Anisha Madhav

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

May 2012

Copyright 2012 Anisha Madhav
ii

Acknowledgements
I would like to give a sincere thank you to my mentor and advisor, Dr. Amir
Goldkorn for giving me the opportunity to do such exciting research and for all of
his guidance and support in completing this thesis.
I would also like to express my gratitude to all the members of the
Goldkorn laboratory: Dr. Tong Xu, Dr. Yucheng Xu, Roy Lau, Kaijie He, and
Ayesha Bhatia. They have all played such an integral part in the completion of
this thesis and I am so grateful for all of their help.

iii

Table of Contents
Acknowledgements ii
List of Figures iv
Abbreviations v
Abstract vi
Introduction 1
Hypothesis 12
Material and Methods 14
Table 1: List of Primers 20
Results 21
Discussion 35
References 41

iv

List of Figures
Figure 1: Telomerase Components 2
Figure 2: hTERT mRNA Expression 22
Figure 3: TRAP Activity 23
Figure 4: Telomere Lengths 23
Figure 5: MTS Assay 24
Figure 6: FACS CD44+CD24- 25
Figure 7: hTERT and CD44 mRNA Expression 26
Figure 8: Sphere Formation Assay 28
Figure 9: Colony Formation Assay 29
Figure 10: Matrigel Invasion Assay 31
Figure 11: hTERT and E-cadherin mRNA Expression 32
Figure 12: Gene Expression of Wnt/β-catenin Regulated Genes 34

v

Abbreviations
ALT: alternative lengthening of telomeres
CD: Catalytically dead
EGFR: epidermal growth factor receptor
FACS: fluorescence-activated cell sorting
FGF: fibroblast growth factor
HA: hemagglutinin
HMECs: human mammary epithelial cells
M1: Mortality stage 1
M2: Mortality stage 2
TCAB1: telomerase Cajal body protein 1
TERT: telomerase reverse transcriptase
TERC: telomerase RNA component
TRAIL: TNF-related apoptosis inducing ligand
TRAP: telomeric repeat amplification protocol
WT: Wildtype

vi

Abstract
The telomerase reverse transcriptase (TERT) component of telomerase
has reverse transcriptase activity capable of elongating telomeres during each
replication cycle of the cell. TERT plays a dual role in activating normal stem cell
niches and in potentiating tumorigenicity and malignancy. Recently, there has
been mounting evidence that TERT may exert some of its effects in stem cells
through functions independent of its ability to maintain telomere ends. Therefore,
we hypothesized that TERT has a non-canonical (non-telomeric) cancer
signaling role that potentiates and expands a highly tumorigenic and drug
resistant subpopulation of cancer stem-like cells. To test this hypothesis, we
ectopically expressed either a wild type or a catalytically dead TERT (or control
vector) in DU145 prostate cancer cells and measured the effects on a spectrum
of cancer stem-like related phenotypes. We found that ectopically expressing
TERT resulted in a slower overall rate of proliferation with downregulation of the
cell cycle checkpoint proteins c-myc and cyclinD1. At the same time TERT-
expressing DU145 had a significant increase in a CD44
+
CD24
-
subpopulation,
which has been associated with a cancer stem-like tumorigenic, clonogenic, and
metastatic phenotype. TERT-expressing DU145 was also more invasive and
expressed lower transcript levels of E-cadherin, an important mediator of cell-cell
adhesion. Furthermore, TERT-expressing DU145 had an overall trend, although
not significant, in the cancer stem-like ability to form spheres, and had significant
downregulation of axin2, a transcriptional target of β-catenin previously shown to
vii

be regulated by TERT in stem cell models. Most remarkably, this spectrum of
phenotypic changes (surface markers, proliferation, sphere formation,
invasiveness, gene expression) occurred in response to either wild type or
catalytically dead TERT, suggesting that TERT may indeed potentiate a
phenotypic shift towards a cancer-stem like state that is independent of its
enzymatic telomere-lengthening role.

1

Introduction
Telomerase and its Components
In 1978 Elizabeth Blackburn and Joseph Gall published their findings of a
tandem repeat of the sequence TTGGGG on the chromosome end of the ciliate,
Tetrahymena thermophila, the first sequenced telomere. Blackburn and Carol W.
Greider would go on to discover telomerase, the enzyme responsible for
maintaining and lengthening telomeres in eukaryotes (de Lange, 2006).
Telomerase, a ribonucleoprotein, is composed of two components:
telomerase reverse transcriptase (TERT) and telomerase RNA component
(TERC) (Figure 1). TERT, a 127kD protein in humans, has reverse transcriptase
activity allowing the enzyme to add G-rich tandem repeats (TTAGGG) to the 3’
end of the telomeric strand using TERC as a template (Collins et al., 2002;
Cohen et al., 2007; Xu et al., 2011). By adding tandem repeats to the 3’ end of
chromosomes, telomerase is able to resolve the end replication problem: the
progressive shortening of the 3’ ends of chromosomes during each replication
cycle.
The TERC component of telomerase is 153kD and 451nt in humans.
After the primary transcript of TERC is transcribed, TERC goes through a
number of modifications including capping, editing, splicing, and cleavage before
the final product is formed (Collins et al., 2002). The final TERC RNA transcript
for humans contains an H/ACA RNA motif with primary and secondary structures
2

that form a hairpin-hinge-hairpin-ACA configuration, which is recognized by
dyskerin, a pseudo-uridine synthase protein (Collins et al., 2002; Cohen et al.,
2007). Binding of dyskerin to the H/ACA motif is required for cellular
accumulation and stability of TERC; therefore those with mutations in dyskerin
Telomerase Components

3

such as patients with dyskeratosis congenital have difficulty accumulating TERC
(Collins et al., 2002).
Recently, telomerase Cajal body protein 1 (TCAB1) has also been
implicated in forming a complex with TERT, TERC, and dyskerin as well as in
having a role in accumulation of TERC and processing in Cajal bodies
(Venteicher et al., 2009).
The well-recognized canonical function of TERT and its partners (TERC,
dyskerin, TCAB1) is the lengthening and capping of telomeres. Another group of
proteins that contribute to telomere protection and length regulation are the
shelterin proteins: TRF1, TRF2, POT1, TIN1, TPP1, and Rap1. TRF1, TRF2,
and POT1 recognize TTAGGG repeats, while TIN1, TPP1, and Rap1 work to
interconnect these proteins. Shelterin proteins bind either double stranded
telomeres or the single stranded telomeric 3’ overhang and function in telomeric
length regulation by recruitment of telomerase and by stabilization of the terminal
t-loop, a lariat like structure that is formed when the 3’ overhang loops back and
inserts into the double stranded telomeric DNA (de Lange, 2005).
Telomerase’s role in proliferation and immortality
Telomerase’s role in enabling cultured cells to proliferate indefinitely is
well established. When primary cells are cultured in vitro they go through a
number of cell divisions before undergoing growth arrest. This stage is referred
to as mortality stage 1 or senescence (M1) (Zhu et al., 1999; Kim et al., 2003).
Interestingly, this phenomenon can be bypassed by transforming the cells with
4

viral oncogenes such as simian virus 40 (SV40) large T antigen or through the
loss of suppressors such as p53 and pRb. Cells that are thus transformed can
undergo further cell divisions before undergoing mortality stage 2 or crisis (M2)
(Zhu et al., 1999).
However, both M1 senescence and M2 crisis can be bypassed through
the expression of hTERT in human cultured cells, resulting in indefinite cell
proliferation and lengthened telomeres. While expression of wildtype (WT)
hTERT is able to both lengthen telomeres and allow cells to proliferate
indefinitely, experiments using a number of different hTERT mutants and
hemagglutinin (HA) tagged hTERT have shown that hTERT’s ability to bypass
M1 and M2 is not necessarily a function of its telomere-lengthening role (Counter
et al., 1998; Zhu et al., 1999; Kim et al., 2003). In fact, experiments in cultured
human fibroblasts have shown that introduction of a mutant hTERT with 10
additional amino acids to the c-terminal, achieves bypass of M2 crisis despite
progressive shortening of telomeres to lengths that are actually shorter than
those in control cells that had undergone crisis (Zhu et al., 1999; Kim et al.,
2003). Similarly, Mukherjee et al., ectopically expressed a number of different
hTERT mutants (hTERT
IA-
, hTERT
N-DAT 116
, and hTERT
CTerm
), that had catalytic
activity but were unable to maintain telomeres in vivo, in human mammary
epithelial cells (HMECs), and found that while cellular life span was extended
there was subsequent shortening of telomeres (Mukherjee et al., 2011). They
further established that capping of short telomere ends by the shelterin subunit
5

POT1 is necessary for avoidance of senescence. Conversely, another group has
shown that TERT expression in cultured mouse embryonic fibroblasts resulted in
an increase in telomere lengths, but the cells were still not able to avoid
senescence, further establishing that TERT’s role in telomere lengthening can be
separated from its role in promoting proliferation by bypassing crisis (Artandi et
al., 2002).
Experiments using different TERT variants have shown a difference in
functions; therefore Mukherjee et al., sought to elucidate whether certain
domains of hTERT were associated with specific functions by using 15 different
variants of hTERT. While the WT hTERT has a number of functions that include
catalytic activity, the ability to synthesize telomeres, the capacity to bypass
senescence, a proliferative advantage in the absence of exogenous mitogens,
and the capability to regulate DNA damage response signaling, the 15 hTERT
variants had different mutations that rendered them incapable of doing one or
more of these functions; enabling them to use the different variants to separate
their functions. They were able to show that TERT’s growth advantage in the
absence of exogenous mitogens was dependent on catalytic activity, but could
be separated from cellular life span extension, telomere maintenance, and DNA
damage response (Mukherjee et al., 2011).
TERT in Cancer
In vitro experiments using cultured cell lines have shown that ectopically
expressing TERT can lead to a number of events such as activation of
6

oncogenes, diminished requirement for mitogens, ability to grow in unfavorable
conditions, and ability to inhibit DNA damage response (Hahn et al, 1999; Wang
et al., 2000; Gonzalez-Suarez et al., 2001; Stampfer et al., 2001; Smith et al.,
2003; Beliveau et al., 2007)
Expression of TERT in cultured primary human cells, normal human
epithelial and fibroblasts, can work with oncogenes, such as simian virus 40
large-T and H-ras, to produce neoplastic cells (Hahn et al, 1999). It has also
been shown that cell proliferation due to ectopic expression of TERT in human
mammary epithelial cells (HMEC) results in activation of the c-myc oncogene
(Wang et al, 2000). In HMEC, when TERT was ectopically expressed, the cells
requirement for exogenous mitogens and genes promoting cell growth were
diminished. TERT microarray data showed an increase in the expression of a
number of genes important in proliferation including the epidermal growth factor
recteptor (EGFR) and fibroblast growth factor (FGF) as well as a decrease in a
number of genes important in regulation of genes that control growth and
apoptosis including TNF-related apoptosis inducing ligand (TRAIL) (Smith et al.,
2003).
In a study where mTERT was overexpressed in basal keratinocytes of
mice, mTERT made the skin more susceptible to tumors upon chemical
carcinogenesis and increased the ability of these mice to heal wounds
(Gonzales-Suarez et al., 2001). Similarly, another study using mTERT over
expression in mice showed that TERT expression correlated with spontaneous
7

development of mammary intraepithelial neoplasia and invasive mammary
carcinomas (Artandi et al., 2002). Therefore, even in vivo studies show that over
expression of TERT directly correlates with increased malignancy.
Given the copious data, reviewed above, supporting the role of telomerase
in immortalization and transformation, it comes as no surprise that telomerase is
very active in many cancers and stem/progenitor cells. In fact, about 90% of all
cancers have telomerase activity to a degree that generally correlates with
aggressive or metastatic potential (Shay et al., 1997; Xu et al., 2011).
TERT’s ability to transform cells into more malignant metastatic
phenotypes can be dissociated from its role in maintaining telomeres, just as
TERT’s ability to overcome senescence can be dissociated from its role in
maintaining telomeres. When TERT was expressed in GM847 fibroblasts that
utilize alternative lengthening of telomeres (ALT), a homologous recombination-
based lengthening mechanism found in telomerase-negative tumors of
mesenchymal origin, the cells became significantly more tumorigenic (Stewart et
al., 2002; Cesare et al., 2010). The tumorigenic phenotype was also augmented
by a TERT c-terminal tagged HA, which is catalytically active but cannot maintain
telomeres in vivo, further suggesting that TERT’s role in malignancy and
tumorigenicity is not dependent upon its role in telomere lengthening (Stewart et
al., 2002).

8

TERT in stem cells
Low levels of telomerase activity have been found in a number of human
adult stem cell types including haematopoietic, neuronal, skin, intestinal crypt,
mammary epithelial, pancreas, adrenal cortex, kidney, and mesenchymal stem
cells (MSCs) (Hiyama and Hiyama, 2007). In comparison, telomerase is absent
in most normal somatic and benign tissues (Kim et al., 1994).
In mouse models, telomere shortening inhibited mobilization of hair follicle
stem cells out of their niche, impaired their growth, and resulted in suppression of
stem cell proliferation capacity in vitro (Flores et al., 2005). Conversely, TERT
overexpression promoted stem cell mobilization, hair growth, and stem cell
proliferation in vitro (Flores et al., 2005). Interestingly, the ability of stem cells to
mobilize, proliferate, and grow hair required both TERT and TERC as
mTERT/TERC-/- mice did not show these effects (Flores et al., 2005). In
contrast, another report from the same year showed that hair follicle activation
was achieved even with a catalytically inactive TERT or in the absence of TERC
(TERC-/-) (Sarin et al., 2005). Microarray analysis showed TERT mediated the
cycling from telogen to anagen through the activation of c-myc and wnt signaling,
two proteins that are important in embryogenesis and oncogenesis (Choi et al.,
2008). Further studies showed that this process was mediated by TERT binding
to a β-catenin Brg-1 (a SWI/SNF related chromatin remodeling protein) complex
and that this complex regulates Wnt-dependent genes through binding to
TCF/LEF binding sites (Park et al., 2009).
9

TERT in Cancer Stem Cells (CSCs)
Many tumor types contain a small subpopulation of cells referred to as
cancer stem cells, defined as pluripotent cells that can differentiate, form new
tumors, and are drug resistant to many therapies (Keith et al., 2007; Tang et al.,
2007).
Frequently, cancer stem cell populations have been shown to express
drug efflux pumps such as MDR-1 and ABCG2 that allow the cells to pump out
many conventional therapies; these pumps can also efflux the DNA binding
Hoechst dye allowing for fluorescence-activated cell sorting (FACS)
quantification and sorting of side population (Hoechst-negative) and non-side
population (Tang et al., 2007; Xu et al., 2011). However, this is not an
universally applicable marker; for example, our lab and others have found that
this technique cannot be employed for the prostate cancer cell line DU145 due to
inaccuracy and very low number of side population in DU145 (Tang et al., 2007).
Other markers available to detect subpopulations that are more
proliferative and invasive including cell surface markers (CD133, CD44, and
integrinα2β1) (Tang et al., 2007). Prostate cancer cells expressing the adhesion
molecule CD44 were shown to be more proliferative, clonogenic, tumorigenic,
and metastatic than CD44
-
prostate cancer cells. Furthermore, CD44
+
cells were
enriched for progenitor cells stemness genes such as Oct-3/4, bmi1, β-catenin,
and SMO (Patrawala et al., 2006). Populations of CD44
+
prostate cancer cells
were found to have a heterogenic population of both primitive stem cells and late
10

progenitor cells, suggesting that another marker along with CD44 is necessary to
separate primitive stem cells from late progenitor cells (Patrawala et al., 2006).
Interestingly, integrinα2β1
high
CD44
+
cells in both human prostate tumors and cell
lines were shown to have increased telomerase expression by our lab,
suggesting that telomerase expression maybe a necessary requirement to
potentiate this more invasive, metastatic phenotype (Marion et al., 2010, Xu et
al., 2011).
Recently a subpopulation of CD44
+
prostate cells that are CD24
-
were
identified as being more clonogenic, tumorigenic, and had more CSC-like
characteristics, similar to studies done on CD44
+
CD24
-
populations in breast
cancer (Al-Hajj et al., 2003; Ponti et al., 2005; Hurt et al., 2008). CD44
+
CD24
-

prostate cancer cells also had high expression of stemness genes, including
Oct3/4, Bmi1, SMO, and β-catenin (Hurt et al., 2008).
The CD44
+
CD24
-
subset of prostate cells also had the capability for
anchorage independent growth in spheres, similar to mammospheres formed by
CD44
+
CD24
-
breast cancer cells or neurospheres formed by CD133
+
glioma cells
(Singh et al., 2004; Ponti et al., 2005). Sphere-derived cells are enriched in stem
cell like characteristics (tumorigenicity, drug resistance, self renewal), and when
serum is added to the prostatospheres, mammospheres, or neurospheres, the
cells revert back into a parental-like differentiated phenotype (Singh et al., 2004;
Ponti et al., 2005; Hurt et al., 2008). Prostatospheres isolated from malignant
and benign prostatectomy tissue have stem like properties such as the ability to
11

regenerate tissue and self-renew (Garraway et al., 2010). Therefore, in prostate
cancer cells, enrichment for CD44
+
CD24
-
is a reasonable approach to identify a
cancer stem cell-like subset of cancer cells.

12

Hypothesis
Telomerase’s role in immortalizing cultured cell lines, stem cells, and
tumors through telomere maintenance is well established (Kim et al., 1994; Shay
et al., 1997; Counter et al., 1998; Hahn et al., 1999; Zhu et al., 1999; Wang et al.,
2000; Gonzales-Suarez et al., 2001; Stampfer et al., 2001; Artandi et al., 2002;
Collins et al., 2002; Kim et al., 2003, Smith et al., 2003; Flores et al., 2005; Sarin
et al., 2005; Ju et al., 2006; Beliveau et al., 2007; Cohen et al., 2007; Hiyama et
al., 2007; Mukherjee et al., 2011). Moreover, telomerase activity appears to
reside preferentially in progenitor cells: high in stem cells versus barely detected
in differentiated somatic cells or non-malignant tissues (Kim et al., 1994; Wright
et al., 1996; Forsyth et al., 2002; Flores et al., 2005; Sarin et al., 2005; Hiyama et
al., 2007). This observation may also be true in cancer, where a subset of the
cancer cells that are more tumorigenic and malignant may have higher
telomerase than the rest of the population. In prostate cancer, it has already
been shown that TERT activity is much higher in prostate cancer progenitor cells
than the rest of the prostate cancer cell population both in established cultured
cells and human patient samples (Marion et al., 2010; Xu et al., 2011).
Interestingly, recent data is suggesting that TERT may facilitate a role in
increasing cancer tumorigenesis and malignancy through a non-canonical
function that does not require its ability to maintain telomeres (Artandi et al.,
2002; Stewart et al., 2002; Park et al., 2009; Cesare et al., 2010; Mukherjee et
al., 2011). Therefore, we hypothesized that TERT functions in potentiating a
13

more malignant, metastatic, tumor-initiating cancer stem cell like phenotype and
that this function may be dissociated from its canonical role of maintaining
telomeres. To answer this question, we infected DU145 prostate cancer cells
with either a lentiviral wild type (WT) hTERT or a catalytically dead (CD) hTERT
harboring a mutation at D868A (Goldkorn et al., 2006). The WT hTERT and CD
hTERT DU145 cells were then used for a number of readouts to explore their
cancer stem cell like phenotype; these readouts included fluorescence activated
cell sorting (FACS) for CD44
+
CD24
-
populations, sphere formation assay, colony
formation assay, matrigel invasion assay, and qPCR for a number of genes
associated with a stem-like phenotype.

14

Material and Methods
Cell culture
DU145 human prostate cancer cell line was cultured in RPMI 1640
supplemented with 10% fetal bovine serum (Omega), penicillin (100 units/mL,
Invitrogen), and streptomycin (100ug/mL). DU145 cells were maintained at
37°C, 5% CO2.
Lentiviral production
Lentivirus was generated by co-trasfecting 293T with 12ug of lentiviral
vector, 3 ug of pMD.G and 9 ug of pCMV plasmids using calcium phosphate co-
precipitation method. The virus media was then harvested 48hrs and 72 hrs after
transfection. Collected media was filtered through a 0.45um filter.
Lentiviral Infection
DU145 cells were seeded at 10
4
per well in a 6 well plate 1 day before
infection. Either empty vector puromycin, WT hTERT-flag hygromycin, or CD
(D868A) hTERT-flag hygromycin lentivirus was added to the plates and
supplemented with 8 ug/ml polybrene. Media was changed the next morning.
Cells were then collected for either the 72 hour time point without selection or
selected using puromycin or hygromycin and collected at 2 weeks post-infection.
For cells collected at 72 hours post infection, the lentivirus was concentrated by
ultracentrifugation at 28,000 rpm for 2 hours. For cells collected at 2 weeks post
infection, lentivirus was not concentrated.

15

q-PCR-TRAP Activity
Telomerase activity was analyzed using real-time PCR-based telomeric
repeat amplification protocol (TRAP). Counted cells were lysed using TRAPeze
1 x CHAPS Lysis Buffer (Millipore). The MyiQ single color Real-Time PCR
Detection System was used to run the samples (Bio-Rad) and the program iQ5
(Bio-Rad) was used to analyze the data.
TRAP is essentially a 2 step process; during the first step, telomerase
from the lysed cell extract is used to add TTAGGG repeats to a TS
oligonucleotide, producing an extension product. The extension product is then
amplified in the second step by the addition of Taq and measured by qPCR.
Since the extension product is dependent on TERT’s ability to elongate the TS
oligonucleotide, the qPCR data indirectly measures TERT’s activity (Kim et al.,
1994).
Telomere Length Assay
To analyze telomere lengths, DNA was extracted from cells using Qiagen
DNeasy Blood and Tissue Kit (Qiagen). Telomere lengths were then measured
by real-time PCR (Bio-Rad MyiQ and iQ5) using T and S primers; primer
sequences for T and S primers can be found in table 1.
Measurement of telomere lengths requires two separate reactions, each
reaction requiring a separate primer set. The T primer is a primer for telomeric
DNA while the S primer acts as a reference for the 36B4 gene, a gene that
encodes the acidic ribosomal phosphoprotein PI. Since telomeres consist of
16

tandem repeats, the T reaction will measure the number of tandem repeats that
are present in the telomere and therefore the length of the telomere. The S
reaction will measure the single copy gene 36B4 and can be used as a control to
calculate a T/S ratio or a telomere repeat copy number to single gene copy
number ratio (Cawthon, 2002).
MTS Growth Assay
The MTS growth assay was conducted by plating 200 cells per well in a
96 well plate. Samples for each day of the growth assay were plated in
triplicates. To analyze the growth each day, 20 uL of MTS mixture (Promega)
was added to each well containing 100uL of media and cells. The plate was then
incubated for an hour at 37°C. Absorbance was then read at 490nm using Hidex
Multilable Detection Program and MikroWin 2000 to analyze data.
FACS CD44+/CD24-
Fluorescence activated cell sorting (FACS) was used to analyze
CD44
+
CD24
-
population of cells. A total of 10
6
DU145 cells were collected and
centrifuged at 1,000 rpm for 5 minutes; the cells were then washed with staining
buffer, PBS supplemented with 2% FBS and 5mM EDTA, twice. DU145 cells
were then incubated with 5uL PE conjugated anti-human CD24 (12-0247-42
eBioscience) and 20uL FITC mouse anti-human CD44 (661609 BD Pharmingen)
for 20 minutes in the dark, on ice. The stained cells were then centrifuged at
1,000 rpm for 5 minutes after incubation and washed with staining buffer twice to
remove any unbound antibody. CD44CD24 stained cells were then analyzed by
17

Flow Cytometry using FACSLSR-II (Becton Dickinson Immunocytometry
Systems).
Colony Formation Assay
To investigate the colony formation ability of infected cells, 1,000 cells
were seeded into a 10cm plate. The cells were then cultured at 37°C, 5% CO
2
.
After one week, the plates were washed 3 times with PBS and fixed with a 100%
methanol for 15 minutes at room temperature. The plates were then stained with
7mL of Giemsa stain (Sigma-Aldrich) for 1 hour at room temperature. The stain
was then removed and the plates were washed 3 times with water and air dried
overnight.
Sphere Formation Assay
To test the sphere formation ability of infected cells, cells were infected
and selected using hygromycin or puromycin 48 hours after infection. One week
after infection cells were collected and 10
4
cells per a well in a 6 well low
attachment plate were grown in MEBM Basal Medium (Lonza) with 1mL B-27
serum free supplement (Invitrogen), 20ng/mL basic human fibroblast growth
factor bFGF (Sigma), 20ng/mL human recombinant epidermal growth factor EGF
(BD bioscience) and 4ug/mL insulin from bovine (Sigma) at 37°C, 5% CO
2
. After
one week, the number of spheres was counted and images were taken using
Carl Zeiss AxioVision Rel 4.6.

18

Matrigel Invasion Chamber Assay
BD Matrigel Invasion Chambers were used to test the invasive capability
of infected cells. Infected cells were serum starved for 24 hours to allow
adequate removal of all serum for optimum chemotaxis and then collected and
counted. Matrigel-coated 24 well inserts (BD Pharmigen) were rehydrated for 2
hours and RPMI 1640 supplemented with 10% FBS (chemoattractant) was
placed into wells or lower chamber of a 24 well plate. Then 4 x 10
4
cells were
seeded onto inserts (BD Pharmigen) or upper chamber using serum free RPMI
1640 and incubated for 48 hrs at 37°C, 5% CO
2
. After 48 hours, non-migrated
cells were removed from the upper chamber by scraping using a cotton swab.
Migrated cells were then fixed with 100% methanol for 15 minutes at room
temperature and stained with Geimsa stain (Sigma-Aldrich) for 1 hour at room
temperature. The inserts were washed with water and air dried overnight. Cells
were counted using Carl Zeiss AxioVision Rel 4.6.
Quantitative Real Time PCR, qRT-QPCR
After 48 hours post infection, cells were collected and RNA was extracted
using RNA-Bee and RETROscript (Ambion). RNA was reverse transcribed into
cDNA using BluePrint RT reagent Kit for Real Time (Takara). The cDNA was
then used for real-time PCR amplification with specific gene primers and Quanta
B-R Syber Green qPCR supermix (Bioscience) using Bio-Rad MyiQ single color
Real-Time PCR Detection System (Bio-Rad) and Bio-Rad iQ5 (Bio-Rad). The
housekeeping gene β-actin was used as a loading control, hTERT was used to
19

quantify the up regulation of TERT, and genes CD44, Oct-3/4, Nanog, and Bmi1
were used as indicators of stemness. Primers for c-myc, axin2, and cylcinD1
were used to elucidate the effect of TERT on the Wnt pathway. A list of primer
sequences can be found in Table 1.
Drug Resistance
48 hours post infection, cells were collected and re-plated at a density of
3,000 cells per a well in a 96-well plate. The next day, cisplatin was added to the
cells at a concentration of 0uM, 1.5uM, 3uM, 4.5uM, and 6uM. The wells were
then analyzed by MTS assay by addition of 20uL of MTS mixture (Promega) to
each well containing 100uL of media and cells, 48 hours and 72 hours after
treatment. The plate was then incubated for an hour at 37°C. Absorbance was
then read at 490nm using Hidex Multilable Detection Program and MikroWin
2000 to analyze data.
Statistical Analysis
Experiments were conducted in triplicates and represented as means.
Error bars represent the standard deviation derived from triplicates. Statistical
significance was determined by the Student’s t test and used to compare
uninfected to WT hTERT-infected and CD hTERT-infected DU145 mean values.
Significance was determined by p-values ≤ 0.05.
20

21

Results
TERT mRNA expression, telomerase activity, and telomere lengths
DU145 cells infected with two different telomerase reverse transcriptase
(TERT) constructs were analyzed to verify the TERT mRNA expression levels,
the telomerase reverse transcriptase activity, and their ability to lengthen
telomeres. DU145 cells were infected with empty vector, WT hTERT, or CD
hTERT and antibiotic selected after 48 hours post infection. Two weeks after
infection, DU145 control uninfected, empty vector-infected, WT hTERT-infected,
or CD hTERT-infected were collected; RNA was isolated to quantify TERT
mRNA levels, cells were lysed in CHAPS buffer to quantify telomerase activity
using quanitative qPCR telomeric repeat amplification protocol (qPCR TRAP), or
DNA was extracted to verify telomere lengths.
Data from qPCR showed levels of hTERT mRNA expression were
significantly higher, approximately 200 fold, after infection with WT hTERT or CD
hTERT than uninfected cells (Figure 2).
TRAP assay data showed an 18 fold increase in telomerase activity in WT
hTERT-infected DU145 compared to uninfected and empty vector-infected
(Figure 3); similarly telomere lengths were also elongated in WT hTERT-infected
cells compared to uninfected and empty vector-infected cells (Figure 4). CD
hTERT-infected DU145 did not show any telomerase activity and telomeres were
significantly shorter suggesting that the CD hTERT mutant was in fact
catalytically dead and unable to maintain telomeres. CD hTERT-infected DU145
22

had shorter telomeres compared to uninfected or empty vector-infected cells due
to CD hTERT possibly displacing and out competing endogenous TERT to bind
to telomeric ends. While CD hTERT is able to bind to telomeric ends, it does not
have reverse transcriptase activity capable of lengthening telomere; so there is a
shortening of telomere ends during each replication cycle.

23

24

MTS Growth Assay
MTS growth assay was used to measure the proliferation rate of
uninfected, empty vector-infected, WT hTERT-infected, and CD hTERT-infected
DU145 cells. Cells were selected with either puromycin or hygromycin, re-plated
6 days post-infection in 96-well plates, and were assayed for 7 days starting at 7
days post-infection. MTS data showed that uninfected and empty vector-infected
cells proliferated at about the same rate (Figure 5). Both WT hTERT-infected
and CD hTERT-infected cells grew at a slower rate than uninfected and empty
25

vector-infected cells, with CD hTERT-infected growing slower than WT hTERT-
infected.
CD44+CD24- population size
The CD44
+
CD24
-
population of DU145 cells were quantified using
fluorescence-activated cell sorting (FACS). Uninfected, empty vector-infected,
WT hTERT-infected, and CD hTERT-infected DU145 cells were collected 2
weeks post infection, after selection was complete, and stained with CD44 and
CD24 antibodies to test for a subpopulation of cancer cells with a CD44
+
CD24
-

phenotype. The stained cells were then analyzed for FACS (Figure 6); WT
hTERT-infected and CD hTERT-infected cells had a 30%-40% increase in

26

CD44
+
CD24
-
population in comparison to uninfected and empty vector-infected
cells. CD44 upregulation also was measured at the transcriptional level. DU145
cells were infected with concentrated lentivirus of empty vector, WT hTERT, or
27

CD hTERT. 72 hours and 2 weeks post-infection cells were collected and lysed
for RNA extraction and reverse transcription to cDNA. The cDNA was then
analyzed using qPCR for the expression of CD44 mRNA normalized to β-actin
and to uninfected controls (ΔΔCt fold expression). Cells infected with WT hTERT
had approximately 1500-fold increase in hTERT mRNA associated with
approximately 11-fold increase in CD44 mRNA levels, 72 hours post infection
(Figure 7a). Similarly, cells infected with CD hTERT had approximately 600-fold
increase in hTERT mRNA and approximately 3-fold increase in CD44 mRNA. A
similar trend was seen 2 weeks post infection (Figure 7b).
Sphere Formation
We tested the ability of hTERT to induce sphere formation, a property
associated with the highly tumorigenic cancer stem-like phenotype. Control
uninfected, empty vector-infected, WT hTERT-infected, and CD hTERT-infected
cells were seeded in a 6 well low attachment plate with MEBM media
supplemented with B-27 serum free supplement, basic human fibroblast growth
factor bFGF, human recombinant epidermal growth factor EGF, and insulin. One
week after seeding, the number of spheres were counted and imaged. The
spheres were counted based on size and delineated either large or small, larger
spheres were those that were approximately 50 times the size of a single cell.
Uninfected, empty vector-infected, WT hTERT-infected and CD hTERT-infected
celled produced approximately 70, 83, 92, and 95 spheres total, respectively,
with a corresponding standard deviations of ±9.9, ±9.2, ±2.5 and ±6.6 (Figure 8).
28

The total number of spheres formed by WT hTERT-infected and CD hTERT-
infected cells was not significantly greater compared to the number of spheres
formed by empty vector infected cells. While on average WT hTERT-infected
and CD hTERT-infected cells induced a trend of more total sphere formation than
29

uninfected and empty vector-infected, the number of larger spheres was
significantly greater in uninfected and empty vector-infected. Uninfected and
empty vector both had 18 and 17 large spheres on average with a standard
deviation of ±2.1 and ±2.3. In comparison, WT hTERT and CD hTERT both had
approximately 5 and 4 large spheres with a standard deviation of ±1.5 for both.

30

Colony Formation Ability
TERT’s impact on clonogenicity, a hallmark of cancer stem cells, was
tested through a colony formation assay. After DU145 cells were infected and
selected, 1,000 cells were seeded onto 10cm dishes. After one week, the cells
were stained with Giemsa stain and colonies were counted. Both uninfected and
empty vector-infected cells had significantly more colonies than WT hTERT-
infected and CD hTERT-infected cells (Figure 9). Uninfected, empty vector-
infected, WT hTERT-infected, and CD hTERT-infected cells had approximately
230, 187, 72, and 49 colonies with a standard deviation of ±22.7, ±35.2, ±14.8,
and ±6.4.
Invasive Potential
The matrigel invasion chamber was used to quantify the invasive potential
of WT hTERT-infected and CD hTERT-infected cells compared to uninfected and
empty vector-infected cells. After infection and selection, cells were plated on
matrigel invasion chambers and cells that had invaded the matrigel were stained
48 hours after plating. WT hTERT-infected and CD hTERT-infected cells were
more invasive than uninfected and empty vector-infected cells (Figure 10). On
average, 255 uninfected cells invaded the matrigel chamber and 222 empty
vector-infected cells invaded. WT hTERT-infected and CD hTERT-infected cell
invaded at higher rates of approximately 980 cells and 1024 cells respectively.

31

To further investigate the invasive potential of WT hTERT-infected and CD
hTERT-infected cells, gene expression levels were measured for E-cadherin, an
important molecule in cell-cell recognition and adhesion; decreased E-cadherin
has been implicated previously in a more invasive and metastatic population in
32

DU145 cells (Chunthapong et al., 2004). The qPCR data from cells 72 hours and
2 weeks post-infection and selection showed a decrease in E-cadherin levels in
both WT hTERT-infected and CD hTERT-infected cells compared to uninfected
and empty vector-infected cells. At 72 hours, hTERT fold change of 1, 0.551,
33

1398.825, and 541.193 corresponded to E-cadherin fold change of 1, 0.48971,
0.075, and 0.176 for uninfected, empty vector-infected, WT hTERT-infected, and
CD hTERT-infected cells (Figure 11a). Similarly, for cells collected 2 weeks
post-infection, uninfected, empty vector-infected, WT hTERT-infected, and CD
hTERT-infected fold changes of 1, 0.588, 307.740, and 164.573 in hTERT mRNA
correlated with a fold change of 1, 1, 0.356, and 0.335 in E-cadherin mRNA
(Figure11b). At both time points, there was a marked decrease in E-cadherin
expression when hTERT was ectopically expressed.
Expression of ‘stemness’ genes
RNA was extracted for qPCR from cells 72 hours post infection to quantify
the ability of WT hTERT or CD hTERT to induce expression of Oct3/4, nanog,
and bmi1, well studied genes whose expression has been measured previously
in a number of studies to reflect a “stem-like” transcriptional profile. In WT
hTERT-infected and CD hTERT infected DU145 cells, expression of Oct3/4,
nanog, and bmi1 was not significantly different than in uninfected or empty
vector-infected cells.
Expression of Wnt/β-catenin regulated genes
Recently, Park et al. have shown that TERT can regulate the expression
of Wnt/β-catenin genes through interaction with Brg-1 (a chromatin remodeling
complex) and β-catenin (Park et al., 2009). To test this in DU145, cells were
collected 72 hours and 2 weeks post infection and used for qPCR analysis of the
34

Wnt/β-catenin regulated genes c-myc, cyclinD1, and axin2. 72 hours post-
infection, there was no significant change observed in the transcript levels of
these genes; however, after 2 weeks, WT hTERT-infected and CD hTERT-
infected cells had a significant downregulation of c-myc, cyclinD1, and axin2
gene expression (Figure 12). Uninfected, empty vector-infected, WT hTERT-
infected, and CD hTERT-infected cells had a 1-fold, 1.222-fold, 0.611-fold, and
0.620-fold change of c-myc expression. 1-fold, 1.357-fold, 0.766-fold, and 0.917-
fold of cyclin D1 expression; and 1-fold, 0.911-fold, 0.107-fold, and 0.107-fold
axin2 expression.

35

Discussion
Recently, there has emerged increasing evidence that the TERT
component of telomerase can facilitate tumorigenicity and malignancy in a
number of different systems that is independent of its well known role of
lengthening telomeres (Artandi et al., 2002; Stewart et al., 2002; Park et al.,
2009; Cesare et al., 2010; Mukherjee et al., 2011). Our lab also recently
published data suggesting that TERT is not expressed homogenously in prostate
cancer, but that TERT is preferentially higher in prostate cancer progenitor cells
(Xu et al., 2011). Therefore, we sought to determine whether TERT can
potentiate a more cancer stem-like phenotype and if this function can be
dissociated from its conventional role of lengthening telomeres. In order to
answer this question, we used DU145 an extensively studied prostate cancer cell
line with well characterized markers for enrichment of cancer stem-like cells.
Use of Du145 also allowed us to manipulate the cells by lentiviral introduction of
empty vector, wild type (WT) hTERT, or catalytically dead (CD) hTERT, with a
mutation at D868A.
Infected DU145 were quantified for their hTERT mRNA levels, telomerase
activity, and telomere lengths. The hTERT mRNA levels in WT hTERT-infected
and CD hTERT-infected cells were significantly higher than uninfected or empty
vector-infected cells. Recently, Nguyen et al. have shown that cells ectopically
expressing a mutant (D712A) hTERT had lower levels of mRNA expression
compared to WT hTERT due to the mutant hTERT being exported to the
36

cytoplasm and rapidly degraded (Nguyen et al., 2009). However, our qPCR
results showed that levels of CD (D868A) hTERT were approximately the same
as WT hTERT in infected cells. Difference in results could be due to the different
mutations sites used to render the hTERT catalytically dead; it is possible that
D712A site may be specifically recognized for export to the cytoplasm or by
degradation machinery. Intoduction of CD hTERT resulted in shorter telomeres
as expected, likely due to displacement of active WT hTERT from telomeres in
these cells.
Normal adult stem cells are characterized by slow proliferation rates, a
characteristic thought to be common to cancer stem cells (CSCs) (Moore et al.,
2011). Infection of cells with WT hTERT or CD hTERT caused a significant
decrease in proliferation rates of DU145. Introduction of the large TERT-lenti
plasmid may have caused slow proliferation independently of the TERT protein
expression; therefore proliferation was controlled for by introducing an empty
vector-lenti plasmid. Slow proliferation could also be indicative of a quiescent
stem-cell like phenotype; as a result, further experiments were done to establish
whether TERT potentiated a change in phenotype.
WT hTERT-infected and CD hTERT-infected cells had a significant
increase in CD44
+
CD24
-
population percentage and an increase in CD44 mRNA
expression. CD44
+
CD24
-
population has previously been associated with a
cancer stem-like phenotype such as being more proliferative, clonogenic,
tumorigenic, and metastatic in prostate cancer cells (Patrawala et al., 2006).
37

CD44
+
expression has also been implicated with a quiescent stem cell,
progenitor cell-like phenotype due to negative BrdU labeling in CD44
+
prostate
cancer cells in holoclones or prostatospheres (Tang et al., 2007). Therefore, one
explanation for the slow growth rates of WT hTERT and CD hTERT DU145 may
be a transition to a more cancer stem-like phenotype, as further evidenced by
their increased expression of CD44
+
.
WT hTERT-infected or CD hTERT-infected cells had increased sphere
forming ability and invasive ability. The ability to form spheres is an important
determinant in identifying cancer stem-like cells. In our study, WT hTERT-
infected and CD hTERT-infected DU145 were able to form significantly more
prostatospheres than uninfected DU145. Uninfected and empty vector-infected
had more larger sized spheres than WT-hTERT-infected and CD-hTERT infected
cells, which may be a product of WT-hTERT-infected and CD-hTERT infected
cells proliferating at a slower rate than uninfected and empty vector-infected
cells. Possibly, uninfected and empty vector-infected cells have fewer cancer
stem-like cells capable of forming spheres than WT hTERT-infected or CD
hTERT-infected cells, but uninfected and empty vector-infected cancer stem-like
cells were able to proliferate at a faster rate than WT hTERT-infected and CD
hTERT-infected cells leading to larger sized spheres.
WT hTERT-infected and CD-hTERT-infected DU145 were 4-times more
invasive than uninfected cells and empty vector-infected cells. Notably,
ectopically expressing hTERT also decreased the transcriptional levels of E-
38

cadherin, an important event in the epithelial-to-mesenchymal transition
necessary for metastasis (Chunthapong et al., 2004; Nauseef et al., 2011).
While WT hTERT-infected and CD hTERT-infected cells are more migratory,
further experiments are necessary to elucidate TERT’s role in invasiveness and
metastasis, and to determine whether this is mediated by known regulators of
EMT or by a novel mechanism.
Control and empty vector-infected cells formed more colonies than WT
hTERT-infected and CD hTERT-infected DU145. For the colony formation
assay, colonies were classified based on size (larger than 1mm). Therefore WT
hTERT-infected and CD hTERT-infected cells ability to form clones larger than
1mm may have been hindered by their slow proliferation rates due to a number
of colonies that did not make the 1mm cutoff. Conversely, WT hTERT-infected
and CD hTERT-infected cells may have caused a phenotypic change rendering
the cells less capable of forming colonies than uninfected and empty vector-
infected.
Previous experiments have shown that CD44
+
or CD44
+
CD24
-
populations
in DU145 have higher expression of oct3/4, nanog, notch, and bmi1 (Patrawala
et al., 2006; Hurt et al., 2008). While hTERT increased the population of
CD44
+
CD24
-
, in our studies, we did not see an increase in oct3/4, nanog, notch,
bmi1. There are no published reports directly connecting hTERT to the
regulation of these genes; therefore, any association of hTERT signaling with a
39

cancer stem-like phenotype may not be mediated through these pathways,
although further studies would be necessary to rule out an association.
There has been recent evidence that TERT can regulate Wnt/β-catenin
regulated genes, including c-myc, axin2, cyclinD1, through interaction with β-
catenin and Brg-1, a chromatin remodeling complex (Choi et al., 2008, Park et
al., 2009). Our results demonstrated a correlation between hTERT expression
and c-myc, cyclinD1, and axin2 mRNA expression; introduction of WT hTERT or
and CD hTERT, resulted in a decrease in c-myc, cyclinD1, and axin2 mRNA
levels. Both c-myc and cyclinD1 are important in the regulation of the cell cycle;
decrease in c-myc and cyclinD1 may explain in part the slow proliferation rate of
both WT hTERT-infected and CD hTERT-infected cells. Future studies utilizing
BrdU staining and FACS cell cycle analysis could help to elucidate these
important points.
In summary, ectopic expression of hTERT resulted in slower proliferation
rates, a change in cell surface markers (more CD44+CD24- population), an
increase in sphere formation capability, more invasive cells, a decrease in the
ability to form clones, and a change in gene expression of CD44, E-cadherin, c-
myc, cyclinD1, and axin2. Most importantly, all of the cancer stem-like phenotypic
changes seen in WT hTERT-infected DU145 were reproduced in CD hTERT-
infected DU145, suggesting that hTERT may indeed have a non-canonical
function in potentiating a cancer stem-like phenotypic change that is independent
of its canonical role of maintaining telomeres.
40

In order to further elucidate this potential non-canonical function of TERT,
additional experiments will have to be done. Currently, we are testing the
tumorigenicity potential of WT hTERT and CD hTERT infected DU145 in
immunodeficient (NSG) mice. A characteristic of CSCs is their ability to evade
conventional therapies; therefore, we are currently testing the ability of hTERT-
infected cells to enrich for a CSC population that is resistant to
chemotherapeutics such as docetaxel and cisplatin.
Our current lentiviral hygromycin WT hTERT and CD hTERT are
constitutively active, resulting in a suboptimal experimental time lines because of
the necessary delays posed by selection. To address this drawback, I recently
cloned WT hTERT and CD hTERT constructs into a tet-inducible vector.
Infecting cells with an inducible vector will be a useful tool for selecting a pure
population prior to expressing the TERT variants, and then expressing them for
short time spans such as 24 hours, 48 hours, and 72 hours prior to measuring
the phenotypes of interest (e.g. gene expression, sphere formation, cell surface
markers, etc).
These experiments will all be conducted in a number of different cancer
cell lines including DU145 and ultimately in vivo or in primary tissue to better
identify and mechanistically study the possible non-canonical signaling role of
TERT in the cancer-stem-like phenotype.

41

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Abstract (if available)

Abstract The telomerase reverse transcriptase (TERT) component of telomerase has reverse transcriptase activity capable of elongating telomeres during each replication cycle of the cell. TERT plays a dual role in activating normal stem cell niches and in potentiating tumorigenicity and malignancy. Recently, there has been mounting evidence that TERT may exert some of its effects in stem cells through functions independent of its ability to maintain telomere ends. Therefore, we hypothesized that TERT has a non-canonical (non-telomeric) cancer signaling role that potentiates and expands a highly tumorigenic and drug resistant subpopulation of cancer stem-like cells. To test this hypothesis, we ectopically expressed either a wild type or a catalytically dead TERT (or control vector) in DU145 prostate cancer cells and measured the effects on a spectrum of cancer stem-like related phenotypes. We found that ectopically expressing TERT resulted in a slower overall rate of proliferation with downregulation of the cell cycle checkpoint proteins c-myc and cyclinD1. At the same time TERT-expressing DU145 had a significant increase in a CD44+CD24- subpopulation, which has been associated with a cancer stem-like tumorigenic, clonogenic, and metastatic phenotype. TERT-expressing DU145 was also more invasive and expressed lower transcript levels of E-cadherin, an important mediator of cell-cell adhesion. Furthermore, TERT-expressing DU145 had an overall trend, although not significant, in the cancer stem-like ability to form spheres, and had significant downregulation of axin2, a transcriptional target of β-catenin previously shown to be regulated by TERT in stem cell models. Most remarkably, this spectrum of phenotypic changes (surface markers, proliferation, sphere formation, invasiveness, gene expression) occurred in response to either wild type or catalytically dead TERT, suggesting that TERT may indeed potentiate a phenotypic shift towards a cancer-stem like state that is independent of its enzymatic telomere-lengthening role.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Madhav, Anisha (author)

Core Title The noncanonical role of telomerase in prostate cancer cells: exploring a non-telomeric signaling role for telomerase protein (TERT) in a cancer cell line

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 05/04/2012

Defense Date 03/22/2012

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

Tag cancer stem cells,OAI-PMH Harvest,prostate cancer,telomerase,TERT

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair)

Creator Email anishama@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-30249

Unique identifier UC11289506

Identifier usctheses-c3-30249 (legacy record id)

Legacy Identifier etd-MadhavAnis-764-0.pdf

Dmrecord 30249

Document Type Thesis

Rights Madhav, Anisha

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...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA