Attributes of Oct4 in stem cell biology: perspectives on cancer stem cells of the ovary.
ABSTRACT Epithelial ovarian cancer (EOC) remains the most lethal of all the gynaecological malignancies with drug resistance and recurrence remaining the major therapeutic barrier in the management of the disease. Although several studies have been undertaken to understand the mechanisms responsible for chemoresistance and subsequent recurrence in EOC, the exact mechanisms associated with chemoresistance/recurrence continue to remain elusive. Recent studies have shown that the parallel characteristics commonly seen between embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) are also shared by a relatively rare population of cells within tumors that display stem cell-like features. These cells, termed 'cancer initiating cells' or 'cancer stem cells (CSCs)' have been shown not only to display increased self renewal and pluripotent abilities as seen in ESCs and iPSCs, but are also highly tumorigenic in in vivo mouse models. Additionally, these CSCs have been implicated in tumor recurrence and chemoresistance, and when isolated have consistently shown to express the master pluripotency and embryonic stem cell regulating gene Oct4. This article reviews the involvement of Oct4 in cancer progression and chemoresistance, with emphasis on ovarian cancer. Overall, we highlight why ovarian cancer patients, who initially respond to conventional chemotherapy subsequently relapse with recurrent chemoresistant disease that is essentially incurable.
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ABSTRACT: The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.Reproductive Biology and Endocrinology 11/2014; 12(1):114. · 2.41 Impact Factor
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ABSTRACT: Cell differentiation and different pathways of cell death were immunohistochemically analyzed in ovaries of six human embryos, 20 serous borderline tumors (SBT) and ovarian serous carcinomas (OSC) using markers for apoptosis (caspase-3, AIF, TUNEL) and stemness (Oct-4). In the 5-8-week ovaries, caspase-3 was absent in the ovarian surface epithelium (ose) and mildly positive in the ovarian stroma (os), AIF was expressed moderately, while Oct-4 expression gradually decreased during that period. Some ovarian cells expressed only caspase-3 or AIF together with TUNEL, while both caspase-3 and AIF were co-expressed in other ovarian cells. Mild expression of Oct-4 and caspase-3 characterized some cells of SBT, while their expression varied from mild to strong in OSC. AIF displayed mild to strong expression in ose of SBT and moderate to strong expression in OSC, while no expression of AIF was observed in os of both tumors. In the ose of both SBT and OSC, caspase-3 and AIF were co-expressed only occasionally, while AIF and Oct-4 were co-expressed strongly. Our study showed the presence of stemness cells and different pathways of cell death (caspase-3 and AIF-mediated) in the ovarian tissue during development and carcinogenesis, indicating the correlation between developmental plasticity in human embryonic ovaries and OSC.Acta histochemica 09/2013; · 1.61 Impact Factor
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ABSTRACT: Tumor microenvironment plays an important role in cancer progression owing to interactions between the tumor and adjoining cells and, as in bone marrow, the unique ar-chitecture and chemical compounds that characterize it. It has recently been proposed that bone marrow shelters cancer cells in niches which may favor regulation of their quiescence, although the mechanisms involved have yet to be elucidated. We conjectured that a bone mar-row mimicking, calcium providing 3D biolattice recently developed by our group could affect fibrosarcoma cells in various ways, whether via its specific architecture or due to its provi-sion of calcium, an element correlated with many tumorgenic processes. In order to verify our conjecture, we examined the modifications induced in fibrosarcoma cells by this biolattice. We found that its regulatory effects on fibrosarcomas enhanced tumorigenicity, mediated by up-regulated tumorigenesis related genes. We observed decreased proliferation of cancer cells accompanied by up-regulation of genes associated with cancer stem cells, pointing to a process of de-differentiation. In addition, our results revealed up-regulation of Wnt4 and c-Myc in cells cultured on the biolattice, along with down-regulation of AXIN-1 and WIF-1. Taken together these findings suggest that a calcium rich bone marrow-like microenvironment can affect the tumorigenic capacity and fibrosarcoma cells de-differentiation through the mediation of the Wnt signaling pathway.Cancer studies and molecular medicine. 11/2014; 1(1):15-25.
Attributes of Oct4 in stem cell biology:
perspectives on cancer stem cells of the ovary
Chantel Samardzija1,2, Michael Quinn1,2, Jock K Findlay1,2,3and Nuzhat Ahmed1,2,3*
Epithelial ovarian cancer (EOC) remains the most lethal of all the gynaecological malignancies with drug resistance
and recurrence remaining the major therapeutic barrier in the management of the disease. Although several
studies have been undertaken to understand the mechanisms responsible for chemoresistance and subsequent
recurrence in EOC, the exact mechanisms associated with chemoresistance/recurrence continue to remain elusive.
Recent studies have shown that the parallel characteristics commonly seen between embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSC) are also shared by a relatively rare population of cells within tumors that
display stem cell-like features. These cells, termed ‘cancer initiating cells’ or ‘cancer stem cells (CSCs)’ have been
shown not only to display increased self renewal and pluripotent abilities as seen in ESCs and iPSCs, but are also
highly tumorigenic in in vivo mouse models. Additionally, these CSCs have been implicated in tumor recurrence
and chemoresistance, and when isolated have consistently shown to express the master pluripotency and
embryonic stem cell regulating gene Oct4. This article reviews the involvement of Oct4 in cancer progression and
chemoresistance, with emphasis on ovarian cancer. Overall, we highlight why ovarian cancer patients, who initially
respond to conventional chemotherapy subsequently relapse with recurrent chemoresistant disease that is
Keywords: Ovarian carcinoma, Cancer stem cell, Metastasis, Chemoresistance, Recurrence, Embryonic stem cells,
Induced pluripotent stem cells
Ovarian cancer is the fifth leading cause of cancer-related
death in women worldwide and has the highest rate of
cancer-related mortality among all the gynaecological
neoplasms in the Western world . It predominately
204,000 women diagnosed with this disease each year .
While the term ‘ovarian cancer’ encompasses a broad
range of ovarian neoplasms , there are three basic
classes of ovarian malignancies and each arise from the
rapid growth and division of one of three major cell types
found within the ovary . Tumors developing from the
oocyte producing germ cells are known as germ cell
tumors (~5-10%), while those arising from specialised
granulosa, theca and hilus cells are classified as sex cord
stromal tumors (~10-15%) . In comparison however,
these tumors represent a relatively rare group of ovarian
malignancies, with cancers of the ovarian surface epithe-
lium accounting for the largest proportion of all ovarian
cancer cases. Such tumors have been termed Epithelial
Ovarian Cancers (EOCs) and account for 90% of all ovar-
ian tumors . Despite an improved knowledge about
EOCs and advances in existing treatments, more than
70% of EOC patients succumb to the disease within
5 years of their initial diagnosis , contributing annually
to more than 125,000 deaths worldwide . If however
the disease is detected at an early stage, the five year sur-
vival rate dramatically increases to 95% . Unfortunately,
the asymptomatic nature of the disease combined with
the lack of specific screening techniques for early detec-
tion, means most women are diagnosed when the disease
has progressed to an advanced metastatic stage (Stages
* Correspondence: Nuzhat. Ahmed@thewomens.org.au
1Women’s Cancer Research Centre, Royal Women’s Hospital, 20 Flemington
Road, Parkville, VIC 3052, Australia
2Department of Obstetrics and Gynaecology, University of Melbourne,
Melbourne, VIC 3052, Australia
Full list of author information is available at the end of the article
© 2012 Samardzija et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Progression of epithelial ovarian cancer
Advanced stage EOC is a highly metastatic disease com-
monly characterised by widespread peritoneal dissemin-
ation and ascites . Due to the lack of anatomical
barriers surrounding the ovaries, ovarian carcinomas are
easily capable of disseminating directly from the ovary
and into the peritoneal cavity, thus allowing the direct
attachment of malignant cells to the peritoneum to form
secondary tumors. This occurs by either mechanical or
enzymatic disruption where malignant cells are then able
to form cellular aggregates (spheroids) within the peri-
toneal ascites fluid [6,7]. As a result, the spheroids are
capable of attaching and infiltrating the mesothelial lin-
ing of the peritoneal cavity resulting in the formation of
secondary tumors [7,8]. Currently, this presents a major
problem in the treatment of ovarian cancer as cytore-
ductive surgery is unable to completely eliminate micro-
scopic disease within the peritoneum .
Chemoresistance in epithelial ovarian cancer
Frontline treatment of advanced EOC usually involves
cytoreductive surgery followed by systemic administra-
tion of chemotherapy [5,6]. Currently, the standard
chemotherapy regime is a combination of a platinum
compound such as cisplatin or carboplatin synergised
with a taxol-based agent, usually paclitaxel [5,6]. Overall,
this has achieved an initial patient response rate of
70-80% . However, even in patients who initially re-
spond well, most relapse in a relatively short period of
time and the overall prognosis continues to remain poor
. This can be partly attributed to the advanced-stage
of the disease at diagnosis and the highly aggressive na-
ture of the disease. However, one of the most important
causes of failure in EOC treatment is the development
of residual and recurrent tumor cells that are resistant
to cisplatin and paclitaxel treatment . It has recently
been shown that while platinum-based treatments are
extremely efficient in removing the bulk of the ovarian
tumor mass, it fails to eliminate a core of highly specia-
lised CSC-like cells, which are not only highly invasive
but are capable of initiating new tumor growth [9,10].
Recurrent ovarian tumors are known to be enriched
with CSC-like cells and stem cell pathway mediators in-
cluding ALDH1, CD44, CD133, Notch, Wnt and TGFβ,
suggesting that CSCs may contribute to recurrent dis-
Oct4 and its role as an embryonic stem cell factor in adult
Oct4 (Oct3/4 or POU5F1) is a member of the POU fam-
ily of transcription factors and is known to play a pivotal
role in the maintenance of self-renewal and pluripotency
in ESCs. It is commonly expressed in unfertilized
oocytes, the inner cell mass (ICM) of a blastocyst, germ
cells, embryonic carcinoma cells and embryonic germ
cells . While upregulation of Oct4 sustains an undif-
ferentiated pluripotent stem cell state, a loss of Oct4
induces stem cells to undergo differentiation, producing
a heterogenous population of highly specialised daughter
cells. This is evidenced by the loss of pluripotency in the
ICM cells of Oct4−/−mouse embryos, where loss of
Oct4 results in the differentiation of embryonic stem
cells into a trophoblast lineage . Studies have also
demonstrated that a two-fold increase in Oct4 expres-
sion results in the conversion of ESCs towards a primi-
tive endoderm and mesoderm state . Conversely, a
50% decrease in Oct4 expression can induce differenti-
ation of ESC into trophectoderm . This suggests that
the precise level of Oct4 protein expression in ESCs is
crucial to maintain lineage-specific ESC differentiation
and different developmental fates. Little is known about
the exact regulation of Oct4 protein in ESCs, although it
has been suggested that a highly sensitive sensor mech-
anism exists that is capable of detecting and regulating
Oct4 levels within ESCs .
Interestingly, although Oct4 is primarily expressed in
primitive ESCs and its expression is lost with differenti-
ation during the developmental process , it has been
shown that a minor population of very small embryonic-
like stem cells (VSELs) with pluripotent potential and
positive for Oct4, stage specific embryonic antigen-
(SSEA)-3/4 (human), Sca-1 and Nanog [15,16] are
present in the bone marrow, cord blood, epidermis,
heart, pancreas, testis, bronchial epithelium and ovaries
[15,17]. It has been hypothesized that VSELs expressing
both epiblast and germ line markers are deposited in
developing tissues and organs during early gastrulation
. The Oct4 promoter in these cells has been shown
to have an open chromatin structure which can be ac-
tively transcribed, suggesting the transcription abilities
of these cells . However, these cells are protected
from uncontrolled proliferation and teratoma formation
by a unique DNA methylation pattern in some develop-
mentally crucial imprinted genes which show a hypo-
methylation pattern in paternally methylated genes
[insulin-like growth factor 2 (Igf2) and Rasgrf1] and
hypermethylation in the maternally methylated genes [of
H19, Igf2 receptor (igf2R) and p57Kip2 (also known as
Cdkn 1c)] [18,19]. It has been demonstrated that, rever-
sal of these epigenetic changes in VSELs may result in a
greater expansion of these cells , and a few recent
studies have demonstrated that both murine and human
Oct4 positive VSELs exhibit characteristics of long-term
repopulating hematopoetic stem cells  and may also
differentiate into organ-specific cells (such as cardio-
myocytes) . Consistent with that, a gradual decrease
in the number of Oct4 positive VSELs has been shown
to be an important mechanism of aging, as evidenced in
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 2 of 12
a recent murine model . These results suggest that
isolated Oct4 positive VSELs may serve as a good source
of pluripotent stem cells in adult tissues and have a po-
tential application in regenerative medicine . It has
been hypothesized that in pathological conditions the
tissue-specific VSELs may undergo mutation in the ‘qui-
escence associated genetic imprints’ which may initiate
the development of tissue-specific malignancies .
Oct4 and its role as an induced pluripotent stem cell
The past decade has shown that somatic cells can be re-
programmed into induced pluripotent stem cells (iPSCs)
by transient ectopic expression of a cocktail of transcrip-
tion factors such as Oct4, Sox2, Klf4 and c-Myc [23-25].
For example, human fibroblasts can be reprogrammed
by the ectopic expression of Oct4, Sox2, Nanog and
Lin28 , or similarly by the over expression of only
Oct4 and Bmi1 . To date, iPSCs have been derived
from numerous human somatic cell populations and
closely resemble human ESCs in gene expression, pro-
moter methylation and differentiation potential .
However, among the several combinations of transcrip-
tion factors needed to make iPSC, Oct4 is the only one
that has been shown to be required exogenously, sug-
gesting that this transcription factor may act as a ‘gate-
keeper’ of pluripotency in somatic cells . Nonetheless,
some recent studies have shown that while Oct4 can be
replaced by the overexpression of E-cadherin or the orphan
nuclear receptor Nr5a2 together with Sox2, Klf4 and c-Myc
for reprogramming of mouse embryonic fibroblasts, the
reprogramming efficiency decreases when Oct4 is not
present [30,31], emphasizing once again the absolute
requirement of Oct4 for the efficient reprogramming of
somatic cells into iPSCs.
Synergies of cancer cells with ESC and iPSC in the context
of Oct4 expression
Cancer cells exhibit traits that are commonly associated
with ESCs or iPSCs including immortal cell growth and
high proliferation rates under appropriate culture condi-
tions . Both iPSCs and cancer cells are characterised
as having high telomerase activity  and genomic in-
stability resulting in chromosomal aberrations .
Changes in gene expression profiles and corresponding
epigenetic changes have been observed in cancer cells,
ESCs and iPSCs [35-37]. Additionally, similar to cancer
cells, both ESCs and iPSCs give rise to teratomas after
transplantation into immunocompromised mice . In
this context it should be mentioned that the common
reprogramming factors such as c-Myc, Klf4, Sox2, Lin28
and Oct4 are highly expressed in many cancer cell types
[39,40], suggesting that reprogramming of somatic cells
and tumorigenesis rely on common mechanisms.
Role of Oct4 in tumor progression
The first involvement of Oct4 in cellular transformation
was observed when ectopic dose-dependent expression
of Oct4 was shown to increase the malignant potential
of ESCs . Exogenous expression of Oct4 has been
shown to mitigate dysplasia in the epithelial tissues of
adult mice . Consistent with that, Oct4 is expressed
in a number of malignant neoplasms and the expression
profile has been correlated with tumor grade and disease
progression [43-46]. Compared to tumors with low Oct4
expression, elevated levels of Oct4 have been associated
with metastases and shorter patient survival rates,
[47-49]. A recent study on breast cancer has demon-
strated that ectopic expression of Oct4 in normal breast
cells led to the generation of cells with tumor-initiating
and colonization abilities . These cells developed
high-grade, poorly differentiated breast carcinomas in
nude mice and demonstrated a genomic profile enriched
in an embryonic transcription factor network, suggesting
that Oct4-transduced cells may represent a patient-
specific model system for the discovery of novel onco-
genic targets . Furthermore, ectopic Oct4 expression
has been shown to enhance the features of cancer stem
cells in a mouse model of breast cancer . Oct4 ex-
pression has also been shown to maintain CSC-like
properties in CD133-derived lung cancer cells .
Overall, these studies highlight the importance of sus-
taining Oct4 expression by tumors in order to maintain
the tumorigenic stem cell-like characteristics.
Epithelial to mesenchymal transition (EMT) is a vital
process for morphogenesis during embryonic develop-
ment , and also for the conversion of early stage
tumors to invasive neoplasms . Recent studies have
demonstrated that EMT also plays a critical role in
tumor recurrence which is believed to be tightly linked
with the CSC phenotype [55,56]. A recent study on
prostate cancer has demonstrated that prostate cancer
cell lines that acquired EMT phenotype shared a stem
cell-like signature including enhanced expression of
Oct4 and increased tumorigenicity in mice . In
addition, ectopic expression of Oct4 and Nanog in lung
adenocarcinoma cell line has been shown to increase the
percentage of sub-population cells expressing CD133,
drug resistance and promote EMT . In contrast,
down regulation of Oct4 in a breast cancer cell line
which has a high endogenous level of Oct4 has been
shown to promote invasion and metastasis by inducing
EMT . These contradictory results suggest that the
reprogramming-competent Oct4 can differentiate cancer
cells to either an epithelial or mesenchymal state of plas-
ticity. In these scenarios one can expect that the Oct4-
initiated invasive phenotype (EMT) or Oct4-silenced
EMT may be dictated and tightly regulated by endogen-
ous Oct4 expression.
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 3 of 12
Role of Oct4 in drug resistance
As a regulator of pluripotency and self-renewal, it is
believed that Oct4 plays a crucial role in the survival of
a population of CSCs with drug resistance phenotype
. This has been supported by a study in liver cancer
cells, where Oct4 over expressing cells were found to be
more resistant to cisplatin and doxorubicin treatment
compared to control cells both in vitro and in vivo .
In oral cancer, Oct4 along with Nanog was shown to be
significantly expressed in cisplatin resistant patients .
Treatment of oral cancer cells with cisplatin resulted in
a population of resistant cells enriched in stem/progeni-
tor cells which displayed increased migratory and inva-
sive capabilities both in vitro and in vivo . This
suggests that cancer cells that express Oct4 and survive
treatment with cisplatin could develop into a heteroge-
neous population of differentiated cells that have the
increased ability to become metastatic. In support of this
proposal, drug resistant prostate cancer cell lines have
been shown to have enhanced expression of Oct4 and
several target genes (MIDI, MYB, IL1RN, RPS27 and
CUGBP2) . These cells exhibited enhanced invasive
potential by in vitro assays and tumorigenic potential by
in vivo mouse xenograft models . Knocking down
Oct4 expression by specific small hairpin (sh) RNA atte-
nuated the growth of drug-resistant cells in vitro and
in vivo, suggesting that Oct4 expression in cancer cells
not only plays an important role in tumorigenesis but is
also essential for acquiring/maintaining a drug-resistant
Evidence of Oct4 in normal ovaries
The literature on human ovarian tissues is limited due
to the difficulties in obtaining normal ovaries for re-
search. Scraped human surface epithelium is commonly
used to study the biology of epithelial ovarian cells [4,7].
Recent studies have shown the existence of adult human
ovarian stem cells in the ovarian surface epithelium of
postmenopausal women and women with premature
ovarian failure [17,63]. These women had no oocytes or
follicles. The ovarian surface epithelium of these women
expressed small round bubble-like stem cells with ex-
pression of early embryonic developmental markers such
as SSEA-4, Oct4, Nanog, Sox-2 and c-kit . In cell
culture some of these cells grew in size into oocyte-like
cells which expressed Oct4, c-kit, VASA and ZP2 tran-
scription factors specific for early oocytes [17,64]. Later
studies have shown the presence of two distinct popula-
tions of stem cells in adult mammalian ovarian surface
epithelium (including rabbit, sheep, monkey and meno-
pausal women), the very small (1–3 μM) embryonic-like
stem cells (VSELs) which are quiescent and pluripotent
(PSCs) and express Oct4, Nanog, Sox2, telomerase
(TERT) and signal transduction and activation of
transcription factor 3 (STAT3) . These small PSCs in
culture undergo spontaneous differentiation into larger
(4–7 μM) oocyte-like cells, parthenote and embryoid-
like structures with neuronal and mesenchymal pheno-
types [64-66]. These studies confirm the presence of
putative stem cells in the ovaries of adult and older
women and have important implications for the treat-
ment of infertile women in the field of reproductive
Evidence for Oct4 in EOC
The current literature on the expression and role of
Oct4 in EOCs is relatively sparse, with the transcription
factor primarily being used as marker to detect CSC-like
populations in CSC-enriched ovarian cancer cell lines
and tumors . The expression of Oct4 was first
described in an ovarian dysgerminoma, a tumor of the
ovary that is composed of primitive, undifferentiated
germ cells . However, these authors failed to detect
the expression of Oct4 in other non-germ ovarian
tumors such as granulosa cell tumors, Brenner tumors,
serous and endometrioid adenocarcinomas and ovarian
stromal carcinomas. Recently, Oct4 expression has been
described in immature teratoma of the ovary , in Fal-
lopian tube epithelium and serous and mucinous epithe-
lial ovarian tumors of different histological grades using
immunohistochemical analysis . In this study, Oct4
expression was shown to be significantly increased from
normal ovarian surface epithelium/Fallopian tube epithe-
lium to benign/borderline tumors to high grade serous
carcinomas, suggesting that the expression of Oct4 is
associated with the initiation and progression of serous
ovarian cancer . However, this study found no sig-
nificant difference among normal, benign, borderline
and malignant tumors in the mucinous group, and did
not study the endometrioid and clear subtype of EOC
. The differences in the expression of Oct4 between
serous and mucinous EOC may be due to the differences
in the genetic makeup of serous and mucinous subtypes
of ovarian tumors . Therefore, while the study
included a substantial number of human specimens (495
cases, including 35 normal Fallopian tube samples and
40 normal ovaries) no clear distinction of Oct4 expres-
sion could be obtained between the different histological
sub-types of ovarian tumors. A more recent study on
one case report has demonstrated the presence of
SSEA-4, Sox2, VASA and ZP2 positive oocyte-like cells
on the ovarian surface epithelium of women diagnosed
with serous papillary adenocarcinoma . The authors
in this case report suggest an association between the
pathological condition of serous papillary adenocarcin-
oma and the presence of primitive oocyte-like cells
which may have persisted from foetal period of life of
that particular patient or potentially may have developed
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 4 of 12
from putative stem cells (VSELs) of ovarian surface epi-
The first involvement of Oct4 in EOC stem cells was
demonstrated after a single tumorigenic clone was iso-
lated from the ascites of a patient with advanced EOC
using serial dilution . This clone demonstrated self-
renewal characteristics by forming spheroids in culture
and displayed differentiation properties by forming
multi-cellular colonies in agar. Furthermore, on serial
xenograft implantation, isolated clones continued to es-
tablish tumors in nude mice similar to primary human
EOC tumors. In subsequent studies, CSCs have been
isolated from ovarian tumors and cell lines based on
their abilities to differentially efflux the DNA binding
dyes, commonly known as side population by flow cyto-
metric pattern [74,75]. This population of stem cells dis-
played the classical stem cell property in tumorigenicity
assays, had an enhanced expression of Oct4, and were
extracted from an ovarian cancer cell line were found to
be enriched for the ATP-binding cassette (ABC) trans-
porter (ABCB1) and histone methyltransferase (EZH2 a
member of polycomb family with stemness property)
and Oct4 after chemotherapy treatment . In the
same context, side population enriched cells have been
isolated from the ascites of ovarian cancer patients and
these have shown an enhanced stemness profile com-
pared to non-side population cells . In addition, gene
expression analysis have shown that the side population
cell signature was enriched in patients with early recur-
rence (1–12 months) compared to those with a later
(13–24 months) recurrence . Hence, the expression
of Oct4 in the side population of ovarian cancer patients
may have an important clinical application.
In recent studies several cell surface and non-surface
markers have been used to isolate ovarian CSCs. CSCs
in these studies have been isolated depending on the dis-
tinct pattern of surface markers (i.e. CD44, EpCAM,
CD133, CD117, Thy1, CD24) [50,78-80], and non-
surface markers (i.e. aldehyde dehydrogenase activity)
 The CSCs sorted on the basis of these markers have
shown the potential to have ‘CSC characteristics’ (ability
to self renew, resistance to therapy, develop tumors in
very small numbers ~100 cells, etc.), and almost all of
them had relatively high expression of Oct4. A recent
study has demonstrated the combined expression of
Oct4 and Lin28 in ovarian tumors which correlated that
with advanced tumor grade . They also demon-
strated that the repression of Oct4 together with Lin28
in ovarian cancer cell lines by RNA interference reduced
the survival of cancer cells. The most recent study from
our group has demonstrated enhanced mRNA expres-
sion of invasive and CSC-like markers (EpCAM, CD44,
STAT3, Oct4, MMP2 and MMP9) in the ascites-derived
tumor and stromal cells isolated from the ascites of che-
moresistant versus chemonaive patients . These
studies suggest that over expression of Oct4 may be one
of the defining features of ovarian cancer stem cells
which may regulate cancer progression, drug resistance
and recurrence. Hence, Oct4 may be a promising target
for therapy in EOC.
Correlation of Oct4 with Nanog in the context of stem
Like Oct4, Nanog is also essential for the maintenance
of embryonic stem cell fate . Nanog transcript first
appears at the ICM of blastocyst after compaction, and
is no longer detectable at implantation , while Oct4
is expressed prior to compaction in all blastomeres .
The expression of both Nanog and Oct4 remains
restricted to epiblast as embryonic development pro-
gresses . Although both Oct4 and Nanog have inde-
pendent roles in different cell types, a part of their
function in pluripotent cells is driven by a synergistic
interaction that drives the transcription of target genes
[87,88]. A recent study has demonstrated a cooperative
interaction between Nanog, Sox-2 and Oct4 by identify-
ing a composite sox-oct cis-regulatory element within
the Nanog proximal promoter . Using chromatin
immunoprecipitation, this study showed that Oct4 and
Sox-2 bind to the promoter region of Nanog in living
mouse and human ESCs, and specific knockdown of
Oct4 and Sox2 mRNA by RNA interference reduces
Nanog promoter activity to almost that of the back-
ground levels, suggesting a genetic link between the
pluripotent activity of Nanog promoter and the levels of
Oct4 and Sox-2 . In another recent study, endogen-
ous Oct4 and Nanog have been shown to interact and
form multiple repression complexes to control gene ex-
pression in mouse ESCs, suggesting that these two es-
repressors complexes on their target genes to control
the fate of ESC .
The pluripotent potential of Nanog along with Oct4
has been evidenced by the expression of these transcrip-
tion factors in VSELs in adult tissues . Mobilization
of VSELs expressing Nanog and Oct4 into peripheral
blood has been observed in patients with acute myocar-
dial infarction  and acute burn injury , suggest-
ing the pluripotent potential of these cells in tissue
repair. However, how these two embryonic markers con-
tribute to the development of tumor is still not clearly
understood. It has been suggested that epigenetic
changes/mutations in the genes that maintains the qui-
escence of VSELs could potentially lead to tumor forma-
tion . Therefore, it will be important in future
studies to investigate whether the genomic imprinting
pattern differs between VSELs isolated from normal
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 5 of 12
versus tumorigenic populations. In this context, co-
expression of Oct4 and Nanog in heptocellular ,
pancreatic  and oral  cancers has been predictive
of a worse clinical outcome. Both Nanog and Oct4 in
two independent studies have been shown to be highly
expressed in ovarian carcinomas [69,94].
Confusion with Oct4 Isoforms and problems in analysing
data from current literature
The POU family transcription factors regulate genes
containing an octamer motif (ATGCAAAT) in their pro-
moter or enhancer regions . The human Oct4 gene
is located on chromosome 6 and consists of five exons.
Oct4 encodes for three main variants generated by alter-
native splicing known as Oct4A, Oct4B and Oct4B1 
(Figure 1). At the nucleotide level, both Oct4A and
Oct4B are identical in exons 2–5, the differences how-
ever lie in exon 1 . Exon 1 is missing in the trun-
cated Oct4B and it specifically consists of exon 2a.
OctB1 is identical to Oct4B except it has an additional
exon 2c . Human Oct4A and Oct4B are composed
of 360 and 265 amino acids respectively and the last 225
C-terminal amino acids are identical in both splice var-
iants  (Figure 1). The protein product of OctB1 has
not been identified yet. An in-frame stop codon TGA is
located in the additional exon 2c of Oct4B1 which is
spliced out in Oct4B mRNA . Hence, Oct4B1 cannot
encode the full length Oct4B-265 product.
Oct4A is specifically expressed in the nucleus of ESCs,
human somatic stem cells, somatic tumor cells and at a
basal level in some adult stem cells . The functional
protein for Oct4A has not been reliably detected in the
non-pluripotent cells, and it is still not clear if the basal
expression of Oct4A in non-pluripotent cells endows
any biological function. However, a high expression level
of Oct4A protein is found in pluripotent cells .
Oct4B is expressed at low levels in human somatic
stem cells, tumor cells, adult tissues as well as pluripo-
tent cells. The expression of Oct4B is generally localized
to cytoplasm , and currently there is no evidence to
suggest that the Oct4B isoform may be involved in the
generation of iPSC. Oct4B has been shown to play a role
in the stress response , and more detailed biological
studies are need to characterize this transcription factor
further. However, Oct4B1 has been associated with
stemness , and further investigations on Oct4B1 are
also needed to establish its role in stem cell biology. In
spite of this variability and differences in the biological
functions of Oct4 isoforms, most studies in the literature
do not discriminate between Oct4A, Oct4B or OctB1 at
the protein or RNA levels . The fact that the three
isoforms are identical at the C-terminal end of the splice
variants increases the risk of obtaining false positive sig-
nals at the protein and mRNA levels. Immunohisto-
chemistryand immunofluorescence techniques can
discriminate between Oct4A and Oct4B by the differ-
ences in the nuclear and cytoplasmic localization. How-
ever, comparison of the isoforms by Western blot and
fluorescence activated cell sorting cannot discriminate
between the isoforms by product size or differences in
the localization of the fluorescence . Therefore, it is
vital to chose isoform- specific antibodies to interpret
results relating to stemness. In a similar fashion, much
of the data available on Oct4 expression at the RNA
level should be interpreted with caution due to possible
false positive artefacts which may result from false
Intron 1-2 Exon 2Exon 3
Exon 4Exon 5
Exon 2Exon 3Exon 4Exon 5
Figure 1 A schematic diagram representing the human Oct4 isoforms. Both Oct4A and Oct4B share identical exons 2–5. The differences
between the two isoforms lie in exon 1. The self-renewal and pluripotent properties of Oct4 encoded in exon 1 (Adapted from  and ).
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 6 of 12
amplification of the transcripts resulting from improper
primer design. To add to the complexity of Oct4 iso-
forms, six known Oct4 pseudogenes have recently been
described . Due to the high homology of pseudo-
genes to their parental genes, the possibility of amplify-
ing pseudogene-derived PCR product is very high .
Hence, to elucidate the expression pattern and the bio-
logical functions of Oct4A gene in the context of cancer
stemness, it is important to discriminate between the
isoforms and pseudogenes of Oct4A [96,97].
Preliminary data on the expression of Oct4A in EOC
Human ethics statement
Ascites was collected from patients diagnosed with
advanced-stage serous ovarian carcinoma, after obtain-
ing written informed consent under protocols approved
by the Human Research and Ethics Committee (HREC #
09/09) of The Royal Women’s Hospital, Melbourne,
We have previously demonstrated that cisplatin treat-
ment of ovarian cancer cells (primary, ascites tumor cells
and cell lines) treated with cisplatin results in a popula-
tion of residual cells with enhanced stemness including
increased expression of Oct4 compared to untreated
cells . We now provide evidence to suggest that the
mRNA expression of Oct4A was enhanced in the cells
isolated from the ascites of recurrent EOC patients com-
pared to cells isolated from the ascites of chemonaive
patients (patients with primary carcinoma who have not
undergone any treatment). The recurrent patients previ-
ously received combinations of chemotherapy consisting
of paclitaxel, carboplatin and drugs such as doxorubicin,
gemcitabine, docetaxel, cyclophosphamide and topote-
can after each recurrent episode. These patients were
diagnosed with recurrent disease 6–20 months after first
line of chemotherapy. Oct4A expression was signifi-
cantly enhanced in the ascites cells of recurrent patients
compared to chemonaive patients (Figure 2).
Interestingly, the enhanced expression of Oct4A in the
ascites cells of recurrent patients can be related to the
biological actions of Oct4 occurring in developing
embryos . A developing embryo relies on a funda-
mental switch from an undifferentiated to differentiated
state of the inner cellular mass (ICM) of a blastocyst
. While Oct4 expression is uniformly expressed
across all cells of the ICM, a loss of Oct4 expression in
these cells results in spontaneous differentiation to form
cells of the outer protective trophectoderm structure
[13,28]. This loss is known to be imperative for the for-
mation of definitive structures of a developing embryo
such as the outer protective trophectoderm surrounding
the ICM and reinforces the role of Oct4 as a pluripotent
regulator. Interestingly, this scenario of trophectoderm
formation of the developing embryo can be applied to
events that appear during the course of EOC recurrence
involving CSCs. In ovarian cancer spheroids, the ICM
would represent the core of a metastasised tumor spher-
oid containing chemoresistant CSC-like cells that evade
chemotherapy. Following chemotherapy treatment, these
Oct4A expressing residual CSC-like cells would be cap-
able of undergoing self-renewal and differentiation lead-
ing to reformation of ascites tumor masses (Figure 3). If
confirmed, this model would potentially provide the link
between CSCs and chemoresistance in ovarian cancer.
Oct4A as a therapeutic target for EOC
Oct4A is expressed at relatively low levels in normal
somatic tissues compared to their respective tumorgenic
cells, suggesting that targeting Oct4A may be a good
strategy to disable CSCs in EOC. Eliminating the self-
renewing and pluripotent ability of CSCs could prevent
EOC tumor progression and eradicate chemoresistance
and subsequent recurrence. While obvious targeting
Oct-4A mRNA expression
relative to 18S
Figure 2 mRNA expression of OCT4A in isolated cells obtained
from chemonaive and chemoresistant ovarian cancer patients.
Ascites cells were isolated as described previously . RNA
extractions, cDNA synthesis and quantitative determination of mRNA
levels of Oct4A were performed as previously described . Sense
and antisense primers were designed against published human
sequences for Oct4A (Entrez Gene ID 5460, approved symbol
POU5F1): forward- CTCCTGGAGGGCCAGGAATC; reverse-
CCACATCGGCCTGTGTATAT; 18S (Entrez Gene ID 100008588,
approved symbol RN18S1) forward-GTAACCCGTTGAACCCCATT;
reverse-CCATCCAATCGGTAGTAGCG. Gel extraction of PCR products
was performed using the QiaEX II Agarose gel extraction Kit (Qiagen
Australia), as per the manufacturers’ protocol and quantified using
the ND-1000 Nanodrop spectrophotometer (NanoDrop
Technologies Inc Wilmington, DE, USA). Sequences and products
were verified as described previously . Results are expressed as
the difference between the log2 transformed ΔCt values of the
gene of interest to that of housekeeping gene (18S) ±SEM of five
independent samples performed in triplicate. *P<0.05, significantly
different in recurrent versus chemonaive ascites samples.
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 7 of 12
methods include inhibiting the upstream targets of Oct4
such as WNT, AKT and TGFβ , a relatively newer
proposed method would be to target specific miroRNAs
(miRNA) responsible for regulating Oct4A expression in
ovarian cancer progression and chemoresistance.
MicroRNAs associated with Oct4
MicroRNAs (miRNAs) approximately 21–23 nucleotides
long are a group of non-coding RNAs that can regulate
gene expression by degrading their target messenger
RNAs (mRNAs) by binding to the complementary
sequences found in the 3’-untranslated region (UTRs) of
target mRNAs . This result in the modulation of a
cascade of cellular functions, including those related to
ESC self-renewal/differentiation . Recently, deregu-
lation of some miRNAs has been implicated in a number
of human cancers where they can act as either tumor
suppressors or as tumor oncogenes . Interestingly,
an increasing amount of evidence also suggests that
miRNAs play a role in self-renewal and differentiation
with only a few studies describing a role of miRNAs in
reprogramming of somatic cells , and in the regula-
tion of cancer stem cells [101,104].
The let7 family is one of the most extensively studied
and well understood of all miRNAs involved in carcino-
genesis, and has emerged as an important regulatory
factor in a range of cancers including ovarian cancer
. Upregulation of let-7 is a prominent feature of
ESC differentiation, and ESCs are characterised by a
striking down-regulated let-7 expression, which is dom-
inantly expressed in most differentiated cells in the vast
majority of tissues . Lin28 on the other hand is
highly expressed in ESC and cancer cells and has been
demonstrated to be down regulated during differenti-
ation . A high Lin28/low let-7 signature is common
in ESC, iPSC and CSC . A recent study has shown
that overexpression of miR-125b in hESC resulted in the
upregulation of the early cardiac transcription factors,
GATA4 and Nkx2-5, and accelerated the progression of
hESC-derived myocardial precursors to an embryonic
cardiomyocyte phenotype . By using an in silico ap-
proach, let-7, Lin28 and Oct4 were identified as targets
of miR-125b, suggesting that the manipulation of miR-
125b -mediated pathways may be useful for reprogram-
ming ESC to different lineages. In this context, let-7,
miR-125. miR-9 and miR-30 have been shown to repress
Lin28 expression in ESC and cancer cells .
Since 2006, a few studies have shown that the miRNA
profile is different in normal ovaries compared to pri-
mary and recurrent ovarian tumors . Let-7a and
miR-200 families have been shown to be deregulated in
ovarian pathogenesis . Decreased expression of let-7
1. Metastasis of ovarian tumor cells
epithelial and fibroblastic-like ovarian tumor cells
detach from primary ovarian tumor and shed
the into peritoneal cavity
3. Evasion of chemotherapy by CSC-like cells
enriched in Oct4A
4. Spheroid Formation
Oct4A expressing CSC-like cells can
undergo self renewal or
differentiation to form bulk of the
2. Chemotherapy Treatment
with platinum and taxol-based drugs
5. Disease recurrence
Spheroids capable of implanting
at secondary site facilitate
recurrence and treatment failure
Oct4 A expressing
CSCs –“core of the
Bulk of tumor mass
cells lacking Oct4A
and other CSC-like
Figure 3 A model of Oct4A-mediated ovarian cancer evolution and progression in the ascites microenvironment. During the course of
ovarian cancer progression a shedding of tumor cells into the peritoneum occurs. Here tumor cells survive as cellular aggregates/spheroids
where CSC-enriched core cells of the spheroids serve as a niche for regenerating cells. During chemotherapy treatment the bulk of the
differentiated tumor cells on the periphery of the spheroids are eradicated leaving behind CSC-enriched core tumor cells. These cells facilitate the
self-renewal of chemotherapy surviving residual cells resulting in tumor recurrence.
Samardzija et al. Journal of Ovarian Research 2012, 5:37
Page 8 of 12
has been associated with the mesenchymal aggressive
phenotype (C5) of high-grade serous ovarian carcinoma
. Down regulation of let-7 has also been associated
with cisplatin and taxol resistance [111,112], which suggests
that restoring the expression of let-7 may be a useful thera-
peutic option overcoming drug resistance. The combined
expression of Lin28 and Oct4 has been demonstrated in
high-grade ovarian carcinomas . Viral delivery of let-7
has also been shown to suppress the tumor growth in a
mouse model of lung adenocarcinoma . These studies
suggest that increasing the expression of let-7 may be an-
other novel therapeutic option to minimise/eradicate che-
moresistant recurrent ovarian tumors.
Oct4 in transdifferentiation
By introducing specific transcription factors, it is pos-
sible to induce cells into an alternative fate through
transdifferentiation. Transdifferentiation of mouse em-
bryonic fibroblasts into functional cardiomyocytes by
overexpressing Oct4, Sox2, Klf4 and c-Myc under
defined cardiac cell culture conditions has recently
gained attention . Hence, by modifying culture
conditions somatic cells can be induced to undergo
transdiffferentiation into cells of other lineages by intro-
ducing an iPSC cocktail which includes Oct4 . In
this context, the use of specific unsaturated fatty acids,
such palmitic, oleic and, linoleic acid that can trigger
adipocyte differentiation in human cancer cell lines, in-
cluding ovarian cancer is worth considering  This
study demonstrated massive production of lipid droplets
and up regulation of the adipogenic nuclear regulator
PPARγ, which belongs to the Peroxisome Proliferator-
Activated Receptor (PPARs) superfamily. As PPAR γ is
over expressed in ovarian carcinomas , this adipo-
genic transdifferentiation may be a feasible option in
combination with chemotherapy or post-chemotherapy
in a certain sub-set of ovarian carcinomas. In addition,
PPAR γ ligands, drug such as pioglitazone, troglitazone
and ciglitazone have been shown to modulate PPAR γ
activity by effecting the proliferation of ovarian cancer
cells . These differentiation strategies represent
promising non-cytotoxic method of decreasing tumor
burden, but how such an approach will impact on the
Oct4A-enriched CSC pool and activity yet remains to
be determined. We suggest that these transdifferentia-
tion studies can be extended to ovarian cancer, and that
Oct4 is likely to be a key player.
The role of Oct4 in EOC tumorigenesis is still not well
defined. However, while Oct4 appears to be essential
during embryogenesis and reprogramming of somatic
cells, enhanced or overexpression of Oct4A may be a
prime factor for EOC initiation, progression and recur-
rence. Its expression is enhanced in high-grade serous
ovarian carcinomas and consistently associated with
CSC-like populations which are believed to be respon-
sible for recurrent and resistant disease. Therefore, un-
less methods to directly target these specific Oct4
expressing populations can be found, it is believed that
this resistant and recurrent cycle of tumor growth after
debulking surgery and initial chemotherapy will con-
tinue, contributing to the tumor burden which leads to
The authors declare that they have no competing interests.
CS and NA conceived the idea, designed and wrote the manuscript. MQ and
JFK edited the manuscript. All authors read and approved the manuscript.
The authors wish to thank Women’s Cancer Foundation and National Health
and Medical Research Council of Australia (JKF, RegKey#441101) for
supporting this work. CS is a recipient of an Australian Postgraduate Award.
1Women’s Cancer Research Centre, Royal Women’s Hospital, 20 Flemington
Road, Parkville, VIC 3052, Australia.2Department of Obstetrics and
Gynaecology, University of Melbourne, Melbourne, VIC 3052, Australia.
3Prince Henry’s Institute of Medical Research, Melbourne, VIC 3168, Australia.
Received: 15 August 2012 Accepted: 30 October 2012
Published: 21 November 2012
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Cite this article as: Samardzija et al.: Attributes of Oct4 in stem cell
biology: perspectives on cancer stem cells of the ovary. Journal of
Ovarian Research 2012 5:37.
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