Introduction
Epithelial ovarian cancer (EOC) remains the most prevalent of all the gynaecological malignancies with approximately 250,000 women diagnosed worldwide with the disease each year [
1]. Despite extensive treatment regimens including cytoreductive surgery and chemotherapy, the disease carries a poor prognosis with a 5 year mortality rate greater than 70 % [
2]. This is predominately due to exceptionally high rates of disease recurrences driven by widespread peritoneal metastasis and ascites [
3]. Although the metastasis of EOC is a highly complex and multi-stage process [
4], it is further emphasised by the lack of anatomical barriers within the pelvic cavity. Consequently, malignant tumour cells from the primary ovarian site are freely able to exfoliate directly into the peritoneal cavity. Here they directly attach to the mesothelial lining of the peritoneum before invading adjacent pelvic organs including the bowels, bladder and liver. Alternatively, exfoliated tumour cells can also maintain long term tumourigenicity within the peritoneal cavity by forming non-adherent multicellular tumour aggregates (spheroids) in the ascites microenvironment [
5]. This combined with the emergence of drug resistant tumour cells following extensive chemotherapy regimens presents a major challenge in the management of the disease [
6,
7]. In order to reduce recurrent EOC tumour burden and improve overall patient survival rates, the molecular mechanisms involved in ascites-mediated EOC progression must be investigated.
Recent evidence suggests solid tumours including those of ovarian origin contain a sub-population of tumour cells exhibiting unlimited self-renewal and pluripotent abilities [
8]. These cells, termed cancer stem cells (CSCs) have gained considerable momentum in cancer biology over the past decade and are hypothesized to mediate tumour cell survival [
9], sustain cytotoxic pressure [
10,
11] and initiate metastatic recurrence [
12]. Indeed, we and others have shown recurrent chemoresistant ovarian tumour cells to be enriched in CSC-like cells and stem cell pathway mediators, indicating that CSCs may contribute to the progression of disease [
13‐
17]. Overall, this suggests that targeting CSCs may provide an effective and accurate method of treating recurrent EOC disease [
18‐
20]. It is therefore vital that tumour specific CSCs as well as the pathways regulating their survival are identified and explored.
Oct4 (Oct3/4 or POU5F1), an embryonic stem cell factor and member of the POU family of transcription factors, has been extensively studied in solid tumours including those of the lung [
21], brain [
22], and breast [
23] where it has closely been related to tumour progression [
24], self-renewal [
25] and drug resistance [
26]. In developmental biology, it is known to play a pivotal role for the maintenance of totipotency in primary blastomeres and pluripotency of the inner cell mass of developing mammalian embryos [
27,
28]. While up regulation of Oct4 sustains an undifferentiated pluripotent stem cell state, a loss of Oct4 induces stem cells to undergo differentiation, producing a heterogeneous population of highly specialized daughter cells [
29]. Unfortunately, several studies previously investigating Oct4 in relation to tumour progression and chemoresistance have failed to recognize the several Oct4 isoforms generated by alternative splicing [
30] termed Oct4A, Oct4B and Oct4B1. Of these isoforms, only the nuclear-specific Oct4A isoform is known to be capable of regulating the pluripotent nature of stem cells [
31,
32]. This along with the several known pseudogenes [
33], makes investigating and interpreting the current literature on Oct4 complex. Hence, to elucidate the expression and the biological functions of Oct4 in the context of cancer stem cells, it is important to discriminate between the several isoforms and pseudogenes of Oct4.
In this study, for the first time using Oct4A specific antibody and primers we determined the expression of Oct4A in a range of serous ovarian tumours of different histological grades, and in tumour cells isolated from the ascites of recurrent and chemonaive EOC patients. Our results reveal that the expression of Oct4A significantly correlated to serous ovarian tumour dedifferentiation and was significantly elevated in the isolated tumour cells of chemotherapy treated recurrent patients compared to untreated chemonaive patients. Using a highly aggressive and metastatic ovarian cancer cell line (HEY) which sustains high endogenous expression of Oct4A, we show that suppression of Oct4A resulted in the loss of CSC-associated expression of Lin28, Sox-2, EpCAM and CD44. This was associated with a decrease in cellular proliferation, migration, spheroid forming abilities and resulted in enhanced sensitivity to cisplatin in vitro. These results correlated with those obtained from in vivo mouse xenograft studies. Mice transplanted with Oct4A knockdown cells demonstrated significantly reduced tumour burden and abrogation of tumour invasive ability, which overall resulted in significantly increased survival rates compared to mice injected with vector control cells. These data emphasize the need to explore further the effect of Oct4A expression in pre-clinical ovarian cancer models.
Discussion
Despite extensive treatment regimes, ascites-mediated recurrence continues to remain a critical process in the progression of EOC [
36]. Understanding the molecular mechanisms which contribute to the survival of exfoliated primary ovarian tumour cells in the peritoneal cavity therefore remains vital in the management of the disease. During the past few years, the embryonic transcription factor Oct4 has received considerable attention in cancer stem cell biology and its expression has been reported in a wide range of tumours [
45]. However, unfamiliarity with the several known Oct4 isoforms, as well as the existence of a high number of pseudogenes [
30] has led to the misinterpretation of several results surrounding Oct4A in the context of stem cell biology [
30]. A more rigorous analysis of the specific Oct4 isoforms is therefore required to elucidate their functional roles in cancer.
Although the expression of Oct4 has previously been reported in ovarian tumours [
46] and patient ascites samples [
47], this is the first study to our knowledge to demonstrate the expression of the specific Oct4A isoform in serous ovarian tumours and chemotherapy-treated recurrent serous patient ascites samples. We demonstrate that Oct4A is significantly elevated in serous borderline tumours compared to normal ovarian tissues and that this elevated expression is retained throughout the histological spectrum of serous ovarian tumours. Due to a small sample size used in the current study, no elucidation between the elevated expression of Oct4A and patient’s prognosis could be determined. However, this combined with the significant elevation of Oct4A expression in the tumour cells isolated from the ascites of recurrent patients compared to those from chemonaive patients suggests that elevated expression of Oct4A may not only have a role in ovarian cancer progression but may also facilitate recurrence. Interestingly and in this context, the Oct4A-abundant HEY cell line has previously been shown to form highly robust and aggressive tumours in mouse models [
15]. Alternatively, the Oct4A low expressing OVCA433 is not tumourigenic in
in vivo mouse models [
48]. Such observations may potentially correlate to the proposed CSC-like functional role of Oct4A in regulating ovarian tumour growth and survival [
35,
49].
The current literature on the functional role of Oct4 in epithelial ovarian cancer is relatively sparse, with the transcription factor primarily being used as a marker to detect CSC-like cells in primary ovarian tumours and patient ascites samples [
50]. In this study using shRNA-mediated knockdown of Oct4A, we provide evidence to support the concept that the Oct4A isoform may be a key regulatory factor associated with cancer stem cell-like properties in serous ovarian tumours. It has previously been shown that cancer cells cultured as spheroids exhibit enhanced tumourigenic and metastatic ability combined with increased expression of CSC-like genes [
36]. Here we demonstrate that knockdown of Oct4A in HEY ovarian cancer cell line not only significantly diminishes the anchorage-independent growth of ovarian cancer cells as spheroids, but results in the decreased expression of oncogenic and CSC-like Lin28 and Sox-2 expressions compared to vector control cells. Since both Lin28 and Sox2 function to maintain self-renewal in pluripotent stem cell populations [
51,
52], this result further emphasizes the role of Oct4A as a master regulator of self-renewal in stem cell-like populations. It is also the first study to suggest the regulation of Lin28 and Sox2 by the specific Oct4A splice variant in EOC. The expression of both EpCAM and CD44 were also significantly reduced in knockdown cells compared to vector control cells, suggesting that the loss of anchorage independent state as spheroids in Oct4A knockdown cells may be due to the loss of cell-cell adhesive molecules such as EpCAM and CD44. The interaction between CD44 and Oct4 has previously been described in squamous cell tumours [
53], and EpCAM and CD44 have previously been implicated in malignant ovarian tumours [
54,
55]. However, the association between EpCAM and CD44 with regards to the specific Oct4A isoform remains to be identified. We also demonstrate that loss of Oct4A expression resulted in a significant reduction in proliferation and migration of HEY cells, emphasizing the vital role of Oct4A in tumour cell survival and migration. Loss of Oct4A expression also correlated with significantly reduced expression of MMP2 suggesting that the invasive ability of HEY cells has been reduced following Oct4A knockdown. These observations were further validated in mouse xenograft experiments which showed significantly reduced tumour burden, tumour organ invasion and overall significantly enhanced survival of mice injected with Oct4A knockdown cells compared to vector control cells. In addition, tumour xenografts derived from Oct4A knockdown cells displayed relatively lower abundance of markers associated with ovarian cancer including oncogenic markers Lin28 and Sox2, along with proliferation maker Ki67 and anti-apoptotic Bcl-2 expression. This indicates that reduced tumourigenic ability, combined with a reduction in proliferative ability mediated by a loss of Oct4A may have slowed or abrogated tumour formation and growth. Interestingly, tumour xenografts generated from Oct4A knockdown HEY cells exhibited significantly lower CA125 expression. Elevated levels of CA125 are a hallmark characteristic in ovarian cancer diagnosis and is frequently observed in relapsed ovarian cancer patients [
56] and suggests that the tumours generated by Oct4A knockdown cells are overall less tumourigenic and aggressive compared to those with abundant Oct4A expression.
Overexpression of Oct4 has previously been implicated with chemoresistance in tumours [
57]. Here we demonstrate significant elevation of Oct4A expression in established EOC cells which survived cisplatin treatment. This correlated with significantly elevated expression of Oct4A in the isolated tumour cells derived from the ascites of chemoresistant recurrent EOC patients compared to those of chemonaive patients. This highlights the potential role that Oct4A may have in the resistant phenotype currently exhibited by tumour cells of EOC patients. Interestingly, this observation was further reinforced after loss of Oct4A expression in HEY cells which resulted in the increase in the sensitivity to cisplatin treatment, suggesting a direct role of Oct4A in cisplatin-mediated drug resistance which is critical for ovarian tumour recurrence.
Materials and methods
Patient samples
Tissue collection
Primary serous epithelial ovarian tumours and normal ovarian tissues were obtained from patients requiring surgical resection after obtaining written informed consent under protocols approved by the Human Research and Ethics Committee (HREC approval # 09/09) of The Royal Women’s Hospital, Melbourne, Australia. The histopathological diagnosis, tumour grades and stages were determined by anatomical pathologists at the Royal Women’s Hospital as part of clinical diagnosis (Tables
1&2). Patients who were treated with chemotherapy prior to surgery were excluded from specimen collection. Tissues were paraffin embedded or snap frozen at the time of collection and stored at −80 °C until processed.
Ascites collection
Ascites was collected from patients diagnosed with Stages III-IV ovarian serous cystadenocarcinoma or adenocarcinoma Not Otherwise Specified (NOS) after obtaining written informed consent under the protocols approved by the HREC approval #09/09. The histopathological diagnosis, tumour grades and stages were determined by anatomical pathologists at the Royal Women’s Hospital as part of clinical diagnosis (Table
3). Ascites collected from patients at the time of diagnosis and prior to the commencement of treatment were termed chemonaïve (CN). Ascites were also collected from patients at the time of recurrence (CR). Patients in this group had previously received different chemotherapy combinations as described in Table
3. All patients have been classified according to their survival status as either 'alive' or 'deceased' at the time of manuscript preparation. Those labeled as 'At Last Contact (ALC)' were no longer receiving treatment at The Royal Women's Hospital at the time of manuscript preparation. Consequently, their current status remains unknown.
Preparation of tumour cells from ascites of ovarian cancer patients
Tumour cells from ascites were isolated as previously described [
14].
Four established human epithelial ovarian cancer cell lines SKOV3, OVCAR5, OVCA433, and HEY were used in this study. The growth conditions of these cell lines have been described previously [
58]. The human ovarian surface epithelial cell line (IOSE398) transfected with the SV-40 antigen was obtained from Dr Nelly Auersperg, University of British Columbia, Canada [
59]. Cells were routinely checked for mycoplasma infection.
Antibodies
Monoclonal antibody against human Oct4A and the secondary anti-mouse IgG HRP antibody was obtained from R&D Systems (Minneapolis, Minnesota, USA). Monoclonal and polyclonal antibodies against human Sox2 and Lin28 were obtained from Cell Signalling Technology (Danvers, Massachusetts, USA). The polyclonal antibody against GAPDH was obtained from IMGENIX (San Diego, CA, USA). Secondary anti-rabbit IgG antibody was obtained from Millipore (Billerica, MA, USA). DAPI Nucleic acid stain and Alex Fluor® 488 goat anti-mouse IgG were obtained from Life Technologies (Carlsbad, CA, USA).
Immunohistochemistry
Immunohistostaining of primary human tissue specimens for Oct4A was performed using components from the CINtek® p16 Histology Kit (Roche) as described by the manufacturer. Sections were assessed microscopically for positive DAB staining and quantified using the open source image processing package Fiji (Fiji Is Just ImageJ) with a plug-in developed to recognize total DAB staining. Refer to the Supplementary Method for a detailed methodology.
RNA extraction and real-time PCR
Quantitative real-time PCR was performed as described previously [
14]. Relative quantification of gene expression was normalized to 18S and calibrated to the appropriate control sample using the SYBR Green-based comparative CT method (2
-ΔΔCt). The primer sets for Oct4A Lin28, Sox2, EpCAM and CD44 are described in Table
4. The probe for 18S has been described previously [
46].
Table 4
Primer sequences of oligos used in quantitative Real-Time PCR
Oct4A | F | CTC CTG GAG GGC CAG GAAT C |
R | CCA CAT CGG CCTG TGT ATA T |
Lin28 | F | CAA AAG GAA AGA GCA TGC AGA AG |
R | GCA TGA TGA TCT AGA CCT CCA CA |
Sox2 | F | ATG CAC CGC TAC GAC GTG A |
R | CTT TTG CAC CCC TCC CAT TT |
CD44 | F | CCA ATG CCT TTG ATG GAC CA |
R | TGT GAG TGT CCA TCT GAT TC |
EpCAM | F | CGT CAA TGC CAG TGT ACT TCA GTT G |
R | TCC AGT AGG TTC TCA CTC GCT CAG |
shRNA transfection of HEY ovarian cancer cell line
The HEY-Oct4A shRNA clones (Oct4A KD1 and Oct4A KD2) were generated by transfecting cells with the pLKO.1 Mission shRNA custom DNA construct (Sigma-Aldrich) using FuGENE HD transfection reagent (Roche) following the manufacturer’s protocol and using Puromyocin selection. The target sequence for the Oct4A construct was based on published genomic sequences of Oct4A [
31] on human chromosome 6 (accession no. NM_002701) and retrieved from the public databases at the National Center for Biotechnology Information (
www.ncbi.nlm.nih.gov). The target sequence is as follows: CCTTCGCAAGCCCTCATTTCA. Vector control HEY cells were generated by stably transfecting a non-target shRNA negative control.
Western blotting
Cell lysates were extracted using the NU-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Waltham, MA, USA) as per manufacturer's instructions. SDS-PAGE and Western blot was performed on the cell lysates as described previously [
13]. All membranes were probed for 48 h with primary Oct4A, Lin28 or Sox2 antibodies set at 1:1000 dilutions.
The spheroid forming ability of cells was determined by seeding cells in ultra-low attachment 6 well culture plates as described previously [
13]. Cellular aggregates with a diameter greater than 200 μm were classified as spheroids.
Spheroid viability assay
The viability of 18 day spheroids was assessed by directly seeding spheroids onto 6 well plastic culture plates and culturing for 24 h at 37 °C in the presence of 5 % CO2. Adherent spheroids were formalin fixed, stained with 5 % crystal violet and counted manually.
Cell migration assays
The migratory ability of cells was assessed by wound healing assay as described previously [
13]. Quantitative analysis of cell migration was determined by measuring the width of the wound at 10 randomly selected points at t = 0 h and t = 24 h post wound incision using an ocular micrometer. Migration was expressed as a percentage of 24 h wound closure compared to 0 h.
Proliferation assays
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich) assays were used to quantify cell proliferation rates as described previously [
45]. Briefly, 4 x 10
3 cells were seeded in 96-well plates and cultured at 37 °C in the presence of 5 % CO
2 until required for analysis at 24 h, 48 h and 72 h time points. On the day of analysis, the growth media was discarded and MTT (dissolved in 1X PBS solution; final concentration 0.5 mg/mL in 1 X PBS) was added. Cellular metabolism of MTT was permitted to occur at 37
0 in the presence of 5 % CO
2. Following incubation, the MTT reagent was removed, 100 μL of dimethyl sulfoxide (DMSO) added and cells left to incubate for 10 mins in to dissolve formazan crystals. Samples were read at OD
595nm using the SpectraMax190 Absorbance Microplate Reader and SoftMax® Pro Computer Software (Molecular Devices). Proliferation rates are expressed as a percentage of cell growth compared to 24 h.
Chemosensitivity assays
The 50 % growth inhibition (GI
50) value for cells in response to 72 h cisplatin treatment was performed using MTT assays as described previously [
45]. Briefly, 4 X 10
4 cells were seeded in 96-well plates in appropriate growth medium and incubated for 24 h in humidified atmosphere at 37 °C in the presence of 5 % CO
2. Following 24 h incubation, culture media was removed and substituted with complete growth media containing increasing concentrations of cisplatin. Cells were subjected to 72 h cisplatin treatment and the chemosensitivty of each cell line determined by standard MTT assay as described above. Chemosensitivity is expressed as a percentage of cell survival at 72 h compared to untreated control cells.
Animal studies
Animal ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of the Laboratory Animals of the National Health and Medical Research Council of Australia. The experimental protocol was approved by the Department of Surgery, Royal Melbourne Hospital, Australia Animal Ethics Committee (Project-006/11).
Animal experiments
Animal experiments were performed as described previously [
15]. Metastatic development was documented by a Royal Women's Hospital pathologist according to histological examination (H&E staining) of the organs as described previously [
20].
Immunohistochemistry of mouse tumours
Immunohistochemistry analysis of mouse tumours was performed as described previously [
20]. Primary antibodies against human Oct4, Lin28, Sox2, Ki67, Bcl-2 and CA-125 were diluted according to the manufacturer's instruction. Primary antibody staining was detected using the ultraView Universal DAB detection kit (Roche). Negative controls were prepared for each section throughout the staining process by incubating tumour xenografts in the absence of primary antibodies. Images of immunohistochemical staining were taken using an Aperio ImageScope (Leica Microsystems, Mt Waverly, Australia) and associated digital pathology viewing software. DAB staining was measured by taking a minimum of 10 images per tumour section and running the images through the open source image processing package Fiji (Fiji Is Just ImageJ) with a plug-in developed to recognise DAB staining.
Statistical analysis
All results are presented as the mean ± standard error of the mean (SEM) of three independent experiments. Statistical significance was measured compared to the vector control using one way-ANOVA and Dunnett’s Multiple Comparison test unless otherwise indicated. A probability level of <0.05 was adopted throughout to determine statistical significance.
Acknowledgements
The authors wish to thank the Women’s Cancer Foundation, National Health and Medical Research Council of Australia (JKF, RegKey#441101) for supporting this work. CS is a recipient of Australian Postgraduate Award. RBL is a recipient of the Melbourne Brain Centre Post-Doctoral Research Fellowship from the University of Melbourne. The authors also wish to acknowledge the help of Ms Bronwyn Christensen, Anatomical Pathology, The Royal Children's Hospital, Parkville, Australia for assisting with the mouse xenograft immunohistochemistry analysis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
CS designed the study, performed the experiments and wrote the manuscript. RBL assisted with the animal studies. MV assessed the mouse xenograft histological specimens. MQ and JKF edited the manuscript. NA conceived the idea, designed the study and contributed to the writing of the manuscript. All authors read and approved the final manuscript.