- Research article
- Open Access
- Open Peer Review
Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer
© The Author(s). 2016
- Received: 8 November 2015
- Accepted: 24 June 2016
- Published: 8 July 2016
Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis.
The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice.
We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts.
The expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients.
- Ovarian carcinoma
- Cancer stem cells
Ovarian cancer is a major gynaecological malignancy worldwide with 125,000 deaths reported each year . The development of ascites and peritoneal metastases is a major clinical issue in the prognosis and management of ovarian cancer. A significant proportion of ovarian cancer cells within the peritoneal ascites exist as multicellular aggregates or spheroids which have the capacity to invade nearby organs . The pathology of peritoneal-based metastasis includes the attachment of shed primary ovarian tumor cells onto the mesothelial-lined spaces of the peritoneum in the form of spheroids resulting in multiple tumor masses necessary for secondary growth. Current treatment strategies for advanced-stage ovarian cancer patients results in initial remission in up to 80 % of patients . However, following a short remission period (usually 16–22 months), recurrence occurs in almost all patients ultimately resulting in patient mortality. This high rate of recurrence is largely due to the ability of tumor cells to evade the cytotoxic effects of chemotherapy associated with intrinsic or acquired chemoresistance, a property commonly associated with CSCs [4, 5].
The concept of CSCs supports the existence of a sub-population of tumor cells which drive tumor growth and progression, while also sustaining the cytotoxic pressure imposed by therapy to promote the re-growth of therapy-resistant tumors [6, 7]. In this scenario, it can be postulated that the development of an effective therapy for recurrent ovarian tumors will depend on the identification of tumor specific CSCs, as well as the pathways/regulators controlling their survival and sustenance.
Oct4 (Oct3/4 or POU5F1) is a member of the POU-domain family of transcription factors and has been shown to play an important role in the maintenance of self-renewal and pluripotency in embryonic stem cells (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 . Up regulation of Oct4 expression has been shown to sustain an undifferentiated pluripotent stem cell state, while a loss of Oct4 expression results in the induction of differentiation in stem cells, producing a heterogeneous population of highly specialized daughter cells . Additionally, Oct4 has consistently been shown to be an integral factor necessary for the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Although a cocktail of transcription factors are typically involved in this process (eg Oct4, Sox2, Klf4 and c-Myc), reprogramming efficiency is reduced if Oct4 is not present, thus indicating an absolute requirement for Oct4 in maintaining a stem cell-like state . Importantly however, Oct4 is highly expressed in many tumor types, suggesting that the reprogramming of somatic cells as well as tumor development and progression may share common cellular mechanisms .
The Oct4 gene encodes for three isoforms, generated by alternative splicing of genes, known as Oct4A, Oct4B and Oct4B1 [11, 12]. At the nucleotide level, both Oct4A and Oct4B share exons 2–5. However, exon 1 is missing in Oct4B and is replaced by exon 2a [11, 12]. These differences appear to have significant biological implications on isoform function with Oct4A specifically expressed in the nucleus of ESCs, human somatic stem cells, tumor stem cells and in some adult stem cells [11, 12]. Oct4B on the other hand, is localised to the cytoplasm and expressed at low levels in human somatic stem cells, tumor cells, adult tissues as well as pluripotent stem cells. For investigations in stem cell biology, it is therefore crucial that the Oct4A isoform is specifically targeted.
The interaction between CSCs and the neighbouring microenvironment forms a ‘niche’ which is critical for sustaining the stemness of cancer cells . Integrins are heterodimeric transmembrane receptors composed of a combination of different α and β subunits. They are essential in sensing the microenvironment and triggering cellular responses by bridging physical connections between the interior and exterior environments of cells . This allows the flow of bi-directional signals that control basic cellular functions such as adhesion, migration, proliferation, and survival as well as differentiation . In the context of CSCs, integrin receptors have been shown to promote a more malignant phenotype for tumor promotion and drug resistance [14, 15]. These receptors are highly expressed in stem cell niches and contribute to diverse CSC functions [14–16]. In this study using cancer cell lines, we demonstrate a direct link between the expression of α2, α5 and β1 integrin subunits with Oct4A expression in ovarian cancer and discuss the implications of these findings in relation to CSCs and progression of ovarian cancer in patients.
Primary high grade serous epithelial ovarian tumors and normal ovarian tissues were obtained from patients requiring surgical resection at The Royal Women’s Hospital, Melbourne, Australia. The histopathological diagnosis, tumor grades and stages were determined by anatomical pathologists at the Royal Women’s Hospital as part of clinical diagnosis. 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.
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 . 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 . The development of the vector control, Oct4A KD1 and Oct4A KD2 cell lines and their growth conditions have been described previously . Cells were routinely checked for mycoplasma infection.
Mouse monoclonal anti-human Oct4A and Sox2 were obtained from R&D Systems (Minneapolis, Minnesota, USA) and Cell Signalling Technology (Danvers, Massachusetts, USA) respectively. Rabbit polyclonal anti-human GAPDH was obtained from IMGENIX (CA, USA). Mouse anti-human integrin α2 (CD49b), anti-human α5 integrin (CD49e) and anti-human β1 (CD29) were obtained from Millipore (Billerica, Massachusetts, USA). Goat F(ab')2 anti-mouse IgG was purchased from Southern Biotech (Birmingham, AL, USA). Rabbit polyclonal anti-human cytokeratin 7 (CK-7), anti-human Glut-1, anti-human CD34 and anti-human CD31 were obtained from Ventana (Tucson, USA). The DAPI nucleic acid stain and Alexa Fluor® 488 goat anti-mouse IgG were obtained from Life Technologies (Carlsbad, CA, USA). Ventana antibodies used for the immunohistochemical staining of tumor xenografts were obtained from Roche (Basel, Switzerland) as described previously [19–21].
For primary tissue analysis, paraffin embedded tissue samples were sectioned at 5 μm and deparaffinised by xylene and graded ethanol wash. Slides were blocked for 10 min in CAS-Block™ Histochemical Reagent (Invitrogen Corporation). For non-adherent sphere populations, 100–200 μL of sphere containing media was added per chamber well containing 200 μL appropriate fresh growth media and cultured on 8 well μ-Slides (ibidi, Martinsried, Germany) for 24 h to allow for adhesion to plastic before being fixed with 4 % paraformaldehyde. Monolayer cell lines were seeded at 5 × 103 cells per well onto the 8-well Nunc™ Lab-Tek™ Chamber Slide™ System (Thermo Scientific) and cultured as monolayer in complete RPMI-1640 growth media before being fixed with 4 % paraformaldehyde. Samples were probed overnight at 4 °C with either Oct4A (1:200), integrin β1 (1:200) or integrin α5 (1:200) primary antibodies, detected with Alexa Fluor® 488 Goat-Anti-Mouse antibody (1:200) and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (1:10,000). Fluorescence imaging was visualized and captured using an Olympus CellR fluorescence microscope and associated software (Olympus Corporation, Tokyo, Japan). Semi-quantitative analysis to assess fluorescence intensity of the antibody of interest was performed using the inbuilt CellR software. Results are expressed as a fold change of the protein of interest compared to DAPI for each analysis.
RNA extraction and real-time PCR
Primer sequences of oligos used in quantitative Real-Time PCR
Forward (F) 5’-3’ Reverse (R) 5’-3’
CTC CTG GAG GGC CAG GAAT C
CCA CAT CGG CCTG TGT ATA T
ATC CCA GAG GCT CCA AAG AT
CTA AAT GGG CTG GTG CAG TT
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 .
Sphere forming assay
The sphere forming ability of cells and subsequent sphere adhesion ability was determined as described previously . Cellular aggregates with a diameter greater than 200 μm were classified as spheres.
Flow cytometric analysis
Flow cytometry was used to assess the expression of cell surface makers as described previously . Briefly, cells were grown as monolayer cultures, harvested and 106 cells incubated with primary antibody (1:100) for 30 mins at 4 °C. Cells were washed with 1X PBS, stained with secondary Goat F(ab’)2 anti-mouse IgG antibody conjugated with phycoerythin for 30 mins at 4 °C and resuspended in 200 μL 1XPBS prior to flow cytometry analysis. All data was analysed using Cell Quest software (Becton-Dickinson, Bedford, MA, USA) and expressed as background IgG staining subtracted from the IgG staining of the antibody of interest.
Cell adhesion assays were used to assess the ability of cells to adhere to extracellular matrix proteins. Briefly, 5 × 104 cells were seeded in complete growth media on culture plates pre-coated with 10 μg/mL collagen, Type 1 (Sigma-Aldrich) or 10 μg/mL fibronectin (Sigma-Aldrich) with sterile 1X PBS used as a diluent. Cells were incubated for 90 mins at 37 °C in a humidified atmosphere in the presence of 5 % CO2. The growth media was removed and cells were washed vigorously with 1X PBS using an orbital rocker on full speed twice for 5 mins to remove non-adhering cells. Cells were fixed with 4 % paraformaldehyde before being stained for 10 mins with 5 % Crystal Violet (Sigma-Aldrich) diluted in 0.2 % ethanol. Following crystal violet staining, cells were gently rinsed with 1X PBS and plates allowed to dry at room temperature before performing a dry reading at OD550nm with the SpectraMax190 Absorbance Microplate Reader and SoftMax® Pro Computer Software (Molecular Devices). Adhesion was calculated by subtracting the OD550nm reading of the negative control from the OD550nm reading of coated wells.
This was performed as described previously . Briefly, complete growth medium from cells grown as sub-confluent monolayer cultures was discarded and replaced by serum free medium in a humidified atmosphere at 37 °C in the presence of 5 % CO2. After 48 h, the serum free medium was collected and concentrated using 10 kDa Amicon Ultra-4 spin columns (Merck-Millipore, Billerica, MA, USA). Samples were resolved on 10 % (v/v) Tris–HCl acrylamide gels containing 0.1 % (w/v) gelatin, washed and stained with 0.2 % Coomassie blue. The gel was de-stained and areas void of blue stain indicative of areas of enzyme activity. Semi-quantitative densitometric analysis was performed on all gels to determine the extent of enzymatic digestion using Image Quant software (GE Healthcare) and expressed as the intensity of Pro-MMP9 or Pro-MMP2 bands of interest.
Immunohistochemistry of mouse tumors
All results are presented as the mean ± standard error of the mean (SEM) of three independent experiments unless otherwise indicated. Statistical significance was measured compared to the vector control using one way-ANOVA and Dunnett’s Multiple Comparison test unless otherwise indicated. For primary tissue analysis, Student’s t-test was used to compare normal and high grade tissue samples. A probability level of <0.05 was adopted throughout to determine statistical significance.
Expression of Oct4A in serous ovarian tumors
Association between endogenous Oct4A expression and sphere forming abilities of ovarian cancer cell lines
The positive identification of Oct4A in primary serous ovarian tumors suggests a possible biological role of Oct4A in the disease. Using quantitative real-time PCR analysis, we have previously shown mRNA expression of Oct4A in four ovarian cancer cell lines OVCAR5, SKOV3, OVCA 433 and HEY . All cell lines with the exception of OVCA433 expressed significantly elevated levels of Oct4A mRNA when compared to the immortalized normal ovarian surface epithelium cell line ISOE398 . The HEY cell line was found to express the greatest endogenous level of Oct4A mRNA with a 6-fold increase in Oct4A expression, compared to the normal ISOE398 cell line. OVCAR5 and SKOV3 cells both exhibited a 4-fold increase in Oct4A mRNA expression compared to the normal ovarian cell line ISOE398 .
The viability of 18 day spheres produced by the ovarian cancer cell lines was assessed by their capacity to adhere to plastic. Under light microscopy, the spheres produced by the ovarian cancer cell lines adhered to plastic within 24 h (Fig. 2b). Further microscopic evaluation demonstrated these adhered spheres underwent cellular dispersion away from the sphere core. This effect was predominately seen in spheres produced by Oct4A abundant HEY cells and to a lesser extent in spheres derived from other ovarian cancer cell lines. The expression of Oct4A however, was not related to the degree of adhesion of the spheres, with Oct4A low expressing OVCA433 producing several smaller adhered colonies compared to those produced by high Oct4A expressing HEY cell line (Fig. 2b). OVCAR5 and SKOV3 cell lines which had moderate endogenous level of Oct4A produced several colonies which were both smaller and fewer when compared to those produced by HEY cells.
shRNA mediated knockdown of Oct4A in HEY ovarian cancer cell line
Diminution of Oct4A suppresses integrin β1 and α5 subunits expression in HEY spheres
Given that the integrin β1subunit is known to couple with several α integrin sub-units, the expression of the integrin α5 subunit was also investigated in adhered Oct4A KD spheres using immunofluorescence analysis (Fig. 4d and e). Similar, to that of integrin β1, the integrin α5 subunit was significantly down regulated by 50 % in both Oct4A KD1 and Oct4A KD2 spheres compared to vector control spheres. The localization of integrin α5 remained confined to the cell membranes making up the spheres and migrating cells.
Attenuation of Oct4A suppresses integrin β1, α2 and α5 subunit expression in HEY monolayer cells and affects the adhesive ability of HEY cells to ECM proteins collagen and fibronectin
Diminution of Oct4A reduces secreted levels of pro-MMP2 but not MMP9 in monolayer cultures of HEY cells
Attenuation of Oct4A expression decreases in vivo tumorigenicity of HEY cells in the BALB/c nude mouse model
Knockdown of Oct4A reduces the expression of CK7, Glut-1, CD31 and CD34 in xenografts derived from Oct4A KD cells compared to vector control cells
It is well recognized that the interaction between tumor cells and the ECM in the rapidly evolving tumor microenvironment is essential for the generation of regulatory signals that ultimately determine the fate of tumor cells and influence the evolution of a malignant phenotype . The integrin family of cell surface receptors is an important component of the ECM which sense micro environmental changes and trigger a range of cellular responses by forming a physical link between the inside and outside of tumor cells. Moreover, they allow for the bidirectional regulation of signals necessary to promote tumor progression . However, it is not yet clear if the effects that integrins have in modulating tumor cell behavior are also related to their associated role with CSCs in the tumor microenvironment. In this study we demonstrate a direct association between CSC specific Oct4A transcription factor expression and the β1 family of integrins primarily the α2 and α5 subunits.
In a previous study, we demonstrated significantly enhanced expression of the stem cell specific Oct4A isoform which increased according to histological grades of serous ovarian tumors compared to normal ovarian epithelium by immunohistochemical methods . In this study we further validate these results in high-grade serous ovarian tumor samples using immunofluorescence and Western blot techniques. We demonstrate that the expression of Oct4A is enhanced in high-grade serous samples compared to normal ovaries and localized to the nucleus of tumor cells. These results support the hypothesis that Oct4A has a specific role in ovarian cancer progression.
We have previously demonstrated that suppression of Oct4A is capable of inhibiting sphere forming abilities in the HEY ovarian cancer cell line . To expand on this observation, the expression of Oct4A in a range of ovarian cancer cell lines was seen to be directly correlated to the anchorage independent sphere forming abilities of each cell line. Sphere formation has been identified as a property of CSCs and the phenomenon has been reported in leukemia and in solid tumors of breast, colon and brain . However, the molecular mechanisms of how sphere-forming cells retain their stem-like characteristics remain unknown. Recent studies, have demonstrated that mouse ESCs are capable of maintaining long-term stemness within a 3D scaffold by manipulating integrin signaling [36, 37]. These studies reported greatly increased expression of known stem cell markers Oct4 and Nanog was associated with simultaneous activation of Akt1 and Smad1/5/8 by α5β1, αvβ5, α6β1 and α9β1 integrins within the 3D scaffold. This maintained the self-renewal capacity of ESCs in the absence of leukemia inhibitory factor (LIF) signaling . These studies suggest that a ‘stem cell niche-specific integrin signaling mechanism’ within a 3D microenvironment can sustain the survival of ESCs without signals received from growth factors like LIF which are absolutely essential for the survival of ESCs.
Many of the same integrins which support ESC fate are also markers of CSCs in different cancers . These include α6 integrin enriching for CSCs in breast , prostate , squamous cell carcinoma  and colorectal cancer . Integrin β3 is critical for stemness in breast [42, 43], pancreas  and lung cancer , while β1 integrin is necessary for the stemness characteristics in glioblastomas . These studies are in agreement with our findings which demonstrate that integrins β1, α2 and α5 were down regulated by the knockdown of stem cell-specific Oct4A expression in HEY cell line. These observations were made in cells maintained as monolayer as well as sphere cultures and were consistent with the loss of adhesion of these cells on collagen and fibronectin. However, it should be noted, that integrins can also influence CSC niches independent of their capacity to interact with ECM .
We have previously shown that enhanced expression of α2β1 integrin in ovarian cancer spheres facilitates sphere disaggregation, pro-MMP-2/9 expression and MMP-2/9 activation . In the current study, this is consistent with a decrease in α2 and β1 integrin expressions in Oct4A knockdown HEY cells along with reduced pro-MMP-2 secretion. This data correlates to the loss of sphere disaggregating and migratory ability previously shown in Oct4 KD cells . These observations in our Oct4A knockdown model are consistent with observations in pancreatic cell line models, where loss of Oct4 also resulted in reduced MMP2 expression and subsequent reduced tumor cell invasive ability . Interestingly, MMP2 is known to actively degrade fibronectin and collagen I [32, 33, 47], thus potentially linking these results to the decreased ability of Oct4A KD cells to adhere to fibronectin and collagen I. In the pancreatic cancer cell line model, diminution of Oct4 was associated with a decrease in MMP9 expression . In our study, no such relationship in Oct4A KD cells was observed. Interestingly however, the Oct4B isoform has been identified to regulate both MMP2 and MMP9 expression in cervical cancer, suggesting the lack of MMP9 suppression may be due to the fact that Oct4B expression was not specifically suppressed in the current study .
We also demonstrate that the suppression of Oct4A in HEY cells resulted in a significant reduction in the tumor growth, weight and size in Balb/c nude mouse model. In addition, tumor xenografts derived from Oct4A KD cells displayed relatively lower abundance of markers associated with ovarian cancer (CK7), cancer metabolism (Glut-1) and angiogenesis (CD31 and CD34). This indicates that the suppression of Oct4A not only reduced the tumor initiating ability of cells in vivo but also resulted in a reduction in angiogenic potential which consequently may have resulted in slowed or abrogated tumor growth. These results are consistent with the positive correlation between the expression of Oct4 and vasculogenic mimicry formation and poor prognosis in breast cancer patients . In addition, Oct4 has been shown to promote glioblastoma progression through vascular endothelial growth factor production . Importantly, integrins are well documented to play significant roles in mediating tumor vascularity and angiogenesis . Specifically, integrin α5β1 has been identified to play a role in tumor angiogenesis in in vivo mouse models [53, 54]. Collectively, this tie in with the reduced expression of β1 and α5 subunits demonstrated in Oct4A KD cells and overall reduced tumorigenic and angiogenic profiles in this study.
ANOVA, analysis of variance; CK7, cytokeratin 7; CSCs, cancer stem cells; DAPI, 4’,6-diamidino-2-phenylindole; ECM, extracellular matrix; ESC, embryonic stem cells; IP, intraperitoneal; iPSCs, induced pluripotent stem cells; LIF, leukaemia inhibitory factor; MMP, matrix metalloproteinase; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
The authors 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.
CS is a recipient of the Australian Postgraduate Award. NA is supported by June Wilson Will Trust, BJT Legal, Ballarat, Australia. RBL is a recipient of the Melbourne Brain Centre Post-Doctoral Research Fellowship from the University of Melbourne. This work was made possible through the Victorian State Government Operational Infrastructure Support to the Hudson Institute of Medical Research.
Availability of data and materials
Data and materials are included in the manuscript.
CS designed the study, performed the experiments and contributed to the writing of the manuscript. RBL assisted with the animal studies. JKF contributed to the concept of the study, discussion of the results and editing of the manuscript. MQ and GK 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.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Primary high grade serous epithelial ovarian tumors and normal ovarian tissues were obtained from patients following 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.
Animal ethics approval
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, University of Melbourne and Royal Melbourne Hospital, Australia, Animal Ethics Committee (Project-006/11).
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- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90.View ArticlePubMedGoogle Scholar
- Ahmed N, Stenvers KL. Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research. Frontiers Oncol. 2013;3:256.View ArticleGoogle Scholar
- Ozols RF. Systemic therapy for ovarian cancer: current status and new treatments. Semin Oncol. 2006;33(2 Suppl 6):S3–S11.View ArticlePubMedGoogle Scholar
- Ahmed N, Abubaker K, Findlay J, Quinn M. Cancerous ovarian stem cells: obscure targets for therapy but relevant to chemoresistance. J Cell Biochem. 2013;114:21–34.View ArticlePubMedGoogle Scholar
- Ahmed N, Abubaker K, Findlay JK. Ovarian cancer stem cells: Molecular concepts and relevance as therapeutic targets. Mol Aspects Med. 2014;39:110–25.View ArticlePubMedGoogle Scholar
- Aguilar-Gallardo C, Rutledge EC, Martinez-Arroyo AM, Hidalgo JJ, Domingo S, Simon C. Overcoming challenges of ovarian cancer stem cells: novel therapeutic approaches. Stem Cell Rev. 2012;8:994–1010.View ArticlePubMedGoogle Scholar
- Curley MD, Garrett LA, Schorge JO, Foster R, Rueda BR. Evidence for cancer stem cells contributing to the pathogenesis of ovarian cancer. Front Biosci. 2011;16:368–92.View ArticleGoogle Scholar
- Kellner S, Kikyo N. Transcriptional regulation of the Oct4 gene, a master gene for pluripotency. Histol Histopathol. 2010;25:405–12.PubMedPubMed CentralGoogle Scholar
- Stefanovic S, Puceat M. Oct-3/4: not just a gatekeeper of pluripotency for embryonic stem cell, a cell fate instructor through a gene dosage effect. Cell Cycle. 2007;6:8–10.View ArticlePubMedGoogle Scholar
- Samardzija C, Quinn M, Findlay JK, Ahmed N. Attributes of Oct4 in stem cell biology: perspectives on cancer stem cells of the ovary. J Ovarian Res. 2012;5:37.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang X, Dai J. Concise review: isoforms of OCT4 contribute to the confusing diversity in stem cell biology. Stem Cells. 2010;28:885–93.View ArticlePubMedPubMed CentralGoogle Scholar
- Liedtke S, Stephan M, Kogler G. Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research. Biol Chem. 2008;389:845–50.View ArticlePubMedGoogle Scholar
- Pasquier J, Rafii A. Role of the microenvironment in ovarian cancer stem cell maintenance. BioMed Res Intl. 2013;2013:630782.Google Scholar
- Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010;10:9–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Seguin L, Desgrosellier JS, Weis SM, Cheresh DA. Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance. Trends in Cell Biol. 2015;25:234–40.View ArticleGoogle Scholar
- Raymond K, Deugnier MA, Faraldo MM, Glukhova MA. Adhesion within the stem cell niches. Current Opinion in Cell Biol. 2009;21:623–9.View ArticleGoogle Scholar
- Ahmed N, Riley C, Oliva K, Stutt E, Rice GE, Quinn MA. Integrin-linked kinase expression increases with ovarian tumour grade and is sustained by peritoneal tumour fluid. J Pathol. 2003;201:229–37.View ArticlePubMedGoogle Scholar
- Choi JH, Choi KC, Auersperg N, Leung PC. Overexpression of follicle-stimulating hormone receptor activates oncogenic pathways in preneoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab. 2004;89:5508–16.View ArticlePubMedGoogle Scholar
- Samardzija C, Luwor RB, Volchek M, Quinn MA, Findlay JK, Ahmed N. A critical role of Oct4A in mediating metastasis and disease-free survival in a mouse model of ovarian cancer. Mol Cancer. 2015;14:152.View ArticlePubMedPubMed CentralGoogle Scholar
- Abubaker K, Luwor RB, Zhu H, McNally O, Quinn MA, Burns CJ, et al. Inhibition of the JAK2/STAT3 pathway in ovarian cancer results in the loss of cancer stem cell-like characteristics and a reduced tumor burden. BMC Cancer. 2014;14:317.View ArticlePubMedPubMed CentralGoogle Scholar
- Abubaker K, Latifi A, Chan E, Luwor RB, Burns CJ, Thompson EW, et al. Enhanced activation of STAT3 in ascites-derived recurrent ovarian tumors: inhibition of cisplatin-induced STAT3 activation reduced tumorigenicity of ovarian cancer by a loss of cancer stem cell-like characteristics. J Cancer Stem Cell Res. 2015;3:e1001.View ArticleGoogle Scholar
- Latifi A, Quinn MA, Thompson EW, Findlay JK, Ahmed N. Distinct molecular signature of recurrent ovarian tumor cells isolated from the ascites of advanced-stage serous ovarian cancer patients. J Cancer Stem Cell Res. 2014;2:e1006.Google Scholar
- Ahmed N, Oliva K, Wang Y, Quinn M, Rice G. Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-beta1 integrin complex in colon cancer cells. British J Cancer. 2003;89:374–84.View ArticleGoogle Scholar
- Latifi A, Abubaker K, Castrechini N, Ward AC, Liongue C, Dobill F, et al. Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile. J Cell Biochem. 2011;112:2850–64.View ArticlePubMedGoogle Scholar
- Abubaker K, Latifi A, Luwor R, Nazaretian S, Zhu H, Quinn MA, et al. Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden. Mol Cancer. 2013;12:24.View ArticlePubMedPubMed CentralGoogle Scholar
- Abubaker K, Luwor RB, Escalona R, McNally O, Quinn MA, Thompson EW, et al. Targeted Disruption of the JAK2/STAT3 Pathway in Combination with Systemic Administration of Paclitaxel Inhibits the Priming of Ovarian Cancer Stem Cells Leading to a Reduced Tumor Burden. Frontiers in Oncol. 2014;4:75.View ArticleGoogle Scholar
- Ailles LE, Weissman IL. Cancer stem cells in solid tumors. Current opinion in Biotech. 2007;18:460–6.View ArticleGoogle Scholar
- Bapat SA, Mali AM, Koppikar CB, Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res. 2005;65:3025–9.PubMedGoogle Scholar
- Sodek KL, Ringuette MJ, Brown TJ. Compact spheroid formation by ovarian cancer cells is associated with contractile behavior and an invasive phenotype. Intl J Cancer. 2009;124:2060–70.View ArticleGoogle Scholar
- Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of alpha6beta1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British J Cancer. 2005;92:1475–85.View ArticleGoogle Scholar
- Shield K, Riley C, Quinn MA, Rice GE, Ackland ML, Ahmed N. Alpha2beta1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis. J Carcinogenesis. 2007;6:11.View ArticleGoogle Scholar
- Burleson KM, Hansen LK, Skubitz AP. Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers. Clin & Expet Metastasis. 2004;21:685–97.View ArticleGoogle Scholar
- Kenny HA, Kaur S, Coussens LM, Lengyel E. The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin. J Clin Invest. 2008;118:1367–79.View ArticlePubMedPubMed CentralGoogle Scholar
- Kenny HA, Lengyel E. MMP-2 functions as an early response protein in ovarian cancer metastasis. Cell Cycle. 2009;8:683–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Guo W, Giancotti FG. Integrin signalling during tumour progression. Nature Rev Mol Cell Biol. 2004;5:816–26.View ArticleGoogle Scholar
- Lee ST, Yun JI, Jo YS, Mochizuki M, van der Vlies AJ, Kontos S, et al. Engineering integrin signaling for promoting embryonic stem cell self-renewal in a precisely defined niche. Biomaterials. 2010;31:1219–26.View ArticlePubMedGoogle Scholar
- Lee ST, Yun JI, van der Vlies AJ, Kontos S, Jang M, Gong SP, et al. Long-term maintenance of mouse embryonic stem cell pluripotency by manipulating integrin signaling within 3D scaffolds without active Stat3. Biomaterials. 2012;33:8934–42.View ArticlePubMedGoogle Scholar
- Martin TA, Jiang WG. Evaluation of the expression of stem cell markers in human breast cancer reveals a correlation with clinical progression and metastatic disease in ductal carcinoma. Oncol Reports. 2014;31:262–72.Google Scholar
- Hoogland AM, Verhoef EI, Roobol MJ, Schroder FH, Wildhagen MF, van der Kwast TH, et al. Validation of stem cell markers in clinical prostate cancer: alpha6-integrin is predictive for non-aggressive disease. Prostate. 2014;74:488–96.View ArticlePubMedGoogle Scholar
- Schober M, Fuchs E. Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling. PNAS. 2011;108:10544–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Haraguchi N, Ishii H, Mimori K, Ohta K, Uemura M, Nishimura J, et al. CD49f-positive cell population efficiently enriches colon cancer-initiating cells. Internl J Oncol. 2013;43:425–30.Google Scholar
- Desgrosellier JS, Lesperance J, Seguin L, Gozo M, Kato S, Franovic A, et al. Integrin alphavbeta3 drives slug activation and stemness in the pregnant and neoplastic mammary gland. Dev Cell. 2014;30:295–308.View ArticlePubMedPubMed CentralGoogle Scholar
- Lo PK, Kanojia D, Liu X, Singh UP, Berger FG, Wang Q, et al. CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin-TGFbeta signaling. Oncogene. 2012;31:2614–26.View ArticlePubMedGoogle Scholar
- Seguin L, Kato S, Franovic A, Camargo MF, Lesperance J, Elliott KC. An integrin beta(3)-KRAS-RalB complex drives tumour stemness and resistance to EGFR inhibition. Nature Cell Biol. 2014;16:457–68.View ArticlePubMedPubMed CentralGoogle Scholar
- Carbonell WS, DeLay M, Jahangiri A, Park CC, Aghi MK. Beta1 integrin targeting potentiates antiangiogenic therapy and inhibits the growth of bevacizumab-resistant glioblastoma. Cancer Res. 2013;73:3145–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin H, Sun LH, Han W, He TY, Xu XJ, Cheng K, et al. Knockdown of OCT4 suppresses the growth and invasion of pancreatic cancer cells through inhibition of the AKT pathway. Mol Med Reports. 2014;10:1335–42.Google Scholar
- Karagiannis ED, Popel AS. A theoretical model of type I collagen proteolysis by matrix metalloproteinase (MMP) 2 and membrane type 1 MMP in the presence of tissue inhibitor of metalloproteinase 2. J Biol Chem. 2004;279:39105–14.View ArticlePubMedGoogle Scholar
- Lu Y, Zhu H, Shan H, Lu J, Chang X, Li X, et al. Knockdown of Oct4 and Nanog expression inhibits the stemness of pancreatic cancer cells. Cancer Lett. 2013;340:113–23.View ArticlePubMedGoogle Scholar
- Li SW, Wu XL, Dong CL, Xie XY, Wu JF, Zhang X. The differential expression of OCT4 isoforms in cervical carcinoma. PLoS One. 2015;10(3):e0118033.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu T, Sun B, Zhao X, Li Y, Gu Q, Dong X, Liu F. OCT4 expression and vasculogenic mimicry formation positively correlate with poor prognosis in human breast cancer. Intl J Mol Sci. 2014;15:19634–49.View ArticleGoogle Scholar
- Takahashi H, Inoue A, Kawabe Y, Hosokawa Y, Iwata S, Sugimoto K, et al. Oct-3/4 promotes tumor angiogenesis through VEGF production in glioblastoma. Brain Tumor Pathol. 2015;32:31–40.View ArticlePubMedGoogle Scholar
- Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr Top Dev Biol. 2004;64:207–38.View ArticlePubMedGoogle Scholar
- Kim S, Harris M, Varner JA. Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A. J Biol Chem. 2000;275:33920–8.View ArticlePubMedGoogle Scholar
- Kim S, Bell K, Mousa SA, Varner JA. Regulation of angiogenesis in vivo by ligation of integrin alpha5beta1 with the central cell-binding domain of fibronectin. Am J Pathol. 2000;156:1345–62.View ArticlePubMedPubMed CentralGoogle Scholar