Michael Gallagher

Platelet Adhesion and Degranulation Induce Pro-Survival and Pro-Angiogenic Signalling in Ovarian Cancer Cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol®) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high‐grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression‐free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel‐induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR‐433 binding domain in the MAD2 3′ UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down‐regulated in pre‐miR‐433 transfected A2780 cells. Secondly, pre‐miR‐433 suppressed the activity of a reporter construct containing the 3′‐UTR of MAD2. Thirdly, blocking miR‐433 binding to the MAD2 3′ UTR protected MAD2 from miR‐433 induced protein down‐regulation. Importantly, reduced MAD2 protein expression in pre‐miR‐433‐transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high‐grade serous EOC. Copyright

Developing ovarian cancer stem cell models: laying the pipeline from discovery to clinical intervention.

Despite decades of research, ovarian cancer is still associated with unacceptably high mortality rates, which must be addressed by novel therapeutic approaches. One avenue through which this may be achieved is targeting of tumor-initiating ‘Cancer Stem Cells’ (CSCs). CSCs are sufficient to generate primary and recurrent disease through extensive rounds of asymmetric division, which maintain the CSC pool while producing the tissues that form the bulk of the tumor. CSCs thrive in the harsh tumor niche, are generally refractory to therapeutic intervention and closely-linked to the Epithelial-Mesenchymal Transition process, which facilitates invasion and metastasis. While it is well-accepted that CSC-targeting must be assessed as a novel therapeutic avenue, few ovarian CSC models have been developed due to perceived and actual difficulties associated with the process of ‘CSC Discovery’. In this article we review contemporary approaches to CSC Discovery and argue that this process should start with an understanding of the specific challenges associated with clinical intervention, laying the pipeline backwards towards CSC Discovery. Such an approach would expedite the bridging of the gap between laboratory isolation and clinical targeting of ovarian CSCs.

Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients

BackgroundTumours with high proportions of differentiated cells are considered to be of a lower grade to those containing high proportions of undifferentiated cells. This property may be linked to the differentiation properties of stem cell-like populations within malignancies. We aim to identify molecular mechanism associated with the generation of tumours with differing grades from malignant stem cell populations with different differentiation potentials. In this study we assessed microRNA (miRNA) regulation in two populations of malignant Embryonal Carcinoma (EC) stem cell, which differentiate (NTera2) or remain undifferentiated (2102Ep) during tumourigenesis, and compared this to miRNA regulation in ovarian serous carcinoma (OSC) patient samples.MethodsmiRNA expression was assessed in NTera2 and 2102Ep cells in the undifferentiated and differentiated states and compared to that of OSC samples using miRNA qPCR.ResultsOur analysis reveals a substantial overlap between miRNA regulation in 2102Ep cells and OSC samples in terms of miRNA biosynthesis and expression of mature miRNAs, particularly those of the miR-17/92 family and clustering to chromosomes 14 and 19. In the undifferentiated state 2102Ep cells expressed mature miRNAs at up to 15,000 fold increased levels despite decreased expression of miRNA biosynthesis genes Drosha and Dicer. 2102Ep cells avoid differentiation, which we show is associated with consistent levels of expression of miRNA biosynthesis genes and mature miRNAs while expression of miRNAs clustering to chromosomes 14 and 19 is deemphasised. OSC patient samples displayed decreased expression of miRNA biosynthesis genes, decreased expression of mature miRNAs and prominent clustering to chromosome 14 but not 19. This indicates that miRNA biosynthesis and levels of miRNA expression, particularly from chromosome 14, are tightly regulated both in progenitor cells and in tumour samples.ConclusionmiRNA biosynthesis and expression of mature miRNAs, particularly the miR-17/92 family and those clustering to chromosomes 14 and 19, are highly regulated in both progenitor cells and tumour samples. Strikingly, 2102Ep cells are not simply malfunctioning but respond to differentiation specifically, a mechanism that is highly relevant to OSC samples. Our identification and future manipulation of these miRNAs may facilitate generation of lower grade malignancies from these high-grade cells.

The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Pre-Treatment of Platinum Resistant Ovarian Cancer Cells with an MMP-9/MMP-2 Inhibitor Prior to Cisplatin Enhances Cytotoxicity as Determined by High Content Screening

Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated.

Identifying novel hypoxia-associated markers of chemoresistance in ovarian cancer

BackgroundOvarian cancer is associated with poor long-term survival due to late diagnosis and development of chemoresistance. Tumour hypoxia is associated with many features of tumour aggressiveness including increased cellular proliferation, inhibition of apoptosis, increased invasion and metastasis, and chemoresistance, mostly mediated through hypoxia-inducible factor (HIF)-1α. While HIF-1α has been associated with platinum resistance in a variety of cancers, including ovarian, relatively little is known about the importance of the duration of hypoxia. Similarly, the gene pathways activated in ovarian cancer which cause chemoresistance as a result of hypoxia are poorly understood. This study aimed to firstly investigate the effect of hypoxia duration on resistance to cisplatin in an ovarian cancer chemoresistance cell line model and to identify genes whose expression was associated with hypoxia-induced chemoresistance.MethodsCisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cell lines were exposed to various combinations of hypoxia and/or chemotherapeutic drugs as part of a ‘hypoxia matrix’ designed to cover clinically relevant scenarios in terms of tumour hypoxia. Response to cisplatin was measured by the MTT assay. RNA was extracted from cells treated as part of the hypoxia matrix and interrogated on Affymetrix Human Gene ST 1.0 arrays. Differential gene expression analysis was performed for cells exposed to hypoxia and/or cisplatin. From this, four potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by RT-PCR.ResultsHypoxia increased resistance to cisplatin in A2780 and A2780cis cells. A plethora of genes were differentially expressed in cells exposed to hypoxia and cisplatin which could be associated with chemoresistance. In ovarian tumour samples, we found trends for upregulation of ANGPTL4 in partial responders and down-regulation in non-responders compared with responders to chemotherapy; down-regulation of HER3 in partial and non-responders compared to responders; and down-regulation of HIF-1α in non-responders compared with responders.ConclusionThis study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. We have also identified many potential biomarkers of hypoxia and platinum resistance and provided an initial validation of a subset of these markers in ovarian cancer tissues.

An integrated analysis of the SOX2 microRNA response program in human pluripotent and nullipotent stem cell lines

BackgroundSOX2 is a core component of the transcriptional network responsible for maintaining embryonal carcinoma cells (ECCs) in a pluripotent, undifferentiated state of self-renewal. As such, SOX2 is an oncogenic transcription factor and crucial cancer stem cell (CSC) biomarker in embryonal carcinoma and, as more recently found, in the stem-like cancer cell component of many other malignancies. SOX2 is furthermore a crucial factor in the maintenance of adult stem cell phenotypes and has additional roles in cell fate determination. The SOX2-linked microRNA (miRNA) transcriptome and regulome has not yet been fully defined in human pluripotent cells or CSCs. To improve our understanding of the SOX2-linked miRNA regulatory network as a contribution to the phenotype of these cell types, we used high-throughput differential miRNA and gene expression analysis combined with existing genome-wide SOX2 chromatin immunoprecipitation (ChIP) data to map the SOX2 miRNA transcriptome in two human embryonal carcinoma cell (hECC) lines.ResultsWhole-microRNAome and genome analysis of SOX2-silenced hECCs revealed many miRNAs regulated by SOX2, including several with highly characterised functions in both cancer and embryonic stem cell (ESC) biology. We subsequently performed genome-wide differential expression analysis and applied a Monte Carlo simulation algorithm and target prediction to identify a SOX2-linked miRNA regulome, which was strongly enriched with epithelial-to-mesenchymal transition (EMT) markers. Additionally, several deregulated miRNAs important to EMT processes had SOX2 binding sites in their promoter regions.ConclusionIn ESC-like CSCs, SOX2 regulates a large miRNA network that regulates and interlinks the expression of crucial genes involved in EMT.

Suppression of cancer stemness p21-regulating mRNA and microRNA signatures in recurrent ovarian cancer patient samples.

BackgroundMalignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease.MethodsMicroarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples.ResultsMicroarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component.ConclusionWe present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.