In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI-anchored tumour suppressor OPCML Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol-rich lipid domains, where OPCML resides. Here, phospho-AXL is brought in proximity to the lipid domain-restricted phosphatase PTPRG, which de-phosphorylates the RTK/ligand complex. This prevents AXL-mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho-ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL-dependent oncogenic signalling.

HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the context of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one-way ANOVA and t-tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overexpressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene-signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum-resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials.

Ovarian cancer is a complex disease with heterogeneity among the gene expression molecular subtypes (GEMS) between patients. Patients with tumors of a mesenchymal ("Mes") subtype have a poorer prognosis than patients with tumors of an epithelial ("Epi") subtype. We evaluated GEMS of ovarian cancer patients for molecular signaling profiles and assessed how the differences in these profiles could be leveraged to improve patient clinical outcome. Kinome enrichment analysis identified AXL as a particularly abundant kinase in Mes-subtype tumor tissue and cell lines. In Mes cells, upon activation by its ligand GAS6, AXL coclustered with and transactivated the receptor tyrosine kinases (RTKs) cMET, EGFR, and HER2, producing sustained extracellular signal-regulated kinase (ERK) activation. In Epi-A cells, AXL was less abundant and induced a transient activation of ERK without evidence of RTK transactivation. AXL-RTK cross-talk also stimulated sustained activation of the transcription factor FRA1, which correlated with the induction of the epithelial-mesenchymal transition (EMT)-associated transcription factor SLUG and stimulation of motility exclusively in Mes-subtype cells. The AXL inhibitor R428 attenuated RTK and ERK activation and reduced cell motility in Mes cells in culture and reduced tumor growth in a chick chorioallantoic membrane model. A higher concentration of R428 was needed to inhibit ERK activation and cell motility in Epi-A cells. Silencing AXL in Mes-subtype cells reversed the mesenchymal phenotype in culture and abolished tumor formation in an orthotopic xenograft mouse model. Thus, AXL-targeted therapy may improve clinical outcome for patients with Mes-subtype ovarian cancer.

e17084 Background: Ovarian cancer (OC) is a complex disease demonstrated by the heterogeneity in gene expression molecular subtypes (GEMS). To address the GEMS-specific lethality in OC, we highlight the relevance of how a receptor tyrosine kinase (RTK) differs in the signaling pattern, functional consequences, and therapeutic implications in the context of GEMS. Methods: An enrichment analysis of the human kinome among the OC GEMS was performed to identify top ranking RTKs in each GEMS. A top-ranking RTK for the Mes subtype, AXL, was selected for further studies. AXL activation in GEMS-matched OC cells were analyzed for the downstream signaling cascade by western blotting and Reverse Phase Protein Analysis (RPPA), and in vitro functions. A selective inhibitor to AXL was tested for the attenuation of downstream signaling and functional aggressiveness. Results: AXL is the top-ranking RTK for the poor prognosis Mes subtype. Interestingly, AXL is also expressed, at a lower rank, in the better prognosis Epi-A subtype. Upon ligand stimulation with Gas6, specific to Mes, there is recurrent temporal activation of the extracellular regulated kinase (ERK) signaling downstream of AXL observed by Western blotting. RPPA and proximity ligation assays further show that Gas6/AXL signaling transactivates other RTKs such as cMET, EGFR, and HER2 in Mes, exclusively. This signal amplification downstream to the Gas6/AXL axis alludes to pathway addiction in Mes. Functionally, the recurrent ERK activation results in a motile and invasive phenotype. This further sensitizes Mes to a selective AXL inhibitor, R428, by attenuating the RTK-ERK activation, reducing the in vitro motility and invasion, and inhibiting the in ovo tumor growth in the chick chorio-allantoic membrane (CAM) assay at a lower IC-50 dose compared to Epi-A. Conclusions: Our results imply that the Mes subtype is more sensitive to AXL inhibition, and the RTK crosstalk rewires the system, drastically sensitizing it to RTK node inhibition. The Epi-A subtype retain a linear signaling axis and shows less therapeutic advantage to targeting AXL. Stratifying OC patients based on the GEMS followed by targeting AXL is promising in a prospective clinical setting.

Ovarian cancer is the leading cause of cancer death among all gynaecological cancers. Its aggressive nature is partly due to genetic heterogeneity and the lack of effective treatments strategies. Standard treatment involves cytoreductive surgery followed by chemotherapy using a platinum-based agent. Although initially, patients response well to this treatment, the majority will relapse and develop recurrent disease, predominantly due to the emergence of platinum resistance. Further understanding of the molecular changes which occur during ovarian ontogenesis and in the development of platinum resistance is essential to design new targeted drugs to improve patient prognosis. HOX gene are a family of homeodomain-containing transcription factors that determine cell and tissue identity in the early embryo and are found to be aberrantly expressed in cancer. HOX gene expression in ovarian cancer of different histological subtypes and in primary ovarian tumours were evaluated here. This is the first comprehensive study of HOX gene expression in a cohort of primary ovarian tumours, including statistical analysis of HOX gene expression profiles along with clinic-pathological data of each patient. HOX genes were found to be profoundly dysregulated in ovarian cancer cell lines and primary ovarian tumours, with very little to no expression found in normal ovarian and fallopian tube tissue. A 5-HOX gene signature which predicts poor overall survival in ovarian cancer patients was identified. Platinum resistant disease displayed an overall higher level of HOX gene expression, significantly HOXB4 and HOXB9. This dysregulated HOX expression reported could therefore act as a set of targets for therapeutic intervention. The novel peptide, HXR9, has been developed to block the interaction between HOX proteins and their co-factor, PBX, and therefore subsequent target gene expression. The efficacy of HXR9 treatment of ovarian cancer cells was explored. HXR9 treatment was shown to induce cell death via apoptosis in ovarian cancer cells shown by an increase in apoptotic cells identified by flow cytometric analysis, and increase in caspase-3 activity and the upregulation of pro-apoptotic gene cFos. Enhanced cell cytotoxicity was observed when combining HXR9 with cisplatin to treat platinum resistant cells, revealing a new therapeutic option for drug resistant disease that should be explored further for potential clinical trial investigation. A limiting factor towards the development of new treatment strategies is the lack of a reliable animal model of ovarian cancer. Common methods used to test new drugs involve in vitro investigation and the use of engineered animal models. However, these models do not represent the true heterogeneity and complexity of human ovarian tumours. Therefore, the use of the chicken chorioallontoic membrane (CAM) as a model of ovarian cancer for the testing of anti-cancer drugs was assessed. Cell lines grafted onto the CAM successfully and developing into micro-tumours. Cell cultured from ascites samples of ovarian cancer patients also grafted, but with a less success rate. Morphological and tumour retardation was detected after treatment with HXR9. This demonstrated the potential of this model to be developed for future personalised drug screening.

The HOX genes are a family of homeodomain-containing transcription factors that determine cellular identity during development. Here we review a number of recent studies showing that HOX genes are strongly expressed in ovarian cancer, and that in some cases the expression of specific HOX genes is sufficient to confer a particular identity and phenotype upon cancer cells. We also review the recent advances in elucidating the different functions of HOX genes in ovarian cancer. A literature search was performed using the search terms HOX genes (including specific HOX genes), ovarian cancer and oncogenesis. Articles were accessed through searches performed in ISI Web of Knowledge, PubMed and ScienceDirect. Taken together, these studies have shown that HOX genes play a role in the oncogenesis of ovarian cancer and function in the inhibition of apoptosis, DNA repair and enhanced cell motility. The function of HOX genes in ovarian cancer oncogenesis supports their potential role as prognostic and diagnostic markers, and as therapeutic targets in this disease.