Differential Regulation of Genes by the Glucogenic Hormone Asprosin in Ovarian Cancer.

Background: Ovarian cancer (OvCa) is one of the most lethal forms of gynaecological malignancy. Altered energy metabolism and increased aerobic glycolysis in OvCa are hallmarks that demand attention. The glucogenic hormone asprosin is often dysregulated in metabolic disorders such as insulin resistance, diabetes (type 2 and gestational), and preeclampsia. Despite association with metabolic disorders, its role in energy metabolism within the tumour microenvironment is yet to be explored. Here, we study the role of asprosin in OvCa using transcriptomics and expand on functional studies with clinical samples. Methods: RNA sequencing, functional gene enrichment analysis, Western blotting and ImageStream. Results: Following treatment with 100 nM of asprosin, the serous OvCa cell line, SKOV-3, displayed 160 and 173 gene regulatory changes, at 4 and 12 h respectively, when compared with control samples (p < 0.05 and Log2FC > 1). In addition to energy metabolism and glucose-related pathways, asprosin was shown to alter pathways associated with cell communication, TGF-ß signalling, and cell proliferation. Moreover, asprosin was shown to induce phosphorylation of ERK1/2 in the same in vitro model. Using liquid biopsies, we also report for novel expression of asprosin's predicted receptors OR4M1 and TLR4 in cancer-associated circulating cells; with significant reduction seen between pre-chemotherapy and end of first line chemotherapy, in addition to patients under maintenance with bevacizumab +/− olaparib for OR4M1. Conclusions: In relation to OvCa, asprosin appears to regulate numerous signalling pathways in-vitro. The prognostic potential of OR4M1 in liquid biopsies should also be explored further.

Differential Expression of RAD51AP1 in Ovarian Cancer: Effects of siRNA In Vitro.

BACKGROUND: DNA double strand breaks can affect genome integrity potentially leading to cancer. RAD51-associated protein 1 (RAD51AP1), an accessory protein to RAD51, is critical for homologous recombination, a key DNA damage response pathway. Emerging studies indicate a novel role for RAD51AP1 in carcinogenesis. Here we provide additional insight into the role of RAD51AP1 in ovarian cancer (OvCa). METHODS: Gene expression and patient phenotype data were obtained from TCGA and GTEX project consortia for bioinformatics analysis. Immunohistochemistry of OvCa tissue microarray was undertaken. Functional analyses were performed in a SKOV3 OvCa cell line with down-regulation of RAD51AP1 using siRNA. RESULTS: RAD51AP1 is overexpressed at gene level in primary and recurrent OvCa compared to controls. At protein level, RAD51AP1 was up-regulated in low grade serous tumors compared to high grade OvCa. There was higher expression of RAD51AP1 in OvCa metastatic to lymph nodes compared to primary cancer samples. Gene enrichment analyses identified 12 differentially expressed genes (DEGs) related to OvCa, eight of which are also common in tissue from patients with type 2 diabetes mellitus (T2DM). CONCLUSIONS: RAD51AP1 is overexpressed in OvCa, Given the link between OvCa and T2DM, the eight-gene signature shows potential for predictive value.

A pancancer overview of FBN1, asprosin and its cognate receptor OR4M1 with detailed expression profiling in ovarian cancer.

Ovarian cancer affects >295,000 women worldwide and is the most lethal of gynaecological malignancies. Often diagnosed at a late stage, current research efforts seek to further the molecular understanding of its aetiopathogenesis and the development of novel biomarkers. The present study investigated the expression levels of the glucogenic hormone asprosin [encoded by fibrillin-1 (FBN1)], and its cognate receptor, olfactory receptor 4M1 (OR4M1), in ovarian cancer. A blend of in silico open access The Cancer Genome Atlas data, as well as in vitro reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry and immunofluorescence data were used. RT-qPCR revealed expression levels of OR4M1 and FBN1 in clinical samples and in ovarian cancer cell lines (SKOV-3, PEO1, PEO4 and MDAH-2774), as well as the normal human ovarian surface epithelial cell line (HOSEpiC). Immunohistochemical staining of a tissue microarray was used to identify the expression levels of OR4M1 and asprosin in ovarian cancer samples of varying histological subtype and grade, including clear cell carcinoma, serous ovarian cancer and mucinous adenocarcinoma. Immunofluorescence analysis revealed asprosin expression in SKOV-3 and HOSEpiC cells. These results demonstrated the expression of both asprosin and OR4M1 in normal and malignant human ovarian tissues. This research invokes further investigation to advance the understanding of the role of asprosin and OR4M1 within the ovarian tumour microenvironment.

Identification of Potential Bisphenol A (BPA) Exposure Biomarkers in Ovarian Cancer.

Endocrine-disrupting chemicals (EDCs) can exert multiple deleterious effects and have been implicated in carcinogenesis. The xenoestrogen Bisphenol A (BPA) that is found in various consumer products has been involved in the dysregulation of numerous signalling pathways. In this paper, we present the analysis of a set of 94 genes that have been shown to be dysregulated in presence of BPA in ovarian cancer cell lines since we hypothesised that these genes might be of biomarker potential. This study sought to identify biomarkers of disease and biomarkers of disease-associated exposure. In silico analyses took place using gene expression data extracted from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. Differential expression was further validated at protein level using immunohistochemistry on an ovarian cancer tissue microarray. We found that 14 out of 94 genes are solely dysregulated in the presence of BPA, while the remaining 80 genes are already dysregulated (p-value < 0.05) in their expression pattern as a consequence of the disease. We also found that seven genes have prognostic power for the overall survival in OC in relation to their expression levels. Out of these seven genes, Keratin 4 (KRT4) appears to be a biomarker of exposure-associated ovarian cancer, whereas Guanylate Binding Protein 5 (GBP5), long intergenic non-protein coding RNA 707 (LINC00707) and Solute Carrier Family 4 Member 11 (SLC4A11) are biomarkers of disease. BPA can exert a plethora of effects that can be tissue- or cancer-specific. Our in silico findings generate a hypothesis around biomarkers of disease and exposure that could potentially inform regulation and policy making.

H2A Histone Family Member X (H2AX) Is Upregulated in Ovarian Cancer and Demonstrates Utility as a Prognostic Biomarker in Terms of Overall Survival.

Background: H2AX can be of prognostic value in breast cancer, since in advanced stage patients with high levels, there was an association with worse overall survival (OS). However, the clinical relevance of H2AX in ovarian cancer (OC) remains to be elucidated. Methods: OC H2AX expression studied using the TCGA/GTEX datasets. Subsequently, patients were classified as either high or low in terms of H2AX expression to compare OS and perform gene set enrichment. qRT-PCR validated in-silico H2AX findings followed by immunohistochemistry on a tissue microarray. The association between single nucleotide polymorphisms in the area of H2AX; prevalence and five-year OC survival was tested in samples from the UK Biobank. Results: H2AX was significantly overexpressed in OCs compared to normal tissues, with higher expression associated with better OS (p = 0.010). Gene Set Enrichment Analysis demonstrated gene sets involved in G2/M checkpoint, DNA repair mTORC1 signalling were enriched in the H2AX highly expressing OCs. Polymorphisms in the area around the gene were associated with both OC prevalence (rs72997349-C, p = 0.005) and worse OS (rs10790282-G, p = 0.011). Finally, we demonstrated that H2AX gene expression correlated with γ-H2AX staining in vitro. Conclusions: Our findings suggest that H2AX can be a novel prognostic biomarker for OC.

Identification of Cancer-Associated Circulating Cells in Anal Cancer Patients.

Whilst anal cancer accounts for less than 1% of all new cancer cases, incidence rates have increased by up to 70% in the last 30 years with the majority of cases driven by human papilloma virus (HPV) infection. Standard treatment for localised anal cancer is chemoradiotherapy (CRT). Localised progression is the predominant pattern of relapse but well under 50% of cases are salvaged by surgery, predominantly because confirming recurrence within post-radiation change is very challenging. Identifying cancer-associated circulating cells (CCs) in peripheral blood could offer a corroborative method of monitoring treatment efficacy and identifying relapse early. To study this, nucleated cells were isolated from the blood of patients with anal cancer prior to, during, and after CRT and processed through the Amnis® ImageStream®X Mk II Imaging Flow Cytometer, without prior enrichment, using Pan-cytokeratin (PCK), CD45 antibodies and making use of the DNA dye DRAQ5. Analysis was undertaken using IDEAS software to identify those cells that were PCK-positive and DRAQ5-positive as well as CD45-negative; these were designated as CCs. CCs were identified in 7 of 8 patients; range 60-876 cells per mL of blood. This first report of the successful identification of CCs in anal cancer patients raises the possibility that liquid biopsies will find a future role as a prognostic/diagnostic tool in this patient group.

Surfactant Protein D as a Potential Biomarker and Therapeutic Target in Ovarian Cancer.

Surfactant protein D (SP-D) is an important innate immune molecule that is involved in clearing pathogens and regulating inflammation at pulmonary as well as extra-pulmonary sites. Recent studies have established the role of SP-D as an innate immune surveillance molecule against lung and pancreatic cancer, but little is known about its involvement in signaling pathways it can potentially activate in ovarian cancer. We focused our study on ovarian cancer by performing bioinformatics analysis (Oncomine) of datasets and survival analysis (Kaplan-Meier plotter), followed by immunohistochemistry using ovarian cancer tissue microarrays. SP-D mRNA was found to be expressed widely in different types of ovarian cancer irrespective of stage or grade. These in silico data were further validated by immunohistochemistry of clinical tissues. High transcriptional levels of SP-D were associated with unfavorable prognosis (overall and progression-free survival). We also detected SP-D protein in Circulating Tumor Cells of three ovarian cancer patients, suggesting that SP-D can also be used as a potential biomarker. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis in pancreatic cancer cells via Fas-mediated pathway. In this study, we report that treatment of SKOV3 cells (an ovarian cancer cell line) with rfhSP-D led to a decrease in cell motility and cell proliferation. This was followed by an inhibition of the mTOR pathway activity, increase in caspase 3 cleavage, and induction of pro-apoptotic genes Fas and TNF-α. These data, suggesting a likely protective role of rfhSP-D against ovarian cancer, together with the observation that the ovarian cancer microenvironment overexperesses SP-D leading to poor prognosis, seems to suggest that the tumor microenvironment components manipulate the protective effect of SP-D in vivo.

Involvement of the glutamine RF­amide peptide and its cognate receptor GPR103 in prostate cancer.

Glutamine RF­amide peptide (QRFP) belongs to the RFamide neuropeptide family, which is involved in a wide spectrum of biological activities, ranging from food intake and cardiovascular functioning to analgesia, aldosterone secretion, locomotor activity and reproduction. Recently, QRFP has been demonstrated to exert its effects by activating the G protein­coupled receptor GPR103. QRFP is expressed in the brain and peripherally in the adipose tissue, bladder, colon, testis, parathyroid and thyroid gland, as well as in the prostate gland. Following lung cancer, prostate cancer constitutes the second most frequently diagnosed cancer among men, whilst obesity appears to be a contributing factor for aggressive prostate cancer. In the present study, we sought to investigate the role of QRFP in prostate cancer, using two androgen­independent human prostate cancer cell lines (PC3 and DU145) as in vitro experimental models and clinical human prostate cancer samples. The expression of both QRFP and GPR103 at the gene and protein level was higher in human prostate cancer tissue samples compared to control and benign prostatic hyperplasia (BHP) samples. Furthermore, in both prostate cancer cell lines used in the present study, QRFP treatment induced the phosphorylation of ERK1/2, p38, JNK and Akt. In addition, QRFP increased cell migration and invasion in these in vitro models, with the increased expression of MMP2. Furthermore, we demonstrated that the pleiotropic adipokine, leptin, increased the expression of QRFP and GPR103 in PC3 prostate cancer cells via a PI3K­ and MAPK­dependent mechanism, indicating a novel potential link between adiposity and prostate cancer. Our findings expand the existing evidence and provide novel insight into the implication of QRFP in prostate cancer.

Differential expression of mTOR components in endometriosis and ovarian cancer: Effects of rapalogues and dual kinase inhibitors on mTORC1 and mTORC2 stoichiometry.

Endometriosis is a well­known risk factor for ovarian cancer. The genetic changes that characterise endometriosis are poorly understood; however, the mechanistic target of rapamycin (mTOR) pathway is involved. In this study, we investigated the expression of key mTOR components in endometriosis and the effects of rapalogues using an endometrioid ovarian carcinoma cell line (MDAH 2774) as an in vitro model. Gene expression of mTOR, DEPTOR, Rictor and Raptor was assessed by qPCR in 24 endometriosis patients and in silico in ovarian cancer patients. Furthermore, the effects of Rapamycin, Everolimus, Deforolimus, Temsirolimus, Resveratrol, and BEZ235 (Dactolisib, a dual kinase inhibitor) on mTOR signalling components was assessed. mTOR showed a significant increase in the expression in endometriosis and ovarian endometrioid adenocarcinoma patients compared to non­affected controls. DEPTOR, an inhibitor of mTOR, was downregulated in the advanced stages of ovarian cancer (III and IV) compared to earlier stages (I and II). Treatment of MDAH­2774 cells with the mTOR inhibitors resulted in the significant upregulation of DEPTOR mRNA, whereas treatment with rapamycin and BEZ­235 (100 nM) resulted in downregulation of the mTOR protein expression after 48 h of treatment. None of the treatments resulted in translocation of mTOR from cytoplasm to nucleus. Upregulation of DEPTOR is a positive prognostic marker in ovarian cancer and is increased in response to mTOR pathway inhibition suggesting that it functions as a tumour suppressor gene in endometrioid ovarian carcinoma. Collectively, our data suggest the mTOR pathway as a potential connection between endometriosis and ovarian cancer and may be a potential target in the treatment of both conditions.

Identification of cancer biomarkers of prognostic value using specific gene regulatory networks (GRN): a novel role of RAD51AP1 for ovarian and lung cancers.

To date, microarray analyses have led to the discovery of numerous individual 'molecular signatures' associated with specific cancers. However, there are serious limitations for the adoption of these multi-gene signatures in the clinical environment for diagnostic or prognostic testing as studies with more power need to be carried out. This may involve larger richer cohorts and more advanced analyses. In this study, we conduct analyses-based on gene regulatory network-to reveal distinct and common biomarkers across cancer types. Using microarray data of triple-negative and medullary breast, ovarian and lung cancers applied to a combination of glasso and Bayesian networks (BNs), we derived a unique network-containing genes that are uniquely involved: small proline-rich protein 1A (SPRR1A), follistatin like 1 (FSTL1), collagen type XII alpha 1 (COL12A1) and RAD51 associated protein 1 (RAD51AP1). RAD51AP1 and FSTL1 are significantly overexpressed in ovarian cancer patients but only RAD51AP1 is upregulated in lung cancer patients compared with healthy controls. The upregulation of RAD51AP1 was mirrored in the bloods of both ovarian and lung cancer patients, and Kaplan-Meier (KM) plots predicted poorer overall survival (OS) in patients with high expression of RAD51AP1. Suppression of RAD51AP1 by RNA interference reduced cell proliferation in vitro in ovarian (SKOV3) and lung (A549) cancer cells. This effect appears to be modulated by a decrease in the expression of mTOR-related genes and pro-metastatic candidate genes. Our data describe how an initial in silico approach can generate novel biomarkers that could potentially support current clinical practice and improve long-term outcomes.