Elucidation of SERPINB3, SERPINB11, GAL11, SPP1, and A2M as Prognostic Biomarkers for Epithelial-Derived Ovarian Cancer

Abstract

Elucidation of SERPINB3, SERPINB11, GAL11, SPP1, and A2M as Prognostic Biomarkers for Epithelial-Derived Ovarian Cancer

Whasun Lim WCU Biomodulation Major Department of Agricultural Biotechnology The Graduate School Seoul National University

Ovarian cancer is the most fatal gynecological malignancy leading to cancer-related deaths in women worldwide. Diagnosis of ovarian cancer at an early stage when 90% of patients can be cured is very difficult due to lack of symptoms and early detection markers. Therefore, in most of patients, this disease is detected at an advanced stage (Stage Ⅲ-Ⅳ) which results in a low survival rate (< 30%). More than 90% of ovarian carcinomas originate from ovarian surface epithelial cells

therefore, it is called epithelial-derived ovarian cancer (EOC). The risk of EOC increases with incessant ovulation involving rupture and repair of the surface epithelium of ovaries. For investigation with animal models of EOC, laying hens are the most relevant animal model because they spontaneously develop EOC as occurs in women through ovulating almost every day. As in women, EOC in the hen is age-related and grossly and histologically similar to that in women. Recently studies have shown that ovarian cancer could arise from epithelium from the oviduct as oviduct-related genes are up-regulated in EOC of hens. Therefore, the objectives of this study were to determine: 1) the distribution and localization of developmentally-regulated genes in the oviduct including serpin peptidase inhibior, clade B (ovalbumin), member 3 (SERPINB3), SERPINB11, gallicin 11 (GAL11), secreted phosphoprotein 1 (SPP1) and alpha 2 macroglobulin (A2M) in normal and cancerous ovaries of laying hens

2) the expression pattern of target genes among normal and cancer cells of ovaries from laying hens and human ovarian cancer cell lines

and 3) the functional role of target genes in human EOC.

First, we investigated the functional role of SERPINB3 gene associated with programmed cell death and immune responses related to human EOC using the laying hen animal model. Of 136 laying hens studied, EOC was found in 10 (7.4%). SERPINB3 mRNA was induced in cancerous, but not normal ovaries of laying hens (P < 0.01), and it was abundant only in the glandular epithelium (GE) of cancerous ovaries. Further, several microRNAs, specifically miR-101, miR-1668 and miR-1681 were discovered to influence SERPINB3 expression via its 3′-UTR which suggests that post-transcriptional regulation influences SERPINB3 expression in chickens. SERPINB3 protein was localized predominantly to the nucleus of glandular epithelium in cancerous ovaries of laying hens, and it was also abundant in the nucleus of both chicken and human ovarian cancer cell lines. Of 109 human patients with EOC, 15 (13.8%), 66 (60.6%) and 28 (25.7%) had ovaries with weak, moderate and strong expression of SERPINB3 protein, respectively. Strong expression of SERPINB3 protein was a prognostic factor for platinum resistance (adjusted OR

odd ratio, 5.94

95% Confidence Limits, 1.21-29.15), and for poor progression-free survival (PFS

adjusted HR

hazard ratio, 2.07

95% CI

confidence interval, 1.03-4.41).

Secondly, we identified SERPINB11 as a novel carcinogenesis-related gene in cancerous ovaries of laying hens that had similar characteristics to that detected in women with EOC. SERPINB11 was most abundant in the GE of endometrioid adenocarcinoma of cancerous, but not normal, ovaries of laying hens. In addition, bisulfite sequencing revealed that about 30% of -110 CpG sites are methylated in ovarian cancer cells, whereas those -110 CpG sites are demethylated in normal ovarian cells. On the other hand, in human ovarian cancer cells such as OVCAR-3, SKOV-3 and PA-1 cells, immunoreactive SERPINB11 protein was predominantly in the cytoplasm and had a similar expression pattern to that in ovarian cancer cells of laying hens.

The third candidate gene studied was GAL11 also known as avian beta-defensins (AvBDs). AvBDs are small cationic peptides having three cysteine disulphide bonds between their cysteine residues. They play essential roles in the innate immune system and also stimulate proliferation of epithelial cells and fibroblasts. Although we found the avian homolog of human beta-defensin 11 little is known about its expression in cancerous ovaries of laying hens. Results of this study determined that GAL11 is most abundant in the glandular epithelium of endometrioid-type ovarian tumors, but not normal ovaries of hens. In addition, miRNA-1615 was discovered to influence GAL11 expression via its 3'-UTR which suggests post-transcriptional regulation of GAL11 expression in chickens.

Fourth, we demonstrated expression of SPP1, a highly phosphorylated protein containing a polyaspartic acid sequence and a conserved RGD motif. SPP1 plays important roles in physiological processes such as inflammatory responses, calcification, organ development, immune cell function and carcinogenesis. Results of the present study indicate that SPP1 mRNA and protein are significantly more abundant in GE of ovarian endometrioid carcinomas, but not in other cancerous and normal ovaries of hens. Further, miRNA-140 was discovered to influence SPP1 expression via its 3-UTR which suggests post-transcriptional regulation of SPP1 expression in chickens.

Finally, we identified A2M (also known as ovostatin) that has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In this study, we determined expression patterns of A2M in normal and cancerous ovaries from laying hens. Expression of A2M was most abundant in GE of endometrioid adenocarcinomas of cancerous, but not normal ovaries of laying hens based on results from quantitative RT-PCR and in situ hybridization analyses.

Collectively, the present results indicate that expression of SERPINB3, SERPINB11, GAL11, SPP1 and A2M is clearly associated with the development of ovarian carcinogenesis. These results provide new insights into the prognostic biomarkers for epithelial-derived ovarian cancer to diagnose and to evaluate responses to therapies for treatment of EOC in humans. Therefore, target genes, especially SERPINB3, may play an important role in ovarian carcinogenesis and be a novel biomarker for predicting platinum resistance and a poor prognosis for survival in patients with EOC.

Keywords: epithelial-derived ovarian cancer, SERPINB3, SERPINB11, GAL11, SPP1, A2M

Student Number: 2010-22859