Abstract
Abstract Using transcriptome profiling analysis, we identified a gene signature in the fibroblastic stromal component of high grade serous ovarian tumor tissue. One of the most differentially expressed proteins was microfibrillar associated protein 5 (MFAP5). This study aims at validating the differential expression patterns of MFAP5 in ovarian cancer stroma, evaluating the clinical significance of stromal MFAP5 over-expression and delineating the functional role and mechanism of MFAP5 in modulating angiogenesis and tumor progression. Quantitative RT-PCR analysis was first performed on RNA isolated from microdissected normal and malignant ovarian tissue samples and immunolocalization of MFAP5 protein was performed on paraffin tissue sections (n=102) to validate differential stromal MFAP5 expression. Both demonstrated significant higher levels of MFAP5 expression in the fibroblastic stromal component of cancer tissue samples. Kaplan Meier analysis demonstrated a significant correlation between stromal MFAP5 expression and poor overall patient survival (p<0.001). Cox regression analysis showed a hazard ratio of 2.603 (p<0.001). Further correlation studies showed that MFAP5 expression correlated with CD34 positive microvessel densities, suggested a pro-angiogenic role of MFAP5. Thus, functional studies of exogenous MFAP5 on angiogenesis modulation and ovarian cancer cell growth were performed. Motility assay and tube formation assay were conducted on human microvascular endothelial cell 1 (hMEC-1) and telomerase immortalized microvascular endothelial cells (TIME). Increased motility and tube formation were observed in the presence of exogenous MFAP5 in both cell lines. In addition, both anti-αVα3 integrin antibody and cell permeant calcium chelator, BAPTA, AM, attenuated MFAP5 stimulated motility. These suggested that MFAP5 exerts its biological activity through the αVα3 integrin receptor and calcium signal transduction. The hypothesis is further supported by the successful detection of calcium influx and increased amplitude and change of distribution of traction force exerted by endothelial cells after treatment with MFAP5. In vivo, extensive CD34 positive tubular network was formed in the matrigel mixed with recombinant MFAP5 injected subcutaneously into mice when compared to the control. Co-injection of MFAP5 overexpressing ovarian fibroblasts and cancer cells subcutaneously into mice led to increased tumor size and increased number of CD34 positive microvessels within the tumor tissue. Finally, in vivo silencing of stromal MFAP5 using MFAP5 specific siRNA delivered in chitosan nanoparticle significantly decreased tumor sizes and intratumor microvessel densities. To conclude, stromal MFAP5 is a novel angiogenic factor that acts on αVα3 integrin receptor. It modulates endothelial cell behavior through calcium signaling which may lead to tumor progression and subsequent poor patient survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4270. doi:1538-7445.AM2012-4270
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