Application of RNA-Seq transcriptome analysis: CD151 is an Invasion/Migration target in all stages of epithelial ovarian cancer

Abstract

Background: RNA-Seq allows a theoretically unbiased analysis of both genome-wide transcription levels and mutation status of a tumor. Using this technique we sought to identify novel candidate therapeutic targets expressed in epithelial ovarian cancer (EOC).

Methods: Specifically, we sought candidate invasion/migration targets based on expression levels across all tumors, novelty of expression in EOC, and known function. RNA-Seq analysis revealed the high expression of CD151, a transmembrane protein, across all stages of EOC. Expression was confirmed at both the mRNA and protein levels using RT-PCR and immunohistochemical staining, respectively.

Results: In both EOC tumors and normal ovarian surface epithelial cells we demonstrated CD151 to be localized to the membrane and cell-cell junctions in patient-derived and established EOC cell lines. We next evaluated its role in EOC dissemination using two ovarian cancer-derived cell lines with differential levels of CD151 expression. Targeted antibody-mediated and siRNA inhibition or loss of CD151 in SKOV3 and OVCAR5 cell lines effectively inhibited their migration and invasion.

Conclusion: Taken together, these findings provide the first proof-of-principle demonstration for a next generation sequencing approach to identifying candidate therapeutic targets and reveal CD151 to play a role in EOC dissemination.

Figure 1

CD151 is expressed in EOC tumors and normal ovary surface epithelial cells. a-d) Membranous (arrows) and cytoplasmic (arrowheads) CD151 staining in early stage epithelial ovarian cancer. e-f) Primarily membranous (arrows) staining in the ovarian surface epithelial cell layer of a normal ovary. Insets in a) and e) show higher magnification and staining detail. Insets show higher maginifcation images.

Figure 2

CD151 qRT-PCR mRNA expression levels in patient-derived ascites cell lines. Patient-derived ascites cell lines, immortalized OSE, and EOC cell lines express CD151.

Figure 3

Ascites cells and EOC cell lines express CD151 in vitro. CD151 localizes to the cell membrane with specificlocalization to cell-cell junctions (white arrowheads) and cell membrane extensions (white arrows) in IOSE397 (a), IOSE527 (b), A2780 (c), OVCAR3 (d), OVCAR5 (e), and SKOV3 (f) and patient derived cells 50A (g) and 54A (h). Original magnification 60X.

Figure 4

Silencing and/or blocking of CD151 impedes SKOV3 and OVCAR5 migration and invasion. A-C) CD151 knockdown with siCD151 transfection at the mRNA (a) and protein (b-c) levels. d) Reduction of SKOV3 and OVCAR5 migration through transwell membranes in response to either siCD151 transfection or αCD151 antibody treatment. e) Reduction of SKOV3 and OVCAR5 invasion through matrigel and transwell membranes in response to either siCD151 transfection or treatment with αCD151 antibody. *p < 0.01. Results are representative of 3 separate experiments performed in triplicate.