Reduced RBPMS Levels Promote Cell Proliferation and Decrease Cisplatin Sensitivity in Ovarian Cancer Cells

Abstract

Worldwide, the number of cancer-related deaths continues to increase due to the ability of cancer cells to become chemotherapy-resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front-line therapy, cisplatin. Although many mechanisms of cisplatin resistance have been proposed, the key mechanisms of such resistance remain elusive. The RNA binding protein with multiple splicing (RBPMS) binds to nascent RNA transcripts and regulates splicing, transport, localization, and stability. Evidence indicates that RBPMS also binds to protein members of the AP-1 transcription factor complex repressing its activity. Until now, little has been known about the biological function of RBPMS in ovarian cancer. Accordingly, we interrogated available Internet databases and found that ovarian cancer patients with high RBPMS levels live longer compared to patients with low RBPMS levels. Similarly, immunohistochemical (IHC) analysis in a tissue array of ovarian cancer patient samples showed that serous ovarian cancer tissues showed weaker RBPMS staining when compared with normal ovarian tissues. We generated clustered regularly interspaced short palindromic repeats (CRISPR)-mediated RBPMS knockout vectors that were stably transfected in the high-grade serous ovarian cancer cell line, OVCAR3. The knockout of RBPMS in these cells was confirmed via bioinformatics analysis, real-time PCR, and Western blot analysis. We found that the RBPMS knockout clones grew faster and had increased invasiveness than the control CRISPR clones. RBPMS knockout also reduced the sensitivity of the OVCAR3 cells to cisplatin treatment. Moreover, β-galactosidase (β-Gal) measurements showed that RBPMS knockdown induced senescence in ovarian cancer cells. We performed RNAseq in the RBPMS knockout clones and identified several downstream-RBPMS transcripts, including non-coding RNAs (ncRNAs) and protein-coding genes associated with alteration of the tumor microenvironment as well as those with oncogenic or tumor suppressor capabilities. Moreover, proteomic studies confirmed that RBPMS regulates the expression of proteins involved in cell detoxification, RNA processing, and cytoskeleton network and cell integrity. Interrogation of the Kaplan–Meier (KM) plotter database identified multiple downstream-RBPMS effectors that could be used as prognostic and response-to-therapy biomarkers in ovarian cancer. These studies suggest that RBPMS acts as a tumor suppressor gene and that lower levels of RBPMS promote the cisplatin resistance of ovarian cancer cells.

Increased Expression of the RBPMS Splice Variants Inhibits Cell Proliferation in Ovarian Cancer Cells

“…RBPMS knockout has been associated with increased invasion ability in ovarian cancer [ 17 ]. We assessed the effect of RBPMSA and RBPMSC overexpression in the migration and invasiveness potential in ovarian cancer cells.…”

“…The PCR data showed that five out of eight genes correlated well with the RNAseq data ( Supplementary Table S4 and Figure S6 ). Deregulation of most of these genes has already been associated with cancer progression, metastasis, and immune system response [ 10 , 17 , 21 , 22 , 23 ]. For example, interferon induced protein 44 (IFI44), one of the most increased transcripts upon A2780CP20-RBPMSA overexpression, has been linked to the suppression of the proliferation of human melanoma cell lines [ 24 ] as well as immune response to autoimmune disease [ 25 ].…”

“…It is speculated that each RBPMS splice variant binds and process its own group of RNAs [ 76 ]. We previously reported that CRISPR-mediated RBPMS knockdown reduced the sensitivity of ovarian cancer cells to cisplatin treatment [ 17 ]. However, the role of each RBPMS splice variant in ovarian cancer cells had not been studied previously.…”

“…Evidence indicates that RBPMS binds to the nascent RNA transcripts and regulates their processing, including the pre-mRNA splicing and the transport, localization, and stability of the RNA molecule [ 16 ]. RBPMS is also thought to bind to transcription factors to regulate gene expression [ 17 ]. For example, reports have shown that RBPMS interacts with Smad2, Smad3, and Smad4, promoting Smad-mediated transcriptional activity signaling pathways linked to cell growth, proliferation, and cell survival in vitro and in vivo [ 18 , 19 ].…”

“…In cultured cells and mouse xenograft models, RBPMS inhibited the growth and migration of breast cancer cells through its interaction with c-Fos or Smad3 [ 19 ]. Recently we published that reduced RBPMS levels increase the sensitivity of ovarian cancer cells to cisplatin treatment [ 17 ]. However, the role of each RBPMS splice variant in ovarian cancer cells has not been previously studied.…”