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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.

SUBMITTER: Rabelo-Fernandez RJ

PROVIDER: S-EPMC8745614 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of family proteins that bind to nascent RNA transcripts and regulate their splicing, localization, and stability. Evidence indicates that RBPMS controls the activity of transcription factors associated with cell growth and proliferation, including AP-1 and Smads. Three major RBPMS protein splice variants (RBPMSA, RBPMSB, and RBPMSC) have been described in the literature. We previously reported that reduced RBPMS levels decreased the sensitivity of ovarian cancer cells to cisplatin treatment. However, little is known about the biological role of the RBPMS splice variants in ovarian cancer cells. We performed RT-PCR and Western blots and observed that both RBPMSA and RBPMSC are reduced at the mRNA and protein levels in cisplatin resistant as compared with cisplatin sensitive ovarian cancer cells. The mRNA and protein levels of RBPMSB were not detectable in any of the ovarian cancer cells tested. To better understand the biological role of each RBPMSA and RBPMSC, we transfected these two splice variants in the A2780CP20 and OVCAR3CIS cisplatin resistant ovarian cancer cells and performed cell proliferation, cell migration, and invasion assays. Compared with control clones, a significant reduction in the number of colonies, colony size, cell migration, and invasion was observed with RBPMSA and RBPMSC overexpressed cells. Moreover, A2780CP20-RBPMSA and A2780CP20-RBPMSC clones showed reduced senescence-associated β-galactosidase (β-Gal)-levels when compared with control clones. A2780CP20-RBPMSA clones were more sensitive to cisplatin treatment as compared with A2780CP20-RBPMSC clones. The A2780CP20-RBPMSA and A2780CP20-RBPMSC clones subcutaneously injected into athymic nude mice formed smaller tumors as compared with A2780CP20-EV control group. Additionally, immunohistochemical analysis showed lower proliferation (Ki67) and angiogenesis (CD31) staining in tissue sections of A2780CP20-RBPMSA and A2780CP20-RBPMSC tumors compared with controls. RNAseq studies revealed many common RNA transcripts altered in A2780CP20-RBPMSA and A2780CP20-RBPMSC clones. Unique RNA transcripts deregulated by each RBPMS variant were also observed. Kaplan-Meier (KM) plotter database information identified clinically relevant RBPMSA and RBPMSC downstream effectors. These studies suggest that increased levels of RBPMSA and RBPMSC reduce cell proliferation in ovarian cancer cells. However, only RBPMSA expression levels were associated with the sensitivity of ovarian cancer cells to cisplatin treatment.

Project description:Aquaporins (AQPs) are important mediators of water permeability and are closely associated with tumor cell proliferation, migration, angiogenesis and chemoresistance. Moreover, the chemosensitivity of tumor cells to cisplatin (CDDP) is potentially affected by osmotic pressure. The present study was undertaken to determine whether hyperosmosis regulates ovarian cancer cell sensitivity to CDDP in vitro and to explore whether this is associated with AQP expression. The hyperosmotic stress was induced by D-sorbitol. 3AO ovarian cancer cells were treated with different concentrations of hypertonic medium and/or CDDP for various times, followed by measuring the inhibition rate of cell proliferation using an MTT assay. In addition, AQP expression in response to osmotic pressure and/or CDDP was measured by reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation in response to hypertonic stress was also measured when AQP5 was knocked down by small interfering (si)RNA. 3AO cell proliferation was inhibited by hyperosmotic stress, while the expression of AQP5, but not that of AQP1, AQP3 or AQP9, was increased in a dose- and time-dependent manner in hypertonic sorbitol-containing medium. When AQP5 was silenced by siRNA, cells were susceptible to hypertonic stress. MTT analyses showed that the inhibition of cell proliferation by a low dose of CDDP increased significantly with exposure to a hyperosmotic stimulus, and this effect was reduced when a high dose of CDDP was used. AQP5 expression was induced by a low dose of CDDP, but was reduced by a high dose of CDDP. However, hyperosmosis enhanced AQP5 mRNA expression at every dose of CDDP tested, compared with isotonic medium. With prolonged treatment time, AQP5 expression was reduced by CDDP in hypertonic and isotonic culture medium. Thus, the effects of hyperosmosis on cell sensitivity to CDDP were associated with AQP5 expression. These results suggest that AQP5 expression in ovarian cancer cells is induced by hypertonic medium, and that the sensitivity of ovarian cancer cells to CDDP can be regulated by hyperosmosis associated with AQP5 expression.

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

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