Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells

Abstract

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High-grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR-433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR-433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p-Rb), and an increase in β-galactosidase activity. Furthermore, in silico analysis identified four possible miR-433 target genes associated with cellular senescence: cyclin-dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene. Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR-433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

Keywords: CDK6; chemoresistance; miR-433; ovarian cancer; senescence.

Figure 1

Stable expression of miR-433 in A2780 cells attenuates cellular apoptosis and results in a distinct morphological and proliferative change. (A) Quantitative real-time PCR (qRT-PCR) values for miR-433 in the miR-433-stable A2780 cells compared to miR-433-stable cells showing a significant (P < 0.05) fold increase in miR-433 levels. (B) Western blot analysis showing downregulation of two miR-433 targets, MAD2 and HDAC6 in the A2780 miR-433-stable expressing cell line. (C) Histogram representation of the response of the stable miR-433 versus control-miR cell lines post 24 h paclitaxel (10, 25, 50 nmol/L) treatment. Student's t-test was used for the comparison of means and demonstrates an increased resistance of the miR-433-stable cell line compared to controls at 25 and 50 nmol/L paclitaxel (P < 0.05). (D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage in the miR-433-stable and miR-control-stable cell line treated with 50 nmol/L paclitaxel for 24 h demonstrating a decreased PARP cleavage in the miR-433-stable expressing A2780 cells. (E) Increased miR-433 expression influences the cellular morphology of A2780 cells appreciated by green fluorescent protein (GFP) expression with a resultant flattened and enlarged cellular morphology. Magnification 20×. (F) Colony forming assay of miR-control and miR-433 stable tranfected A2780 cells stained with crystal violet (magnification 4×) demonstrating a significantly lower colony formation ability in the miR-433 stable transfectants. Error bars represent SEM. *P < 0.05, ***P < 0.001.

Figure 2

Bioinformatic analysis highlights miR-433 potential influence on cellular senescence. (A) Venn diagram showing overlapping genes (CDK6, MAPK14, CDKN2B, and E2F3) between senescence-associated genes and miR-433 target genes. (B) Overview of the interaction between miR-433 target genes (blue nodes) and senescence-associated genes (blue nodes), overlapping nodes are colored yellow. (C) Focus on overlapping genes (yellow nodes) and first degree neighbors in both the senescence-associated genes (green nodes) or predicted miR-433 targets (blue nodes). Blue edges indicate experimentally validated protein–protein interactions, magenta edges indicate predicted interactions with miR-433 (red node).

Figure 3

miR-433 stable expression induces cellular senescence. (A) Western blot analysis for p-Rb, p21, and p16 in miR-433-stable and miR-control-stable cell lines showing downregulation of p-RB with no demonstrable upregulation of p16 and p21. (B) Western blot analysis for β-galactosidase activity in miR-433-stable and miR-control-stable cell lines demonstrating an increase in β-galactosidase activity in miR-433 cells. (C) Senescence-dependent β-galactosidase activity staining in the miR-433-stable and miR-control-stable cell lines showing significant upregulation of positively stained cells in miR-433 cell line. (D) qRT-PCR determination of the baseline levels of miR-433 in the A2780, PEO1, and PEO4 ovarian cancer cell lines. (E) Western blot analysis of the baseline levels of expression of p-Rb, p21, and p16 in the A2780, PEO1, and PEO4 ovarian cancer cell lines showing a demonstrable decrease in p-Rb expression in the PEO1 and PEO4 paired cell line independent of p21 and p16 compared to A2780 cells. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (F and G) Analysis of cytokine secretion of interleukin-6 (IL-6), and interleukin-8 (IL-8) in the growth media of A2780, PEO1, and PEO4 cells, showing significant increases in IL-6 in PEO1 and PEO4 cells and IL-8 in PEO1 cells when compared to miR-433 negative A2780 cells. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

miR-433 induces senescence by targeting CDK6. (A) Schematic representation showing the published evidence of the phosphorylation of Rb by three independent cyclin-dependent kinase (CDK)/Cyclin complexes . (B) Western blot analysis for CDK6 expression in the miR-433-stable line demonstrating downregulation of CDK6. (C) Western blot analysis for CDK6 expression in the clonal derivative miR-433-stable line demonstrating downregulation of CDK6. (D) Western blot analysis for CDK6 reexpression in PEO1 cells transfected with anti-miR-control and anti-miR-433 for 96 h demonstrating an upregulation of CDK6.

Figure 5

High miR-433 endogenous expression attenuates apoptosis with a resultant chemotherapy-induced senescence allowing cells to survive chemotherapy. (A) MTT viability assay demonstrating the % of growth inhibition of the A2780, PEO1, and PEO4 cells treated with paclitaxel (10, 25, and 50 nmol/L) for 48 h, demonstrating an increased resistance to paclitaxel treatment in PEO1 and PEO4 cells. (B) qRT-PCR analysis of miR-433 expression in PEO1 and PEO4 cells either untreated or treated with 50 nmol/L paclitaxel for 72 h, washed with PBS, and then cultured for 8 days in full growth medium showing an increased expression in cells treated with paclitaxel comparing to initial expression in untreated cells. Error bars represent SEM. *P < 0.05, **P < 0.01.

Figure 6

miR-433 expression has the potential to induce cellular senescence in the tumor microenvironment. (A) MTT viability assay demonstrating a decreased viability in miR-control-stable cells treated with growth conditioned medium (GCM) from stable miR-433 expressing cells compared to being cultured in control medium. (B) Western blot analysis of the expression of senescent markers of A2780 cells treated with GCM from A2780, PEO1, or PEO4 cells demonstrating a marked reduction in p-RB and Ki67 when A2780 cells are grown in conditioned medium from PEO4 cell line known to express the highest baseline levels of miR-433 (Fig.3C). (C) Relative miR-433 expression in exosomes derived from GCM harvested from A2780, PEO1, and PEO4 cells was assessed by TaqMan® qRT-PCR using the comparative C_T (ΔΔC_T) method. miR-433 expression was present in the exosomes derived from all cell lines. (D) Fluorescent micrograph showing a successful incorporation of Dil-labeled vesicles from A2780, PEO1, or PEO4 GCM (shown in red) inside the recipient A2780 cells, counterstained with DAPI nuclear staining (shown in blue) and microtubules with Alexa Fluor 488 α-tubulin staining (shown in green). Error bars represent SEM. *P < 0.05.