PLA2G3-deficient OC cells showed attenuated tumorigenesis.
Immunoblot analysis showed PLA2G3 is highly expressed in several OC cells compared to normal fallopian tube epithelial FTEs 190 and 194 and is not expressed either in FTE240 [34] or normal ovarian fibroblast NOF151hTERT cells (Fig. 1A). TCGA analysis of high-grade serous subtype showed 2.26% cases with altered PLA2G3 expression (@cbioportal, Fig. S1A). To better understand the role of PLA2G3, we generated PLA2G3 knockout (KO) clones in OVCAR8 cells and two shRNA-mediated stable PLA2G3-KD clones of OVCAR5 (sh33/sh35) along with scrambled RNA (SCG) and non-targeted control (NTC) transduced cells as controls respectively. Efficient PLA2G3 downregulation in the KO and KD cells was verified by immunoblot analysis (Fig. 1B).
Clonogenic survival assays of OVCAR8-KO and OVCAR5-sh33/sh35 KD cells showed significantly reduced number of colonies compared to respective controls (Fig. 1C-D). Additionally, wound-healing assay showed significant reduced migration in both KO and KD cells compared to respective controls (Fig. 1E-F, S1B-C). We reported that group-IVA cytosolic phospholipase A2 is a critical regulator for LD biogenesis in cancer [35]. To determine whether PLA2G3 affects lipid metabolism in OC cells, we assessed LD formation by Bodipy staining and found a decrease in the number of LDs in KO and sh35/33 KD cells compared to respective controls (Fig. 1G, S1D). Consistent with these results, TEM analysis also showed a significant reduction in the numbers of LDs in the OVCAR8-KO cells compared to SCG-transfected cells (Fig. 1H). Collectively, these results suggest that PLA2G3 abrogation impairs the lipogenesis pathway and attenuates OC tumorigenesis.
PLA2G3 KD cells are sensitive to Platinum based-drug treatment.
Given that LD-rich cancer cells exhibit chemo-resistive properties, we investigated whether PLA2G3 KD sensitizes cancer cells to cisplatin/carboplatin-induced cytotoxicity. The OVCAR8 KO and SCG-control cells treated with increasing concentrations of cisplatin for 24hrs showed a significant reduction in IC50 value from 9.91 µM in SCG-transfected cells to 4.75 µM in the KO cells (Fig. 2A). Similarly, stable HeyA8MDR PLA2G3-KD cells (Fig. S2A) treated with increasing CBP doses for 24hrs showed a decrease in IC50 value to 95.4 µM compared to 170 µM in the NTC cells (Fig. S2B-C). Additionally, the KD cells showed an improved dose-dependent decrease in cell survival upon CBP treatment compared to NTC cells (Fig. S2D). Together, these results substantiate that PLA2G3 downregulation sensitizes cells to platinum-drug mediated cell death.
Likewise, to understand whether induction of autophagy is essential for sensitizing OC cells to cisplatin-induced cytotoxicity, we pretreated OVCAR8 cells with BafA1 (inhibitor of autophagolysosome formation) for 2hr followed by cisplatin treatment for 24hrs. Results showed that inhibition of autophagy diminished the cytotoxic effect of cisplatin in OC cells (IC50: 13 µM to 28 µM, Fig. 2B), which suggests that autophagy-mediated cytotoxicity is critical for sensitizing cancer cells to the platinum drug-induced cell death. To validate the role of PLA2G3 in sensitizing cells to platinum drug-induced cytotoxicity, IF analysis for GFP-LC3B puncta expression and its co-localization with lysosomal associated membrane protein 2 (RFP-LAMP2) upon cisplatin treatment in both KO and SCG-transfected OVCAR8 cells was performed. Results showed increased expression and co-localization of RFP-LAMP2 and GFP-LC3B in the KO cells compared to control upon cisplatin treatment (Fig. 2C). Likewise, Cyto-ID staining used as a read-out for autophagic induction, showed a significant increase in fluorescent signal in KO cells compared to SCG-transfected cells upon treatment with 5 µM cisplatin (Fig. 2D). Immunoblot analysis also revealed an increased expression of LC3BII and cleaved PARP1 with reduced p62/SQSTM1 levels in KO cells compared to SCG-transfected cells upon cisplatin treatment (Fig. 2E). Pretreatment for 2hr with BafA1 inhibited the cisplatin-induced autophagy with increase of the LC3BII and rescue of the p62/SQSTM1 levels respectively and a decreased induction of the cleaved PARP1 in the cells (Fig. 2E). Collectively, these results suggest that PLA2G3 KD increased sensitivity of the cancer cells to autophagy-induced cytotoxicity upon treatment with platinum drugs.
Aberrant PLA2G3 expression impairs PC formation in OC cells.
Since aberrant lipogenic signaling is associated with distortion of PC [26] we assessed whether LD deregulation due to aberrant PLA2G3 expression is involved in regulation of ciliogenesis in OC cells. Immunoblot analysis of OVCAR5-KD and OVCAR8-KO cells showed a significant increase in expression of acetylated α-tubulin (a marker for PC) compared to respective controls (Fig. 3A-B). Likewise, IF study using fluorescently tagged-acetylated α-tubulin revealed an increase in percent ciliation in OVCAR5 sh35-KD and OVCAR8-KO cells compared to controls (Fig. 3C-D respectively) with efficient downregulation of PLA2G3 under similar conditions. Together these data validate the importance of PLA2G3 in the regulation of lipid metabolic pathway and PC in OC. To determine the role of PC in OC progression, we transiently knockdown IFT88, a key factor regulating ciliogenesis, and as shown in fig. S3A, a decrease in acetylated α-tubulin in KD cells was confirmed by immunoblot. IFT88 KD cells showed a significant increase in colony forming ability and increased migration of OVCAR5 cells (Fig. S3B-C).
Knockdown of PLA2G3 inhibits in vivo tumorigenesis and metastatic spread in OVCAR5 xenograft model.
To support our in vitro findings, the effect of PLA2G3-KO alone and in combination with CBP treatment on tumor growth and metastatic spread was assessed in vivo as described (Fig. 4A). No significant alteration in health condition was observed (data not shown); however, two mice died in the control group due to unknown reasons very early on and therefore had to be excluded in the analysis. PLA2G3-KO tumor-bearing mice showed a significant reduction in tumor growth and metastatic spread compared to the SCG-control group (Fig. 4B). Interestingly, the KO tumor-bearing mice showed almost no tumor burden upon CBP treatment compared to the SCG-control cohort (Fig. 4B). Comparative statistical analysis of the tumor weight and Ki67 staining of tumor tissue sections showed a similar significant reduction in the KO model both with and without CBP treatment (Fig. 4C-D). Immunoblot analysis confirmed downregulated PLA2G3 expression and increased acetylated α-tubulin in the KO-cohort compared to SCG-derived xenografts (Fig. 4E) and an increased expression of LC3BII with p62 downregulation in CBP treated SCG-control and KO cohort of mice (Fig. 4F). Hence, our in vivo data supports the role of PLA2G3 in metastatic spread and its downregulation sensitizes cells to chemotherapy.
Targeting by PFK158 inhibitor restores PC by reducing PLA2G3 in an autophagy-dependent manner.
Although we highlighted the role of PFK158-induced autophagy in regulating lipophagy [35], it did not address if PFK158 regulated ciliation. Driven by our observations, we wondered if inhibition of lipogenic signaling by PFK158 can restore ciliation in OC cells. IF analysis with fluorescently tagged-acetylated α-tubulin, showed that PFK158 treatment significantly restored PC in both OVCAR8 and OVCAR5 cells (Fig. 5A, S4A). Quantitation of increase in percent cilia is shown in Figs. 5B-C. Immunoblot analysis of acetylated α-tubulin also showed increased expression in OVCAR8 and OVCAR5 cells upon treatment with PFK158 (Fig. 5D). To understand if PFK158-induced autophagy plays a role in induction of PC, we monitored induction of autophagic flux by GFP-RFP-LC3B transfection in PFK158 treated OVCAR5 cells. Confocal analysis showed induction of autophagic flux through the formation of increased red puncta in PFK158-treated cells compared to untreated cells (Fig. 5E). Interestingly, pretreatment with BafA1 for 2hr inhibited PFK158-induced increase of acetylated α-tubulin in OVCAR8 cells (Fig. 5F, top-panel). Also, immunoblot analysis showed PFK158-treatment attenuated PLA2G3 expression which was restored when cells were pretreated with BafA1 (Fig. 5F, middle-panel). To understand whether inhibition of autophagy regulates PC levels, we treated OVCAR8 cells with 3MA and BafA1 individually and observed that both early and late stage inhibition of autophagy downregulated acetylated α-tubulin levels (Fig. S4B). Effect of BafA1 as an inhibitor of PFK158-induced autophagy was confirmed by the resulting increases in LC3BII expression and the rescue of p62/SQSTM1 both OC cells (Fig. 5G-H; panels2-3). Under similar conditions, the PFK158-induced reduction of PLA2G3 was restored in both cells in presence of BafA1 (Fig. 5G-H; panel-1).
Consistent with these results, PFK158-induced ciliation was inhibited by BafA1 treatment as determined by levels of acetylated α-tubulin with confocal microscopy (Fig. 6A-B). Under parallel conditions, BafA1 treatment rescued LD formation that was reduced by PFK158 in OVCAR8 cells (Fig. 6C). Taken together, these results mechanistically support that PFK158-induced autophagy-mediated downregulation of PLA2G3 regulates PC in OC. By analyzing percent ciliated cells, we determined that BafA1 treatment downregulated acetylated α-tubulin in OVCAR5-sh35KD cells confirming the role of autophagy (Fig. 6D-E), which was also validated by western blot analysis, that showed a rescue of both LC3BII and p62 levels (Fig. 6F). To validate the role of autophagy in regulating ciliogenesis, we determined acetylated α-tubulin levels in WT and in autophagy compromised Atg5−/− MEFs, by IF. Atg5−/− MEFs showed reduced percent ciliated cells compared to their WT counterpart (Fig. 6G), which was also corroborated by western analysis (Fig. 6H, panel-2). Further, PFK158 treatment did not show a significant change in the expression of acetylated α-tubulin in Atg5−/− MEFs (Fig. 6I, lower panel-1). In contrast there was a significant up-regulation in WT cells (Fig. 6I, top panel-1). Together, these results show PFK158-induced autophagy that leads to PLA2G3 degradation regulates ciliary maintenance.
PFK158-mediates autophagic degradation of PLA2G3 and reduces viability in patient-derived ascites.
To understand the clinical relevance, we determined PLA2G3 expression in 9 patient-derived ascites cells [27, 28]. Immunoblot analysis with human epithelial specific antigen marker (EpCAM) and fibroblast activated protein marker (FAP) showed that the ascitic cells are predominantly epithelial in nature (Fig. S5A) and 5 out of 9 samples expressed PLA2G3 (Fig. 7A-B). A reduction in percent viability was observed in PLA2G3-expressing ascitic cells following PFK158 (0–20 µM) treatment at 24hr with IC50 values ranging between 4.0–9.0 µM (Fig. 7C-D). Immunoblot analysis showed PFK158-induced autophagy, as determined by an increase in LC3BII, decreased p62 levels, and downregulation of PLA2G3 respectively (Fig. 7E-F). Further, IF analysis showed significant increase in percent ciliated cells upon PFK158-treatment in the A7683, KP263 and A4832 ascitic cells model compared to untreated control (Fig. 7G-I, S5B). When cisplatin was combined with 1/2IC50 of PFK158, a substantial reduction in IC50 ranging from 29 µM to 7 µM (AM812), 37.5 µM to 8.5 µM (KP263) and 33 µM to 21 µM (JM076; Fig. 7J-L) was observed, suggestive of the ability of PFK158 to sensitize the cells to chemotherapy. Together PFK158-treatment sensitizes the patient-derived OC ascites to chemotherapy at least in part through the degradation of PLA2G3.