Search

The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancer, which remains the leading cause of death from gynecologic cancer. Several studies have shown that the antiapoptotic proteins Bcl-xL and Mcl-1, as well as the proapoptotic protein Bim, are key elements to be modulated to kill ovarian cancer cells. Pharmacologic inhibition of Bcl-xL is possible by using BH3-mimetic molecules like ABT-737. However, inhibition of Mcl-1 and/or promotion of its BH3-only partners (including Bim, Puma, and Noxa) remains a challenge that may be achieved by modulating the signaling pathways upstream. This study sought whether AZD8055-induced mTOR inhibition and/or trametinib-induced MEK inhibition could modulate Mcl-1 and its partners to decrease the Mcl-1/BH3-only ratio and thus sensitize various ovarian cancer cell lines to ABT-737. AZD8055 treatment inhibited Mcl-1 and increased Puma expression but did not induce massive apoptosis in combination with ABT-737. In contrast, trametinib, which decreased the Mcl-1/BH3-only protein ratio by upregulating Puma and dephosphorylated active Bim, sensitized IGROV1-R10 and OVCAR3 cells to ABT-737. Adding AZD8055 to trametinib further reduced the Mcl-1/BH3-only protein ratio and triggered apoptosis without ABT-737 in IGROV1-R10 cells. Moreover, the AZD8055/trametinib association highly sensitized all cell lines including SKOV3 to ABT-737, the induced dephosphorylated Bim being crucial in this sensitization. Finally, the three-drug combination was also very efficient when replacing AZD8055 by the pan-Akt inhibitor MK-2206. This study thus proposes original multitargeted strategies and may have important implications for the design of novel approaches for ovarian cancer treatment. Mol Cancer Ther; 16(1); 102–15. ©2016 AACR.

The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancers, which remain the leading cause of death from gynecologic cancer. Our previous studies have shown that the anti-apoptotic proteins Bcl-xL and Mcl-1 cooperate to protect resistant ovarian cancer cells from apoptosis as their concomitant inhibition results in massive apoptotic cell death. Moreover, their BH3-only pro-apoptotic partners Bim and Puma are crucial actors in the induced cell death. This suggests that ovarian cancer cell apoptosis can be triggered by inhibiting Bcl-xL and Mcl-1 and/or by promoting their BH3-only partners, especially Bim and Puma. The expression of these Bcl-2 family proteins has been reported to be modulated by HDAC (histone deacetylases) inhibitors in various cancer models. The objective of the present study was to evaluate the efficacy of the FDA-approved pan-HDAC inhibitor belinostat (i) to reduce the [Bcl-xL and Mcl-1] / BH3-only proteins ratio and (ii) to trigger apoptosis in chemoresistant ovarian cancer cell lines, alone or in combination with other pharmacological molecules that modulate these Bcl-2 family proteins. HDAC inhibition by belinostat led to a G2/M blockade and consequently reduced cell proliferation. Moreover, belinostat used at high concentrations (from 1000 to 2000 nM) efficiently triggered apoptosis in chemoresistant ovarian cancer cells. This cytotoxic effect was associated with both an up-regulation of Bim and Puma protein expression and an inhibition of Bcl-xL protein expression. Used at sub-toxic concentrations, belinostat also up-regulated Bim and Puma proteins but failed to down-regulate Bcl-xL and Mcl-1 protein expression. We therefore investigated whether Bcl-xL or Mcl-1 inhibition could sensitize ovarian cancer cells to sub-toxic concentrations of belinostat. Interestingly, the combination of belinostat with the BH3-mimetic ABT-737, that inhibits Bcl-xL, induced massive apoptosis. Moreover, Mcl-1 direct or indirect inhibition was also very efficient to sensitize ovarian cancer cells to belinostat. These results suggest that belinostat, alone or in association with pharmacological molecules that inhibit Mcl-1 or Bcl-xL, represents a promising approach for the treatment of chemoresistant ovarian cancers. Citation Format: Charlène Duboc, Edwige Abeilard, Anne Sophie Voisin-Chiret, Pascal Gauduchon, Laurent Poulain, Marie Villedieu. The sensitivity of ovarian cancer cells to the HDAC inhibitor belinostat is improved by inhibiting Bcl-xL or Mcl-1 anti-apoptotic proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1921.

Objective. Emergence of chemoresistance in the course of treatments with platinum drugs remains a major hurdle to ovarian carcinoma therapy. We have previously shown that acquisition of cisplatin resistance by OAW42-R ovarian carcinoma cells was associated with the loss of ERK activation in response to cisplatin. To try to sensitize this cell line by restoring ERK activation, we tested a new synthetic compound, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE), which was described to induce ERK activation and to display anticancer properties. Methods. We treated four ovarian carcinoma cell lines with 2,3-DCPE, alone or combined with cisplatin. We characterized its effects on apoptosis induction and proliferation and correlated them with molecular modulations. Results. We showed that 2,3-DCPE induced cell death and ERK phosphorylation in a time- and concentration-dependent manner in OAW42-R cells. 2,3-DCPE-triggered apoptosis was also associated with the inhibition of Bcl-2 expression and, to a less extent, with that of Bcl-x L . Treatment with 2,3-DCPE also elicited a strong G0/G1 cell cycle arrest, accompanied with p21 WAF1/CIP1 up-regulation. All of these effects revealed to be irreversible. Moreover, 2,3-DCPE exerted a cytostatic effect on OAW42, IGROV1-R10 and SKOV3 ovarian carcinoma cells, the sensitivity to 2,3-DCPE appearing in particular linked with a low basal level of P-ERK. Finally, we showed that 2,3-DCPE increased the cytotoxic effect of cisplatin in OAW42-R resistant cells. Conclusion. Our results emphasized the potential interest of 2,3-DCPE, used alone or combined with cisplatin, for ovarian carcinoma treatment. The absence of basal P-ERK may constitute a predictive marker of response to this novel therapy.

Objective. Recurrence and cisplatin resistance that progressively develops in the course of treatments are major impediments in ovarian cancer therapy. We investigated the involvement of alterations of different signaling pathways in this acquired chemoresistance. Methods. We studied the activation of these pathways in a model of progressive acquisition of resistance that we established, by discontinuously exposing a sensitive ovarian carcinoma cell line, OAW42, to increasing concentrations of cisplatin. Results. OAW42-T1 and -T2 variants, which emerged after the first two treatments of OAW42 cells with 5 μg/ml cisplatin, showed enhanced but transient resistance. OAW42-R cells, obtained following successive reiterations of the treatment, displayed both a stronger resistance to cisplatin-induced apoptosis and an increased capacity to recover a normal proliferation after treatment. The measurement of DNA adducts demonstrated that the mechanisms leading to a decreased DNA platination could not explain the level of resistance of OAW42-R cells. The simultaneous study of activation pattern of key proteins of different signaling pathways revealed that cisplatin induced both activation of ERK and p38 and inhibition of P-FAK in the sensitive cells, whereas it progressively failed to elicit such a response in the resistant variants. In contrast, STAT3 and Akt did not seem to be involved in the acquired chemoresistance in our model. Conclusions. Our results suggested that emergence of chemoresistance was accompanied with the progressive loss of ERK and p38 activation and with the maintenance of FAK activation in response to cisplatin, and they demonstrated that these alterations were early events in the course of treatments.

We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Here we assessed the anticancer potential of combining ABT-737-induced inhibition of Bcl-xL with Mcl-1 inhibition via PI3K/Akt/mTOR pathway disruption using NVP-BEZ235. NVP-BEZ235 inhibited cell proliferation without inducing apoptosis. It strongly repressed Mcl-1 expression and induced Puma expression in both cell lines tested while differentially modulating Bim between the two. Interestingly, NVP-BEZ235 efficiently sensitized ovarian carcinoma cells to ABT-737, provided that Bim expression was induced. Moreover, inhibiting the ERK1/2 pathway restored Bim expression and sensitized low Bim-expressing cancer cells to the BEZ235/ABT-737 treatment.

Acquisition of resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of estrogen receptor alpha (ERα)-positive breast cancers. The Res-Ana cells, a new model of acquired resistance to anastrozole, were established by long-term exposure of aromatase-overexpressing MCF-7 cells to this drug. These resistant cells developed ER-independent mechanisms of resistance and decreased sensitivity to the AI letrozole or to ERα antagonists. They also displayed a constitutive activation of the PI3K/Akt/mTOR pathway and a deregulated expression of several ErbB receptors. An observed increase in the phospho-Akt/Akt ratio between primary and matched recurrent breast tumors of patients who relapsed under anastrozole adjuvant therapy also argued for a pivotal role of the Akt pathway in acquired resistance to anastrozole. Ectopic overexpression of constitutively active Akt1 in control cells was sufficient to induce de novo resistance to anastrozole. Strikingly, combining anastrozole with the highly selective and allosteric Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings lead to us proposing a model of anastrozole-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to anastrozole and sensitivity to MK-2206. Altogether, our work demonstrated that the Akt/mTOR pathway plays a key role in resistance to anastrozole and that combining anastrozole with Akt/mTOR pathway inhibitors represents a promising strategy in the clinical management of hormone-dependent breast cancer patients.

Background ZNF217 is a candidate oncogene located at 20q13, a chromosomal region frequently amplified in breast cancers. The precise mechanisms involved in ZNF217 pro-survival function are currently unknown, and utmost importance is given to deciphering the role of ZNF217 in cancer therapy response. Results We provide evidence that stable overexpression of ZNF217 in MDA-MB-231 breast cancer cells conferred resistance to paclitaxel, stimulated cell proliferation in vitro associated with aberrant expression of several cyclins, and increased tumor growth in mouse xenograft models. Conversely, siRNA-mediated silencing of ZNF217 expression in MCF7 breast cancer cells, which possess high endogenous levels of ZNF217, led to decreased cell proliferation and increased sensitivity to paclitaxel. The paclitaxel resistance developed by ZNF217-overexpressing MDA-MB-231 cells was not mediated by the ABCB1/PgP transporter. However, ZNF217 was able to counteract the apoptotic signals mediated by paclitaxel as a consequence of alterations in the intrinsic apoptotic pathway through constitutive deregulation of the balance of Bcl-2 family proteins. Interestingly, ZNF217 expression levels were correlated with the oncogenic kinase Aurora-A expression levels, as ZNF217 overexpression led to increased expression of the Aurora-A protein, whereas ZNF217 silencing was associated with low Aurora-A expression levels. We showed that a potent Aurora-A kinase inhibitor was able to reverse paclitaxel resistance in the ZNF217-overexpressing cells. Conclusion Altogether, these data suggest that ZNF217 might play an important role in breast neoplastic progression and chemoresistance, and that Aurora-A might be involved in ZNF217-mediated effects.

In ovarian carcinomas, recurrence and acquired chemoresistance are the first leading causes of therapeutic failure and are responsible for the poor overall survival rate. Cisplatin exposure of sensitive cells has been previously associated with a down-regulation of Bcl-X(L) expression and apoptosis, whereas recurrence was systematically observed when Bcl-X(L) expression was maintained. Bcl-X(L) down-regulation could thus constitute an interesting chemosensitizing strategy. We showed that a Bcl-X(L)targeted RNA interference strategy efficiently sensitized chemoresistant ovarian carcinoma cells to cisplatin, but some of them were still able to re-proliferate. Considering the possible cooperation between Bcl-X(L)and MCL-1, we investigated the possibility to avoid recurrence in vitro using a multi-targeted RNAi strategy directed against these two anti-apoptotic proteins. We showed that their concomitant inhibition lead to massive apoptosis in absence of cisplatin, this multi-targeted RNAi approach being much more efficient than conventional chemotherapy. We thus demonstrated that Bcl-X(L) and MCL-1 cooperate to constitute together a strong molecular "bolt", which elimination could be sufficient to allow chemoresistant ovarian carcinoma cells apoptosis. Moreover, we demonstrated that in presence of a low concentration of cisplatin, the concomitant down-regulation of Bcl-X(L) and MCL-1 allowed a complete annihilation of tumour cells population thus avoiding subsequent recurrence in vitro in cell lines highly refractory to any type of conventional chemotherapy. Therefore, Bcl-X(L) and MCL-1 targeted strategies could constitute an efficient therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in association with conventional chemotherapy.

Objective Recurrence and subsequent acquired chemoresistance to platinum-based treatments constitute major hurdles to ovarian carcinoma therapy. Our objective was to examine the involvement of Bcl-x L anti-apoptotic protein in resistance to cisplatin. Methods We described the effect of cisplatin on cell cycle and apoptosis induction in sensitive (IGROV1 and OAW42) and resistant (IGROV1-R10 and SKOV3) ovarian carcinoma cell lines. We correlated it with Bcl-x L mRNA and protein expression after exposure to cisplatin. We then used bcl - x S gene transfer to impede Bcl-x L activity. Results Our study showed that Bcl-x L basal expression was high in both sensitive and resistant cell lines, as well as in all the studied ovarian tumor samples. Thus, Bcl-x L basal expression could not allow to predict sensitivity. Wondering whether variation of Bcl-x L level in response to cisplatin could be a better determinant of sensitivity, we investigated the expression of this protein in the cell lines after treatment. Cisplatin-induced down-regulation of Bcl-x L was strictly associated with apoptosis and absence of recurrence in vitro. Conversely, the maintenance of Bcl-x L expression in response to cisplatin appeared as a sine qua non condition to escape to treatment. To try to sensitize SKOV3 cells by impeding anti-apoptotic activity of Bcl-x L , we transfected bcl-x S gene in these cells. Bcl-x S exogenous expression was only slightly cytotoxic on its own, but highly sensitized SKOV3 resistant cells to cisplatin-induced apoptosis, and delayed recurrence. Conclusion This work thus provides one more argument to put Bcl-x L forward as a pertinent target of inhibition to overcome chemoresistance of epithelial ovarian carcinoma.