Khurana, Ashwani

Abstract Background: Lysosomes are acidic organelles that contain numerous cathepsins and acid hydrolases. Lysosomal membrane permeabilization (LMP) and the presence of cathepsins in the cytosol triggers intrinsic apoptotic pathway through cathepsin L (CTSL) mediated degradation of critical proteins. This process is mediated by the proteolytic activation of the pro-apoptotic molecule Bid, resulting in mitochondrial outer membrane permeability (MOMP), cyt c release and then, cell death. Based on our previous study that quinacrine (QC) induces autophagy in ovarian cancer, the current study is aimed to investigate the potential role of cathepsins in QC induced LMP and MOMP in ovarian cancer. Methods: MagicRed and Mitotracker Red staining was used to assess the QC induced CTSL activation and LMP. To identify the role of specific cathepsin in QC mediated cytotoxicity, different specific cathepsin inhibitors pepstatin A, CA-074Me, Z-FY(tBU)-DMK were used and cell death was assessed by clonogenic assay, PARP cleavage and annexin v-PI staining. Using ATG5 and/or CTSL knockdown cells and pharmacological inhibition with 3-MA/chloroquine and/or CTSL overexpression clones, QC-induced flux in CTSL activation was determined using several different approaches including evaluating autophagy flux using tandem dual labeled RFP/GFP LC3BII by immunoflourescence analysis. Recombinant CTSL was used to validate identify p62 as novel substrate of CTSL. QC-induced autophagy and CTSL activation were validated by western blot analysis in HeyA8MDR xenograft tumors. Results: QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, MOMP, and mitochondrial cytochrome c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas ectopic expression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL suggesting that CTSL plays a role in QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockdown cells or with autophagy inhibitor chloroquine. Importantly we show upregulation of CTSL in QC treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome c, tBid, cleaved PARP, and caspase3. Conclusion: Taken together, the data suggests that QC-induced autophagy and CTSL upregulation promotes a positive feedback loop leading to excessive autophagic flux, LMP, MOMP to promote QC-induced cell death in ovarian cancer cells. Citation Format: Prabhu Thirusangu, Christopher L. Pathoulas, Upasana Ray, Yinan xiao, Julie Staub, Ashwani Khurana, Vijayalakshmi Shridhar. Quinacrine-induced autophagy in ovarian cancer triggers cathepsin-L mediated lysosomal/mitochondrial membrane permeabilization and cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1937.

We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

Metabolic alterations are increasingly recognized as important novel anti‐cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158‐induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co‐immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158‐mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX‐resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.

Abstract Although the majority of patients with endometrial cancer (EC) are diagnosed early when disease is confined in the uterus and prognosis is excellent, there is a subset of patients with advanced, metastatic, recurrent or persistent after surgery disease with dismal prognosis. Chemotherapy has a critical role for these patients. Carboplatin (carbo) and paclitaxel (taxol) is the standard first-line chemotherapy in EC with response rates as low as 50% and even lower for recurrent disease. Furthermore, many patients will eventually relapse with chemo-resistant disease and very poor prognosis. As the chemotherapeutic options for these patients are limited and of questionable efficacy, it is of paramount importance that novel agents are identified to increase or restore chemo-sensitization to platinum-based chemotherapy. Quinacrine (QC) is an inexpensive antimalarial drug with a predictable safety profile which recently surfaced as a promising anticancer agent thought to be associated with decreased risk of developing chemo-resistance through targeting multiple pathways simultaneously. While prior investigators have provided evidence that QC exerts strong anticancer activity against a wide range of solid tumors, there are no studies focused on QC in EC. In our study, QC exhibited strong synergism in vitro when combined with cisplatin, carbo or taxol with the highest level of synergism being observed in the most chemo-resistant EC cell line. Neither QC monotherapy (QC every other day for 3 weeks) nor standard chemotherapy (carbo+taxol for 3 cycles, on days 3, 7, 11) significantly delayed tumor growth in the mouse xenografts. Only combination (standard chemo + QC every other day during carbo+taxol treatment) and maintenance (combination regimen followed by QC every other day until end of study) significantly augmented carbo+taxol antiproliferative effect as evidenced by the significant decrease in tumor burden and delayed tumor growth in xenografts. Combination treatment was associated with a 14-day prolongation of median survival compared to carbo+taxol (68 vs. 54 days). Maintenance therapy with QC was proven superior to carbo+taxol as it resulted in long-term stabilization of disease and further prolongation of overall survival; in fact, median survival in the maintenance group was not reached as the mice were surviving at the end of study. QC alone, in combination with carbo+taxol or as maintenance was well-tolerated with no issues of weight loss compared to control mice. A yellow skin discoloration was noted during QC treatment which was entirely reversible within few days upon QC discontinuation. In conclusion, QC exhibited significant antitumor activity against EC cell lines in vitro and was successful in stabilizing disease and prolonging survival as maintenance therapy in chemo-resistant EC mouse xenografts. This data suggests that QC may be an important adjunct to standard chemotherapy for patients with recurrent EC. Citation Format: Eleftheria Kalogera, Debarshi Roy, Ashwani Khurana, Susmita Mondal, Xiaoping He, Sean C. Dowdy, Viji Shridhar. Quinacrine in endometrial cancer: repurposing an old antimalarial drug. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 261.

Abstract Resistance to chemotherapy is a major hurdle that results in the increased mortality associated with ovarian cancer (OvCa). New therapeutics that synergize with cis-carboplatin/paclitaxel in recurrent tumors will have an enormous impact in prolonging the survival of OvCa patients. Due to the enormous cost of designing and testing new drugs, we have adopted the drug repurposing approach and tested the effect of the antimalarial drug quinacrine (QC) in sensitizing drug resistant OvCa cells to carboplatin induced cytotoxicity and determined that it is autophagy dependent. We have discovered that QC, an autophagy modulator exhibited distinct anti-cancer properties in OvCa cell lines in contrast to other anti-malarial/autophagy inhibitor drugs such as hydroxyquinacrine, bafilomycin A and 3-MA. It is well established that autophagy can be a survival mechanism under stress in cancer or a death promoting phenomenon that is cell type and contextual dependent. Here we demonstrate that QC acts to promote chemotherapy induced cell death specifically more in resistant cells in an autophagy dependent manner. Our preliminary data both in vitro and in vivo indicate that QC induced autophagy downregulates p62 and upregulates cathepsin L levels to sensitize the chemoresistant cells to autophagic and caspase mediated cell death in a p53 independent manner. These unexpected findings led us to explore the mechanistic basis of QC-induced upregulation of the cell cycle inhibitor p21 in cells bearing mutant p53. We uncovered that irrespective of p53 status, QC induces p21 expression in an autophagy-dependent p62-Skp2 mediated ubiquitin pathway resulting in cell cycle arrest and cell death. Since QC is already in PhaseI Clinical trial, these studies may spur new treatment strategies to improve OvCa patient outcome and patient care. Citation Format: Debarshi Roy, Ashwani Khurana, Eleftheria Kalogera, Xuyang Wen, Susmita Mondal, Viji Shridhar. Can we repurpose anti-malarial drug quinacrine to treat ovarian cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B74.

Abstract Dysregulation of autophagy and altered metabolic pathways are frequently observed in cancer. Due to these alterations, pharmacological targeting of these two pathways simultaneously could provide a viable therapeutic option. Although the association between these two pathways is well characterized in metabolic disorders, it is not well defined in ovarian cancer (OVCA). In this regard, we found that loss of endosulfatase HSulf-1, a known putative tumor suppressor, suppresses LC3-GFP foci formation and promotes increased lipid droplet (LD) biogenesis suggesting that absence of HSulf-1 in OVCA affects both autophagy and lipid metabolism. While isogenic cells with genetic ablation of HSulf-1 (OV202Sh1/2 and TOV2223Sh1 cells) displayed LDs, the nontargeted control transduced (NTC) OV202 and TOV2223 cells had significantly less LDs. In contrast, Transmission Electron Micrographs (TEMs) showed that OV202 and TOV2223 NTC cells had significantly more autophagic vacuoles (AVs) compared to their isogenic ShRNA targeted cells. Conversely, ectopic expression of HSulf-1 in SKOV3 cells decreased the number of LDs and increased the number of AVs compared to vector transfected controls. Here we report that OV202Sh1 cells and HSulf-1 deficient OV2008 cells have increased p-cPLA2α(ser505) levels that are associated with biogenesis of large number of LDs with reduced AVs. Interestingly, pharmacological inhibition of cPLA2α with AACOCF3 in OV202Sh1 cells resulted in reduced LD biogenesis, inhibited colony formation and reduced tumorigenesis in vivo. More importantly, treatment of HSulf-1 deficient cells with HS mimetic PG545 which can compensate for loss of HSulf-1, reduced LD biogenesis, promoted autophagy and inhibited tumor growth in vivo. Collectively, these results show a critical role of HSulf-1 in regulating both autophagy and LD biogenesis in ovarian cancer. Citation Format: Debarshi Roy, Susmita Mondal, Ashwani Khurana, Xiaoping He, Edward Hammond, Keith Dredge, Viji Shridhar. Loss of HSulf-1 promotes defective autophagy and increased lipid droplet biogenesis in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2015-1135