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Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway

Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of... Hindawi Journal of Oncology Volume 2019, Article ID 2343876, 15 pages https://doi.org/10.1155/2019/2343876 Research Article Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway 1 1 1,2 Arpita Kulshrestha , Gajendra K. Katara, Safaa A. Ibrahim, 1 1 3 3 Valerie Riehl, Manoranjan Sahoo, James Dolan, Kyle W. Meinke, 4 1 Michael R. Pins, and Kenneth D. Beaman Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Egypt Department of Obstetrics & Gynecology, Advocate Lutheran General Hospital, Park Ridge, IL, USA Department of Pathology, Advocate Lutheran General Hospital, Park Ridge, IL, USA Correspondence should be addressed to Kenneth D. Beaman; kenneth.beaman@rosalindfranklin.edu Received 1 August 2018; Revised 28 January 2019; Accepted 4 March 2019; Published 1 April 2019 Academic Editor: Srikumar P. Chellappan Copyright © 2019 Arpita Kulshrestha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ovarian cancer (OVCA) patients oen ft develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase ‘a2’ isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible f actor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. eTh apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. ese Th cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2- cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy u fl x. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V- ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer. 1. Introduction platinum-based drugs [1]. In addition to apoptosis inhibition, cisplatin resistant cancer cells rely on mechanisms such Ovarian cancer (OVCA) is hard to treat as it exhibits refrac- as reduced drug uptake, increased drug efflux, enhanced toriness to standard chemotherapy approaches including DNA-repair, and defective signaling pathways to survive 2 Journal of Oncology therapeutic cell death [2]. Nevertheless, an understanding Briefly, A2780 and cis-A2780 cells were cultured in RPMI of the precise molecular mechanism of chemoresistance will 1640 medium (Invitrogen, Carlsbad, CA) supplemented with help design strategies to improve the treatment outcome in 10% (v/v) heat-inactivated fetal bovine serum (Biowest LLC, OVCA patients. MO,USA), 100U/ml penicillin, and 100U/ml strepto- Exposure of cancer cells to cisplatin elicits a stress mycin (Sigma–Aldrich) at 37 C, 5% CO2. TOV112D cell response which induces coping mechanisms that favor can- line (American Type Culture Collection [ATCC], Manas- cer cell survival [3]. Autophagy is the primary protective sas, VA) was cultured in CTOV medium [1:1 mixture of process that enables energy supply during stress such as MCDB 105 medium containing a n fi al concentration of chemotherapy exposure and nutrient depletion [4–6]. The 1.5 g/L sodium bicarbonate and medium 199 containing self-degradative pathway of autophagy involves the forma- a n fi al concentration of 2.2 g/L sodium bicarbonate at tion of double-membrane vesicles (autophagosomes) around 37 C, 5% CO2]. The cells were routinely grown until reach- damaged cellular proteins and organelles [7, 8]. Autophago- ing 80% confluency and then subcultured or plated for somes fuse to endo-lysosomal machinery where sequestered experiments. cellular components are ultimately digested for energy recy- .. Generation of Stable V-ATPase-Va Knockdown Cells. cling [9]. In addition to lysosomal machinery, recent studies suggest the importance of early endosomes in autophagy [10]. The shRNA mediated V-ATPase-V0a2 knockdown was per- It is therefore important to understand how molecular targets formed as described previously [18]. Brieyfl , the cisplatin resistant cells (cis-A2780) were plated overnight and then involved in endosomal machinery can modulate autophagy process. transfected with V0a2 shRNA constructs (Suresilencing Plas- A tightly regulated intracellular pH is critical for mid, Qiagen, Valencia, CA, USA) or a scrambled control autophagy [11]. In mammalian cells, vacuolar ATPase (V- shRNA using the Attractene transfection reagent (Qiagen). ATPase) proton pumps are the primary pH regulators that The cells were treated with the selection antibiotic (1 mg/ml G418) after 24 h after transfection. Medium containing G418 maintain intravesicular and/or extracellular pH. In normal cells, V-ATPases pump protons from the cytoplasm to the was replenished every 72 h. After confirming the knockdown lumen of the acidic organelles [9]. In cancer cells, plasma by Q-RT PCR, the positive transfectants were cloned and used for further experiments. membrane-associated V-ATPases extrude protons and acid- ify the extracellular matrix [12, 13]. V-ATPase inhibition disrupts tumor pH gradients that alters drug retention and .. Drugs. Anticancer drug cisplatin was obtained from Sigma–Aldrich. Cisplatin 1mM stock was prepared in normal trackin ffi g in tumor cells. Many proton pump/V-ATPase saline (0.9% NaCl) and stored as aliquots at -20 inhibitors are showing efficacy in increasing the sensitivity Cup to 3 of tumor cells to cytotoxic agents [14–16]. Unlike chemi- months. V-ATPase inhibitor balfi omycin A (Sigma–Aldrich, M17931) was dissolved in DMSO at a 100𝜇 M stock solution. cal inhibitors, targeting cancer specific V-ATPase isoforms will modulate autophagy and will potentially decrease the Autophagy modulators rapamycin and chloroquine were procured from Enzo Life Sciences, USA. Rapamycin was associated toxicity to normal cells. Our previous work dissolved in DMSO at a 500𝜇 M stock solution. Chloroquine highlighted that, in OVCA cells, ‘a2’ isoform (V-ATPase- V0a2) is overexpressed in cisplatin resistant cells and is a was dissolved in deionized water for a 60 mM stock solution. For long term storage, all stock solutions were stored at -20 C. component of plasma-membrane V-ATPase and the early Selective MEK inhibitor cobimetinib (10mM in DMSO) was endosomal machinery [17, 18]. Inhibition of V-ATPase-V0a2 acidified the cytosol thereby sensitizing the resistant OVCA obtained from ApexBio and the stock solutions were stored at -20 C. cells to platinum mediated DNA damage [18]. However, it is not known how V-ATPase-V0a2 regulates cisplatin sensitivity through the endosome dependent autophagy .. RNA Isolation and Reverse Transcription-PCR. The OVCA cells were washedwith PBS anddetachedusing process. Here, we investigated the relationship between V-ATPase accutase solution (Sigma–Aldrich, St Louis, MO, USA). For RNA extraction, RNeasy mini kit (Qiagen, Valencia, inhibition, cisplatin sensitization, and the autophagy process. CA) was used according to the manufacturer’s instruction. We provide evidence that, in chemoresistant OVCA cells (cis- R), inhibition of V-ATPase-V0a2 blocks the autophagy u fl x Reverse transcription was performed using the high capacity cDNA kit (Applied Biosystems, Foster City, CA) according and suppresses ERK/MEK pathway that promotes cisplatin- to manufacturer’s protocol. All real-time PCR reactions were mediated cell death. Our n fi dings provide a rationale for the utility of V-ATPase-V0a2 inhibitors in combination with performed in triplicate in 10 𝜇 l volume using Universal fast PCR Master Mix reagent (Applied Biosystems, USA) standard drugs as a novel strategy to improve the treatment according to the manufacturer’s instructions. The results efficacy of the chemoresistant ovarian cancer. were analyzed using theΔΔCt method using GAPDH as the endogenous control. For cell death and autophagy pathway 2. Material and Methods analysis (RT2 proler fi , SA Biosciences, Frederick, MD, USA), PCR array-based expression proli fi ng was performed using .. Cell Lines and Cell Culture. Human ovarian carcinoma SYBR-Green method and the results were analyzed using the cell line A2780 (Sigma–Aldrich), its acquired cisplatin resis- tant counterpart cis-A2780, and TOV-112D cell lines were ΔΔCt method using RT profiler PCR data analysis sow ft are employed in this study as described previously [17, 18]. version 3.5 (SA Biosciences). Journal of Oncology 3 .. Antibodies. The following primary antibodies were incubated at 4 C for 30 min, aer ft which the cells were employed in study: rabbit anti-GAPDH [1:400; Cell Signaling centrifuged at 13,000× rpm at 4 C. The supernatant was then Technology (CST); Catalog number-5174S)], rabbit anti- collected. Protein quantification was performed using the LC3B (1:400; CST; 2775S), rabbit anti-beclin1 (1:350; CST; BCA assay (Pierce Protein Biology, USA). The 30 𝜇 gprotein 5174S), rabbit anti-ATG7 (1:400; CST; 8558S), rabbit anti- lysates were boiled with 4X SDS sample bueff r containing 2- P62 (1:400; CST; 5114S), anti-cleaved caspase 8 (1:200; mercaptoethanol and proteins were separated by SDS-PAGE CST; 9748), anti-phospho BRAF (1:1000; CST; 2696T), on 4–20% gradient acrylamide gels. All primary antibody anti-phospho-MEK (1:400; CST; 9154T), Mouse anti-beta incubations were performed 1h at room temperature followed actin (1:10,000; Abcam; ab184220), rabbit anti-LAMP-1(1:250; bysecondaryantibodyincubation(IRdye,Licor)for1hat RT. Abcam; ab25630), rat anti-LAMP-2 (1:250; Abcam; ab25631), The Blots werescanned using theOdyssey  infrared imaging rabbit anti-Fas L (1:100; Abcam; ab15285), rabbit anti-caspase system (LI-COR Biotechnology Lincoln, NE, USA). Blots 3 (1:400; Thermo Fisher; 4331182), rabbit anti-Fas (1:100; were probed with a𝛽 -actin endogenous control antibody to Bio legend; 305611), and mouse anti-a2V (Covance, Denver, confirm equivalent protein loading (Abcam, USA). USA). For isotype-control antibodies, control mouse IgG (R&D Systems) and rabbit IgG isotype (Invitrogen) were . . Immunofluorescence Analysis. For immunouo fl rescence used. Secondary antibodies were as follows: goat anti-rabbit analysis, the cells were plated in 8-well chamber slides (Nunc, IgG-FITC, donkey anti-mouse IgG AF-594, donkey anti- USA) at 3000 cells/well and were incubated overnight at rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC 37 C, 5% CO2. The cells were then washed thrice with PBS (Abcam), donkey anti-rabbit IRDye-800CW, and donkey (containing 0.5% FBS), fixed with 4% paraformaldehyde anti-mouse IRDye-680 CW (LI-COR Bioscience, Lincoln, for 30 min at room temperature (RT), and permeabilized NE). with 0.1% Triton X-100 in PBS for 12 min, 4 C. Block- ing was performed using 3% FBS in PBS for 1 h at RT. .. Immunohistochemical Staining of Ovarian Cancer Tissue. The cells were then incubated with primary antibodies (in To explore the clinical relevance of V0a2 expression in mod- blocking bueff r) for 1h at RT. The cells were then rinsed ulating cisplatin efficacy, we obtained paraffin-embedded thrice with PBST and incubated with secondary antibodies: tissues from ovarian cancer patients who reported to Advo- Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor cate Lutheran General Hospital (ALGH), Chicago, USA. The 594-conjugated goat anti-rabbit secondary antibody (1:200 study was approved by the Ethics Committee of ALGH. Eight dilution) (Invitrogen) dissolved in 3%FBS in PBS for 1 h samples each from the drug responder and nonresponder at RT. The cells were prepared for viewing using ProLong patient group were selected. 5-𝜇 m serially sectioned slides Gold (Invitrogen) mounting medium containing DAPI and were prepared. For normal control tissues, ovarian tissue allowed to polymerize at room temperature for 24 h. For sections from the normal ovary (n=2) were obtained from confocal microscopy, the stained cells were imaged on an Biochain Institute, Inc. (Newark, CA, USA). The horseradish Olympus Fluoview Fv10i confocal microscope. The analy- peroxidase-labeled polymer (EnVision+Dual Link System- sis was performed using Fv10i Flouview Ver.3.0 software. HRP; DAKO, USA) based staining method was used accord- Experiments were repeated at least twice in duplicate. For ing to the manufacturer’s protocol. For antigen retrieval, the immunou fl orescence microscopy, stained cells were imaged sections were boiled in sodium citrate buffer (pH = 6.0) in Olympus microscope and analyzed using NIS-Elements as described previously [18]. The slides were then cooled, sow ft are (Nikon Inc., NY, USA). blocked with 5% BSA in PBS, and incubated with the primary antibody at 4 C overnight. Concurrently, for negative mouse . . Flow Cytometry Analysis. Sh-V0a2 transfected/untrans- isotype-control antibody (R&D systems, USA) was used. The fected cells (2.5 x10 cells/tube) were washed with HBSS anti-rabbit/mouse secondary antibody was then added for 15 containing 0.1% FBS. For surface staining, the cells were min at 37 C. The sections were counterstained with Mayer’s incubated with mouse monoclonal FasL or Fas antibody hematoxylin and mounted in Faramount aqueous mounting conjugated to A or A (Covance, Denver, PA) in PBS for 647 488 medium (Dako). The immunostaining was evaluated by 40 min at RT. For the intracellular staining, the cells were light photomicroscopy (Leica ICC50 W, USA) using a high- fixed and permeabilized using xa fi tion and permeabilization resolution camera. buffer (BD Biosciences, San Jose, CA, USA) and the cells The IHC scoring was performed using the semiquantita- were stained as described above. For the indirect staining, the tive integration method. In this method, vfi e random fields of cells were incubated with unconjugated antibodies (caspase- view were selected for each specimen at high magnification 8) for 1 h at RT and subsequently washed twice with PBS and (×200). The following criteria were employed to generate then stained with conjugated secondary antibody (Abcam, a score: rfi st, staining area score [SAS] ( ≤1%:0;2–25%: 1; USA) for 30 min at RT. Appropriate isotype and unstained 26–50%: 2; 51–75%: 3 and>75%: 4); second, staining intensity controls were used for the experiments. The stained cells were [SI] (light brown: 1; moderate brown: 2 and tan: 3). The IHC analyzed on a BD LSR II flow cytometer with FlowJo software score was calculated using the formula: IHC score= SAS X SI. (Tree Star). Experiments were performed at least twice in duplicate. . . Western Blot Analysis. The harvested cell pellets were resuspended in NP-40 lysis buffer containing protease and .. Assessment of Autophagosomes. For autophagy analysis, phosphatase inhibitors (Pierce Protein Biology, USA) and V0a2 shRNA transfected/untransfected OVCA cells were 4 Journal of Oncology incubated with 20𝜇 M cisplatin for 24h at 37 Cin5% CO2. clinical relevance of V-ATPase-V0a2 in OVCA with varying To determine the induction of autophagy, we used the Cyto- cisplatin sensitivity, we utilized archival OVCA tissues from ID Autophagy detection kit (Enzo Life Sciences, Raams- cisplatin responder (n=8) and nonresponder patients (n=8). donksveer, The Netherlands). The autophagy detection is The OVCA tissue samples were obtained from patients based on monodansylcadaverine dye that specifically stains that were intrinsically responsive/nonresponsive to cisplatin autophagosomes. For positive autophagy controls, the cells (before any treatment). Normal ovarian tissues (n=2) were were treated with mTOR inhibitor rapamycin (0.5 𝜇 M) employed as the control. Both cisplatin responder and or lysosomal alkalizer chloroquine (60𝜇 M) or V-ATPase nonresponder patient tissues exhibited high V-ATPase-V0a2 inhibitor balo fi mycin (50nM). No treatment control wells staining compared to the normal ovary tissues. The V0a2 were also included in each set of experiment. After 24 h, protein expression was higher in cisplatin nonresponder the cells were washed with assay buffer provided by the (IHC score=8.9±1.06) than cisplatin responder patient tissues manufacturer (supplemented with 5% FBS) and stained with (IHC score= 6.5 ± 1.4, p=0.02) or normal ovarian tissues the Cyto-ID green detection reagent for 30 minutes and (IHC score=2.2±0.28; p<0.01) by IHC analysis [Figures 1(a) subsequently washed twice again with assay buffer. The and 1(b)]. The V-ATPase-V0a2 expression thus correlates u fl orescence signal was immediately captured in LSRII flow with drug unresponsiveness in ovarian cancer patients. Our cytometer measuring the intensity in 10,000 cells. FlowJo previous study showed a very poor expression of V-ATPase- software was used to process the imaging data. V0a2 in normal ovary tissues suggesting that V0a2 expression is selectively upregulated during tumorigenesis [17]. Confocal .. Transferrin Internalization Assay. As a measure of early microscopy analysis showed coexpression of V0a2 with endosomal function, the cellular internalization of A - OVCA antigen marker, CA125, conrfi ming V0a2 expression labeled transferrin (Tfn) was assayed. First, the cells were specifically in OVCA cells in drug nonresponder tissues serum starved by rinsing with 37 CHBSS (Invitrogen, USA) [Figures 1(c)(i) and 1(c)(ii)] as well as drug responder tissues and incubated in serum-free RPMI containing 25mM HEPES [Supplementary Figure S1]. These data suggest that V0a2 is and 1% BSA (RPMI-BSA) for 30 minutes at 37 C, 5% CO . a prominent target in ovarian cancer patients with varying Cells were incubated in ice for 10 minutes and then incubated cisplatin sensitivity. Further studies employing tissues from in RPMI-BSA containing 50 𝜇 g/ml of Tfn-A conjugate relapse/posttreatment patients and including more number to allow internalization for upto 30 minutes. Finally, cells of samples will further improve our understanding of V- were quick rinsed at least 10 times with HBSS to remove ATPase role in cisplatin resistance in OVCA. surface labeling. The slides were fixed in 4% formaldehyde for 15 minutes at room temperature. Immune-uo fl rescence .. Cisplatin Resistant Ovarian Cancer Cells Show Low analysis for early endosome labeling (EEA1) was performed Sphere-Forming Ability upon V-ATPase-Va Inhibition. To as described above. The slides were then processed for investigate the possible association of V0a2 with cisplatin fluorescence microscopy. resistance in OVCA, we next performed in vitro assays employing the sh-V0a2-cisR and sh-scr-cisR control cells. .. Cell Cytotoxicity Assay. OVCA cells were seeded into Our previous study showed that stable V-ATPase-V0a2 96-well plate (10,000 cells/well) overnight. The OVCA cells knockdown cells exhibited 3.8-fold inhibition at mRNA level were exposed to cisplatin (0.5, 1, 2.5, 5, 10, 20, and 50𝜇 M) and a 2.5-fold reduction in protein expression compared to and 10nM cobimetinib (MEK inhibitor) for 48h at 37 Cin sh-scr-cisR control cells [18]. 5% CO . After incubation, in vitro cell viability was measured 3D cancer cell spheroids mimic both in vivo architecture using MTS reagent (Promega, USA). Untreated cells were and low drug penetration properties that represent a relevant used as negative control. All experiments were performed model for studying drug resistance [19]. Here, we found in triplicate. The semilog plots of dose-response curves were that, upon V0a2 inhibition, sh-V0a2-cisR cells exhibited a generated using Microsoft Excel (Microso). ft reduced sphere-forming ability compared to control OVCA cells (sh-scr-cis-R) [Figure 2(a)]. Further, upon cisplatin treatment (20𝜇 M, 48h), we observed dissociation of sh-V0a2- 3. Statistical Analysis cisR spheroids while control spheroids remained unaeff cted The means of two data sets were compared and signif- [Figure 2(b)]. There was no dieff rence in the sphere-forming icance was determined by two-tailed Students t-test or ability between cisplatin sensitive (A2780) and cisplatin Mann–Whitney U test. Differences were considered to be sta- resistant cells (cis-A2780; data not shown). tistically significant where p <0.05. The data were graphically represented as the mean± standard deviation of the mean .. V-ATPase-Va Inhibition Enhances Cisplatin-Mediated (SD). Thedata wereanalyzed using GraphPad Prism (version Cell Death by Elevating Both Intrinsic and Extrinsic Apoptosis. 5) statistical sowa ft re. All experiments were repeated at least Upon therapeutic stress, the fate of a cancer cell is decided twice in duplicate. by its apoptotic capacity. Induction of intrinsic apoptosis is the primary mechanism of cisplatin-mediated cell death [20]. To understand the precise mechanism by which V- 4. Results ATPase inhibition leads to sensitization of cisplatin resistant cells, we performed cell death pathway PCR array using .. V-ATPase Va Is Highly Expressed in Cisplatin Non- responder Human Ovarian Cancer Tissues. To elucidate the cisplatin treated sh-V0a2-cis-R and sh-scr-cis-R cells. The Journal of Oncology 5 (i) Cisplatin Non-responder (ii) Cisplatin Responder (iii) Normal ovary 10X Normal ovary cisplatin cisplatin 40X responder non-responder (a) (b) DAPI CA-125 V-ATPase-V0a2 Merge zoom (i) (ii) (c) Figure 1: V-ATPase-Va is highly expressed in cisplatin nonresponder ovarian cancer tissues. (a) Immunohistochemical analysis of V-ATPase- V0a2 expression in tissues from (i) cisplatin nonresponder and (ii) cisplatin responder ovarian cancer patients compared to (iii) normal human ovary tissue. Original magnification × 100 (upper panel) and X 400 (lower panel). (b) The quantitative IHC data expressed as IHC intensity score revealed higher V0a2 expression in ovarian cancer tissues from cisplatin nonresponder patients compared to responder patients and to normal ovarian tissues. (c) Confocal microscopy analysis of V0a2 (green) in nonresponder OVCA tissues ((i) and (ii)) shows its coexpression with ovarian cancer cell marker CA125 (red). Nuclear DAPI staining in blue. Merged areas are shown in yellow. Original magnification: × 600. Zoomed areas represent white boxes in merged figures. Representative images from three independent experiments are shown. proapoptotic genes (caspase 3, FASL, caspase 8, TNF, and mechanism in cisplatin resistant cancer ovarian cancer cells TNFR1) were signicfi antly upregulated (p < 0.05) in V0a2 [21, 22]. For successful the autophagy process, the proton knockdown cells upon cisplatin treatment [Supplementary pumping activity of V-ATPase is necessary for the acidifi- Figure S2]. Flow cytometry analysis revealed that the pro- cation of the endo-lysosomal vesicles mediated degradative tein levels of active caspase 3 were increased in sh-V0a2- stage [23]. Inhibition of autophagy is known to sensitize the cisR cells (p=0.009) relative to sh-scr-cisR. The intrinsic resistant cells to cisplatin treatment [24–26]. We therefore apoptotic proteins, active caspase-9 (p=0.02) and Bax (p studied the effect of blocking V-ATPase-V0a2 on the modu- =0.03) [Figure 2(c)], were also upregulated in sh-V0a2-cis- lation of autophagy in chemoresistant cells. In our previous R. Further, cell membrane-bound FasL and cleaved caspase study, we showed that the sh-V0a2-cisR growth rate was 8 levels, members of the extrinsic apoptotic pathway, were slower than the sh-scr-cisR cells; however, V0a2 inhibition in also elevated [Figure 2(d)] compared to control cells. This itself did not impose any cytotoxicity to the cisplatin resistant indicates that inhibition of V0a2 expression potentiates the cells. In the context of autophagy, here, we found lower cell death activity of cisplatin by stimulating both intrinsic autophagosome numbers in sh-V0a2-cisR compared to sh- and extrinsic apoptotic pathways. scr-cisR cells by confocal microscopy analysis [Figure 3(a)]. Lower autophagosome accumulation was also conrfi med by .. V-ATPase-Va Inhibition Dampens the Protective Auto- flow cytometry analysis [Figure 3(b)]. Further, a significantly reduced LC3B, Beclin-1 [Figure 3(c)], and ATG7 [Figure 3(d)] phagy Levels in Cisplatin Resistant Ovarian Cancer Cells. An levels were observed in sh-V0a2-cis-R compared to control enhanced autophagy process reflects an enhanced survival V0a2 IHC intensity score 6 Journal of Oncology Caspase 3 Caspase 9 Bax 1000 4000 Sh-scr-cis- R Sh-V0a2-cis-R 400 ∗ 800 300 600 200 400 100 200 0 0 0 (a) Sh-scr-cis-R Sh-V0a2-cis-R Sh-scr-cis-R Sh-V0a2-cis-R Sh-scr-cis-R Sh-V0a2-cis-R UT UT UT cisplatin cisplatin cisplatin (c) Sh-scr-cis- R + cisplatin Sh-V0a2-cis-R + cisplatin Caspase 8 Fas Fas-L 800 400 30000 10X 400 200 200 100 0 0 0 Sh-scr-cis-R Sh-V0a2-cis-R sh-scr-cis-R sh-V0a2-cis-R sh-scr-cis-R sh-V0a2-cis-R 40X UT UT UT cisplatin cisplatin cisplatin (b) (d) Figure 2: Inhibition of V-ATPase-Va in resistant ovarian cancer cells blunts spheroid formation and enhances cisplatin-mediated cell death. (a) Photomicrographs showing the effect of shRNA mediated V-ATPase-V0a2 inhibition on the spheroid formation in cisplatin resistant ovarian cancer cells (sh-V0a2-cisR) compared to control cells (sh-scr-cis-R). eTh sh-V0a2-cisR exhibited decreased tumor spheroid formation while control cells formed large clusters of spheroids. (b) Upon cisplatin treatment (20𝜇 M, 48h), an enhanced spheroid dissociation was observed in sh-V0a2-cisR compared to control spheroids. Original magnification: X100, X400. (c) Geometric mean u fl orescence intensity of effector apoptotic protein (cleaved caspase-3), intrinsic apoptotic (active caspase-9, Bax), and (d) of extrinsic apoptotic proteins (cleaved caspase-8, Fas, and FasL) in cisplatin treated sh-V0a2-cisR compared to cisplatin treated sh-V0a2-cisR cells as quantitated by flow cytometry. Each value represents the mean± SD of three independent experiments,∗P< 0.05. cells as determined by western blot analysis indicating that accumulation and a reduction in the amount of internalized the initial autophagy steps are inhibited by V-ATPase inhibi- Tfn compared to control sh-scr-cis-R cells [Figure 4(a)]. tion. This is in contrast to V-ATPase inhibition using chem- When the cells (tfn internalized, 30 min) were xfi ed and ical inhibitors which are known to acutely induce autophagy stained with EEA1 (early endosome marker), an intense as a protective mechanism. Interestingly, autophagy substrate colocalization of Tfn was observed in control sh-scr-cis-R protein P62 was upregulated in sh-V0a2-cisR, suggesting a cells. In contrast, sh-V0a2-cis-R cells showed low transferrin concomitant block in the autophagy u fl x due to interference signal in early endosomes [Figure 4(a)]. Further, LC3B with endosomal function [Figure 3(d)]. stained autophagosomes and EEA-1 exhibited diminished colocalization in sh-V0a2-cis-R cells compared to control cells (sh-scr-cis-R) [Figure 4(b)]. The autophagy vacuoles .. Inhibition of V-ATPase-Va Disrupts Early Endosome (LC3B) colocalized with the late endosomes/lysosomes in Trafficking in Cisplatin Resistant Ovarian Cancer Cells. V0a2 depleted cisplatin resistant cells similar to the control The isoform-specific V-ATPase inhibition impairs specific organellar functions in contrast to the chemical V-ATPase cells [supplementary Figure S3]. Further in-depth studies are required to understand the precise role of functional early inhibitors that target the predominant subunits on cellu- endosomes in autophagy process. lar V-ATPases. For the formation of autophagolysosomes, autophagic vacuole undergoes maturation through fusion with early/late endosomes and lysosomes [27, 28]. Since V0a2 .. Cisplatin Treatment in V-ATPase-Va Inhibited Chem- is primarily localized on the early endosomal membrane to oresistant Cells Induces Autophagy. In the parental cisplatin regulate the vesicular pH, we rfi st analyzed the effect of V0a2 sensitive OVCA cells, autophagy overactivation is a known knockdown on early endosomal trafficking. To measure the contributor to cisplatin-mediated cell death [22]. In line with function of the early endosome, we examined transferrin the previous reports, we observed an enhanced autophagy (Tfn) uptake, using Alexa -labeled Tfn. In sh-V0a2-cis- response in cisplatin sensitive OVCA cells (Cis-S) upon R cells, a 30-min incubation with Tfn showed a surface cisplatin treatment. There were an increased autophagosome Untreated MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) Journal of Oncology 7 sh-V0a2-cis-R sh-scr-cis-R sh-scr-cisR sh-V0a2-cisR (a) (b) Cis-S sh-scr-cisR sh-V0a2-cisR 75kD ATG7 Fold change 0.8 1 0.6 Normal ovary Cis-S sh-scr-cisR sh-V0a2-cisR 19kD LC3I -Actin 17kD LC3 II Fold change 0.16 0.47 1 0.34 62kD P62 60kD Beclin1 1.9 1 3.5 Fold change 0.2 0.44 1 0.21 Fold change 47kD -Actin -Actin (c) (d) Figure 3: Protective autophagy is dampened upon V-ATPase-Va inhibition in cisplatin resistant ovarian cancer cells. (a) Confocal microscopy analysis of the subcellular distributions of LC3B levels (red) in V-ATPase-V0a2 inhibited cisplatin resistant cells (sh-V0a2-cisR) compared to control (sh-scr-cis-R) cells. eTh sh-V0a2-cisR exhibit low LC3B staining compared to control cells (x600 magnification). (b) Geometric mean u fl orescence intensity (MFU) of LC3B levels in sh-V0a2-cisR compared to the levels in control (sh-scr-cisR) cells as quantified by flow cytometry. (c) Western blot analysis of the autophagy associated proteins LC3 (I and II) and Beclin1 in sh-V0a2-cisR shows decreased expression compared to control cells (sh-scr-cis-R), similar to cisplatin sensitive parental OVCA cells (cis-S). (d) Western blot analysis of ATG-7 shows lower expression upon V-ATPase V0a2 inhibition while P62 (autophagy substrate protein) shows higher expression in sh- V0a2-cisR compared to control cells (sh-scr-cis-R). Fold change in band densities were measured relative to the control (sh-scr-cisR) and the samples were normalized to endogenous beta-actin levels. Representative images from three independent experiments are shown here. number and decreased P62 levels [Supplementary Figure S4] there was a concomitant accumulation of autophagosomes in cisplatin treated cis-S cells. However, the cisplatin resistant with high accumulation of P62 suggesting an overall block cells are known to depict a protective autophagy response that in autophagy ux fl that facilitates cisplatin-mediated cell counteracts apoptotic cell death. Moreover, pharmacological death [Figures 5(c)(i) and 5(c)(ii)]. Flow cytometry analysis inhibition of autophagy either by inhibiting ATGs, beclin, or confirmed upregulated expression of autophagy initiation lysosomal inhibitors enhances cisplatin-mediated apoptosis proteins Beclin-1 and ATG7 [p<0.05; Figures 5(d) and [21, 24–26]. Here, sh-V0a2-cisR when treated with cisplatin 5(e)]. showed induction of several autophagy initiation related genessuch asMAPLC3A, IGFR, and Atg7[Supplementary . . Enhanced Cisplatin Sensitization upon V-ATPase In- Figure S5]. The upregulation in the autophagy initiation hibition Involves Suppression of ERK/MEK Pathway in proteins was conrfi med by ow fl cytometry. The sh-V0a2-cisR Resistant Ovarian Cancer Cells. Upregulation of oncogenic cells exhibited higher autophagosome accumulation upon Ras pathway has been observed in chemoresistant cancer cisplatin treatment compared to sh-scr-cisR as determined cells. The Ras pathway enhances DNA-repair through the by confocal microscopy analysis [Figure 5(a)]. This enhanced Ras/PI3K/Rac1 pathway [29, 30] to protect against cisplatin- autophagosome accumulation was further conrfi med by ow fl induced therapeutic stress. In addition, inhibition of Ras cytometry analysis of LC3B protein in sh-V0a2-cisR and sh- may enhance the cisplatin sensitivity of human glioblas- scr-cisR [Figure 5(b)]. x Therefore, upon cisplatin treatment toma [31]. In the present study, the mRNA prolin fi g of the of sh-V0a2-cis-R, in spite of enhanced autophagy initiation, Ras pathway array of the sh-V0a2-cisR revealed significant LC3B LC3B MFU (Mean ± SD) 8 Journal of Oncology EEA1 Transferrin (30 min) Merge + DAPI (a) Rab5 LC3B Merge (b) Figure 4: Inhibition of V-ATPase-Va disrupts early endosome trafficking in cisplatin resistant ovarian cancer cells. (a) Cisplatin resistant OVCA cells were treated with control shRNA (sh-scr-cis-R) or with shRNA against V-ATPase-V0a2-cisR (sh-V0a2-cis-R). eTh cells were incubated with Tf-Alexa at 37 C for 30 min to label the entire early endosomal compartment. eTh cells were fixed before permeabilization and were stained with anti-EEA1 (green). Merged images of shV0a2-cis-R and sh-scr-cis-R cells. Yellow color indicates colocalization between Tf-Alexa and EEA1. (b) Immunofluorescence analysis of the subcellular distributions of LC3B (red) and Rab5 (early endosome marker; green) in sh-V0a2-cis-R compared to control (sh-scr-cis-R) cells. eTh sh-V0a2-cisR exhibited low poor LC3B/Rab5 colocalization compared to control cells (x600 magnification). downregulation of certain Ras pathway genes (EGFR, Fos, cells (cis-A2780) suggesting a varied activation and role of BRAF, GRB2, ELK, Raf1, and Myc) [Figure 6(a)]. The western ERK/MEK pathway in cisplatin resistance. Taken together, blot analysis conrfi med downregulated phosphorylation of these findings indicate that V-ATPase mediated inhibition MEK1/2 and BRAF, thus confirming the suppression of of autophagy ux fl contributed to the reversal of cisplatin ERK/MEK pathway in these cells compared to sh-scr-cisR resistance in resistant ovarian cancer cells. Knockdown of control cells [Figure 6(b)]. Further, the treatment of cisplatin V0a2 or use of MEK inhibitors suppresses ERK activation and resistant ovarian cancer cells (cis-A2780) with the MEK1/2 blocks the autophagy while increasing cisplatin-induced cell inhibitor cobimetinib sensitized the human ovarian cancer death. cell lines to cisplatin-induced cell death. Cisplatin alone did not elicit significant cell death, whereas enhanced cell death 5. Discussion was seen when cells were treated for 48 h with cisplatin in combination with 10nM cobimetinib [Figure 6(c)]. At the Ovarian cancer is the leading cause of cancer-related deaths same concentration of cobimetinib, an enhanced cell death in women due to high treatment failure rates [32]. To improve was observed more prominently in cisplatin sensitive cell the OVCA patient outcome, it is imperative to understand the lines (A2780 and TOV-S) compared to cisplatin resistant chemoresistance associated pathways to identify the mode Sh-V0a2-cis-R Sh-scr-cis-R Sh-scr-cis-R Sh-V0a2-cis-R Journal of Oncology 9 (i) (ii) Untreated + Cisplatin sh-V0a2-cisR sh-scr-cisR 5000 4000 0 0 (a) (b) Beclin1 1800 ∗ Sh-scr-cisR Sh-V0a2-cisR 1600 (i) (ii) UT +cis,10M +cis,20M UT +cis,10M +cis,20M LC3I LC3I 600 LC3II LC3II Fold change 1 0.42 0.28 1 1.1 1.6 Fold change +cis +cis +cis +cis UT UT P62 P62 10M 20M 10M 20M Sh-scr-cisR Sh-V0a2-cisR Fold change Fold change 1 0.8 1.8 1 0.67 0.43 (d) ATG5 ATG5 ATG7 Fold change Fold change 1 0.5 0.42 1 1.1 1.4 -actin -actin (c) UT +cis +cis UT +cis +cis 10M 20M 10M 20M Sh-scr-cisR Sh-V0a2-cisR (e) Figure 5: Cisplatin induces protective autophagy in V-ATPase-Va inhibited resistant ovarian cancer cells with a concomitant block in autophagy flux leading to drug sensitization. V-ATPase-V0a2 inhibited cisplatin resistant ovarian cancer cells (sh-V0a2-cisR) were treated with cisplatin (20𝜇 g/ml, 48h). (a) Confocal microscopy analysis of the subcellular distributions of LC3B levels (red); nucleus is stained with DAPI (blue). Upon cisplatin treatment, there is a higher accumulation of LC3B in sh-V0a2-cisR compared to untreated cells. eTh uo fl rescence signals of LC3B were sequentially acquired using an Olympus FluoView confocal microscope. Representative confocal micrographs (original magnification: 80X) are shown. Bars, 5 𝜇 m. (b) Geometric mean u fl orescence intensity units (MFU) of LC3B levels in sh-V0a2-cisR compared to the levels in untreated cells as quantified by flow cytometry. (c) Western blot analysis of the autophagy associated proteins LC3, P62, and ATG5 in (i) control sh-scr-cisR cells and (ii) sh-V0a2-cisR cells upon cisplatin treatment. Geometric mean u fl orescence intensity of (d) beclin1 protein levels and (E) ATG7 protein levels in cisplatin treated/untreated sh-V0a2-cisR compared to control cells (sh-scr-cisR) as quantified by flow cytometry. ∗ p<0.05. Experiments were repeated twice in duplicate. for sensitizing cancer cells to therapy [20]. The prominent is therefore highly debatable and reports on the anticancer mechanism of chemoresistance includes resistance to apopto- effects upon autophagy inhibition or its overactivation are sis [33]. Alternative cell survival pathways such as autophagy equally available [37, 38]. In some cases, overactive autophagy have become a critical area of research [34, 35]. The process induces cell death necessary for the cytotoxic effect of the of tumorigenesis exhibits varied dependence on autophagy therapy. Therefore, autophagy inducers such as the mTOR during progression from primary tumor to metastatic stage or inhibitors have also been considered as potential cancer during chemoresistance [36]. The role of autophagy in cancer treatments [39–41]. sh-scr-cis-R sh-V0a2-cis-R MFU (Mean ± SD) UT + Cisplatin + Bafilomycin + Chloroquine MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) UT + Cisplatin + Bafilomycin + Chloroquine 10 Journal of Oncology Normal ovary Cis-S sh-scr-cisR sh-V0a2-cisR P-MEK 1/2 Fold change 0.27 0.27 1 1 0. 0.65 65 EGFR Fos GRB2 ELK1 Raf1 Myc B-Raf −5 -actin −10 −15 s sh-s h-scr cr-ci -cisR sR C Cis-S is-S s sh-V0a2-cisR h-V0a2-cisR −20 P-B-RAF B-RAF −25 −30 Fold ch ha ange nge 1 0.85 0.71 −35 -actin −40 (a) (b) TOV-112D Cis-A2780 A2780 100 100 −10 0 0 0.5 1 2.5 5 10 0 0.5 1 2.5 5 10 Cisplatin concentration (M) Cisplatin concentration (M) 0 0.5 1 2.5 5 10 20 50 cisplatin cisplatin Cisplatin concentration (M) cisplatin + cobimetinib(10 nM) cisplatin + cobimetinib(10 nM) cisplatin cisplatin + cobimetinib(10 nM) (c) Figure 6: V-ATPase-VainhibitionsensitizesthecisplatinresistantovariancancercellsthroughdownregulationoftheRaspathway. The shRNA mediated V-ATPase-V0a2 inhibition was carried out in cisplatin resistant ovarian cancer cells (sh-V0a2-cisR). Cisplatin treated (20𝜇 g/ml, 48h), sh-V0a2-cisR and control cells (sh-scr-cisR) were analyzed for Ras pathway. (a) Transcriptional profiling of the Ras pathway array showed significant downregulation of the Ras pathway associated genes (EGFR, Fos2, GRB2, Raf1, Elk-1 Myc, and B-Raf). (b) Western blot analysis showed downregulation of phosphorylated B-Raf and MEK 1/2. Fold change in band densities was measured relative to the control (sh-scr-cisR) and the samples were normalized to endogenous beta-actin levels. (c) Combination of cisplatin and MEK inhibitor cobimetinib (10nM) showed enhanced cell death in three ovarian cancer cell lines (A2780, TOV-112D, and cis-A2780). The chemoresistant cancer cells, however, exhibit a dis- space, primarily driven by proton pumps V-ATPases [45– tinct relation with the autophagy process. Several studies have 47]. This altered pH gradient interferes with drug uptake revealed that cisplatin resistant ovarian cancer cells express and metabolism in cancer cells [48]. Regulated assembly high levels of autophagy as a survival mechanism [21, 22]. The of the V-ATPase V0 and V1 domain occurs in response fact that anticancer drugs frequently induce cytoprotective to glucose starvation and involves both PI3K and AMPK autophagy has provided the basis for combination therapy tri- pathway, which in turn modulate autophagy [49]. In this als using autophagy-blocking agents with standard antitumor context, it will be interesting to examine the long term drugs [42]. Several clinical trials are investigating the efficacy effect of dysfunctional V-ATPase complex and its effect on of autophagy inhibition with conventional chemotherapy AMPK pathway. In the previous studies, we identified V- in various types of cancers [43]. Given that autophagy is ATPase-V0a2 as a tumor-associated isoform that is distinctly also a basic physiological mechanism in all cells [8, 44], overexpressed in cisplatin resistant ovarian cancer cells, the inhibition of which sensitized the cells to cisplatin treatment directly targeting the autophagy in cancer leads to unwanted consequences in normal cells. It is therefore vital to identify [17, 18]. In continuation with our efforts, in the present indirect autophagy modulators specicfi for cancer cells that study, we demonstrate that the inhibition of V-ATPase-V0a2 can be effectively targeted for anticancer therapy. sensitizes cisplatin resistant OVCA cells by direct modulation Cancer drug resistance is associated with an altered pH of autophagy process. Cisplatin treatment induces extrin- gradient between the cytosol and extracellular/intravesicular sic and intrinsic apoptotic pathways upon V-ATPase-V0a2 % cell survival Fold downregulation to control % cell survival % cell survival Journal of Oncology 11 Sh-scr-cisR Sh-V0a2-cisR +cisplatin Sh-V0a2-cisR (a) (b) (c) Cisplatin treatment V-ATPase Cell death inhibition Cisplatin resistant ovarian cancer cells (1) Dampened protective autophagy, early (1) Cisplatin induced autophagy, high endosomal dysfunction, block in autophagy flux Enhanced autophagy acts as cell survival factor autophagosome accumulation (2) Suppressed ERK/MEK pathway (2) Concomitant block in autophagy flux Cisplatin sensitization due to Inhibition of protective autophagy V-ATPase block in autophagy flux, and block in autophagy flux endosomes enhanced cell death Autophagosome V-ATPase-V0a2 inhibition Block in Autophagy flux cisplatin Figure 7: V-ATPase-Va isoform inhibition mediates cisplatin-associated cell death in resistant ovarian cancer through a block in autophagy flux. (a) Cisplatin resistant ovarian cancer cells exhibit enhanced autophagy that favors cell survival under stress. (b) Upon V-ATPase inhibition, the autophagy initiation proteins are dampened. er Th e is a block in autophagy u fl x reflected by high P62 accumulation. Suppressed ERK/MEK pathway is also observed. (c) Cisplatin treatment of V-ATPase inhibited cells triggers autophagy; however, there is a concomitant block in autophagy u fl x, leading to autophagosome accumulation and enhanced cell death. isoform inhibition in resistant OVCA cells with inhibition in OVCA tissue samples from relapse/posttreatment patients of protective autophagy. This makes the chemoresistant cells to improve our understanding of the acquired chemore- susceptible to any further genotoxic stress. Our data is in sistance. Such tools will greatly improve the prediction of line with the previous reports suggesting that inhibition of sensitivity or resistance to chemotherapy and allow treatment autophagy sensitizes acquired cis-R cells to cisplatin [21, 22]. stratification. Upon cisplatin treatment, sh-V0a2-cisR cells show induc- Cisplatin resistant cancer cells exhibit defective steps in tion of autophagy pathway as a prosurvival mechanism; how- apoptosis with decreased expression of proapoptotic proteins such as BAD, Bid, and caspases 4 and 6 [3, 20, 54]. Our ever, the accumulation of autophagy substrate P62 confirmed a concomitant block in the overall autophagy flux that drives previous study demonstrated that the apoptotic rate of V0a2 the cells towards cell death. V0a2 inhibition suppressed the inhibited cisplatin resistance cells was higher than in the ERK/MEK pathway in cis-R cells. Our findings provide key control group [18]. Here, we confirm that cisplatin treatment evidence that the isoform-specific inhibition of V-ATPase- effectively induces both intrinsic and extrinsic apoptosis in V0a2 inhibits autophagy that contributes to cisplatin sensi- resistant OVCA cells upon V0a2 inhibition by the activation tization in resistant OVCA cells [Figure 7]. of caspase-3, caspase-8, caspase-9, and caspase-7. Previous studies have confirmed that V-ATPase proton However, targeting the apoptotic mechanisms does not pump subunits are inducible by cisplatin treatment [50]. This optimally inhibit chemoresistance [55, 56]. It is therefore prevents cytosolic acidification of cancer cells that is a trigger critical to exploit the alternative pathways, such as autophagy of apoptosis [51]. Further, the acquired cisplatin resistant to counter chemoresistance. Targeting the autophagy for reg- ulation of cancer chemoresistance is a therapeutic strategy yet cells show upregulated expression of V-ATPase subunits [50, 52]. V-ATPase driven proton flux causes acidicfi ation of to be properly designed. With regard to cisplatin resistance, intracellular vesicles as well as the acidification of the extra- several reports suggest that protective autophagy inhibition cellular microenvironment of cancer cells. This interferes is particularly helpful in chemosensitization [22, 57–59]. Our with drug-induced cytotoxicity in addition to promoting study confirms that V-ATPase-V0a2 inhibition significantly cancer invasiveness [53]. inhibits autophagy process with downregulation of beclin-1 In clinical tissues, there are no detailed reports on the and LC3 levels compared to control cells. After cisplatin treat- correlation between V-ATPase expression and drug respon- ment, several early autophagy-related genes were induced siveness in ovarian cancer patients. Our study demonstrates in response to drug-induced stress; however, there was a for the first time increased expression of V-ATPase-V0a2 in concomitant accumulation of autophagosomes and elevated clinical tissues obtained from cisplatin nonresponder patients P62 levels suggesting an overall block in autophagy u fl x that facilitates cisplatin-mediated cell death. compared to the treatment-responder patients. The data pro- vided here include samples showing intrinsic unresponsive- For successful the autophagy process, a fully functional ness to the treatment. Further studies are therefore required endo-lysosomal system is highly critical. For autophagic 12 Journal of Oncology vacuole (AV) maturation, a sequential fusion of AVs with dif- previous observations, we observed suppression of activated ferent populations of early and late endosomes and lysosomes MEK1/2 and B-RAF in V-ATPase-V0a2 inhibited cisplatin [27, 60] is essential which highlights the importance of dif- resistant OVCA cells compared to control. The combination treatment of small molecular MEK1/2 inhibitor, cobimetinib ferent vesicles of the endo-lysosomal pathway in autophagy. Chloroquine (CQ) derivative, hydroxychloroquine (HCQ), along with cisplatin, could enhance cisplatin-mediated cell the only clinically approved autophagy inhibitor, is presently death in OVCA cells. The combination of MEK inhibitor with cisplatin, however, is more effective in sensitive cells under clinical trials as mono- or combination therapy against various types of cancers [61]. HCQ gets sequestered in compared to resistant cells, indicating that there are other the acidic vesicles such as autolysosomes making them significant players in chemoresistance. alkaline, thereby hindering the degradative steps. However, Taken together, these findings indicate that inhibition of CQ derivatives are known to produce harmful side effects autophagy contributes to the reversal of cisplatin resistance in ovarian cancer cells. From the results obtained in this study, specifically on heart and kidney. Similarly, bafilomycin is another known chemical that inhibits V-ATPase and disrupts we propose that targeting V-ATPase-V0a2 is an effective lysosomal acidification and autophagy that contributes to strategy in sensitizing the chemoresistant ovarian cancer cells to cisplatin treatment. tumor cell death; however, it also interferes with Ca2+ pump SERCA [62]. Proton pump inhibitors (PPI) such as omepra- zole and pantoprazole induce the early accumulation of Data Availability autophagosomes, with concomitant inhibition of autophagic ufl x [63, 64].Considering thesideeeff ctsofthe knownchem- The data used to support the findings of this study are ical autophagy and V-ATPase inhibitors, V-ATPase-V0a2 available from the corresponding author upon request. isoform-specific targeting for V-ATPase/autophagy inhibi- tion will provide a safer anticancer alternative. In addition, Conflicts of Interest due to the absence of V0a2 isoform on renal cells (where a4 isoform is predominant), targeted V0a2 based therapy may The authors declare no conflicts of interest. have fewer renal associated side effects. Recently, functional early endosomes were found to be essential for successful autophagy. Defects in early endosome Authors’ Contributions function led to an accumulation of autophagosomes and Arpita Kulshrestha and Gajendra K. Katara contributed to inhibition of autophagy [10]. V0a2 is found in the early conception and design. Arpita Kulshrestha, Gajendra K. endosomes and is also known to regulate protein degradative Katara, and Safaa A. Ibrahim contributed to development of pathway through interaction with Arf6 and ARNO [65]. V- methodology. Arpita Kulshrestha, Gajendra K. Katara, Safaa ATPase inhibition led to dysregulation of Notch signaling due A. Ibrahim, and Kyle W. Meinke contributed to acquisition of to endosomal acidification [66]. Here, V-ATPase inhibition data. Arpita Kulshrestha and Gajendra K. Katara contributed by V0a2 knockdown indicates defective early endosomal to analysis and interpretation of data. Arpita Kulshrestha, function that may contribute to modulation of autophagy Gajendra K. Katara, and Kenneth D. Beaman contributed process. Further in-depth studies are required to decipher to writing and review. Arpita Kulshrestha, Gajendra K. the exact mechanism of V0a2 mediated regulation of early Katara, Safaa A. Ibrahim, M. Sahoo, Valerie Riehl, Kenneth endosomal function. D. Beaman, J. Dolan, and Michael R. Pins contributed to The mechanism of chemoresistance is dependent on the administrative, technical, or material support (i.e., report- balance of the activities of different intracellular signaling ing or organizing data and constructing databases). Arpita systems. There are extensive reports on the involvement Kulshrestha and Kenneth D. Beaman contributed to study of survival signals such as MAP kinase subfamilies in supervision. regulating autophagy [67, 68]. Several studies indicate that ERK (extracellular signal-regulated kinase), one of the six known mammalian MAPK pathways, is aberrantly activated Acknowledgments in cancer cells and promotes cancer cell proliferation, sup- presses apoptosis, and enhances metastasis/drug resistance. The authors thank Dr. Patricia Loomis and Robert Dick- ERK (ERK1 and ERK2) is activated upon phosphorylation inson for technical assistance and the Rosalind Franklin by MEK (MEK1 and MEK2), which is activated upon University of Medicine and Science Confocal Microscopy phosphorylation by Raf (Raf-1, B-Raf, and A-Raf). ERK and Flow Cytometry Core Facility. We thank Advocate pathway activation promotes autophagy, leading to cisplatin Lutheran General Hospital James R. & Helen D. Russell resistance. Inhibition of ERK activation enhances cisplatin- Institute for Research and Innovation, Park Ridge, Illinois, induced growth inhibition [69]. In cisplatin resistant squa- for their assistance in obtaining specimens and obtaining mous cancer cells, both ERK (extracellular signal-regulated IRB approval. This work was partially supported by grants kinase) activation and autophagy induction are observed from the ALGH-RFUMS collaborative research involving [70]. 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Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway

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Copyright © 2019 Arpita Kulshrestha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2019, Article ID 2343876, 15 pages https://doi.org/10.1155/2019/2343876 Research Article Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway 1 1 1,2 Arpita Kulshrestha , Gajendra K. Katara, Safaa A. Ibrahim, 1 1 3 3 Valerie Riehl, Manoranjan Sahoo, James Dolan, Kyle W. Meinke, 4 1 Michael R. Pins, and Kenneth D. Beaman Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Egypt Department of Obstetrics & Gynecology, Advocate Lutheran General Hospital, Park Ridge, IL, USA Department of Pathology, Advocate Lutheran General Hospital, Park Ridge, IL, USA Correspondence should be addressed to Kenneth D. Beaman; kenneth.beaman@rosalindfranklin.edu Received 1 August 2018; Revised 28 January 2019; Accepted 4 March 2019; Published 1 April 2019 Academic Editor: Srikumar P. Chellappan Copyright © 2019 Arpita Kulshrestha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ovarian cancer (OVCA) patients oen ft develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase ‘a2’ isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible f actor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. eTh apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. ese Th cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2- cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy u fl x. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V- ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer. 1. Introduction platinum-based drugs [1]. In addition to apoptosis inhibition, cisplatin resistant cancer cells rely on mechanisms such Ovarian cancer (OVCA) is hard to treat as it exhibits refrac- as reduced drug uptake, increased drug efflux, enhanced toriness to standard chemotherapy approaches including DNA-repair, and defective signaling pathways to survive 2 Journal of Oncology therapeutic cell death [2]. Nevertheless, an understanding Briefly, A2780 and cis-A2780 cells were cultured in RPMI of the precise molecular mechanism of chemoresistance will 1640 medium (Invitrogen, Carlsbad, CA) supplemented with help design strategies to improve the treatment outcome in 10% (v/v) heat-inactivated fetal bovine serum (Biowest LLC, OVCA patients. MO,USA), 100U/ml penicillin, and 100U/ml strepto- Exposure of cancer cells to cisplatin elicits a stress mycin (Sigma–Aldrich) at 37 C, 5% CO2. TOV112D cell response which induces coping mechanisms that favor can- line (American Type Culture Collection [ATCC], Manas- cer cell survival [3]. Autophagy is the primary protective sas, VA) was cultured in CTOV medium [1:1 mixture of process that enables energy supply during stress such as MCDB 105 medium containing a n fi al concentration of chemotherapy exposure and nutrient depletion [4–6]. The 1.5 g/L sodium bicarbonate and medium 199 containing self-degradative pathway of autophagy involves the forma- a n fi al concentration of 2.2 g/L sodium bicarbonate at tion of double-membrane vesicles (autophagosomes) around 37 C, 5% CO2]. The cells were routinely grown until reach- damaged cellular proteins and organelles [7, 8]. Autophago- ing 80% confluency and then subcultured or plated for somes fuse to endo-lysosomal machinery where sequestered experiments. cellular components are ultimately digested for energy recy- .. Generation of Stable V-ATPase-Va Knockdown Cells. cling [9]. In addition to lysosomal machinery, recent studies suggest the importance of early endosomes in autophagy [10]. The shRNA mediated V-ATPase-V0a2 knockdown was per- It is therefore important to understand how molecular targets formed as described previously [18]. Brieyfl , the cisplatin resistant cells (cis-A2780) were plated overnight and then involved in endosomal machinery can modulate autophagy process. transfected with V0a2 shRNA constructs (Suresilencing Plas- A tightly regulated intracellular pH is critical for mid, Qiagen, Valencia, CA, USA) or a scrambled control autophagy [11]. In mammalian cells, vacuolar ATPase (V- shRNA using the Attractene transfection reagent (Qiagen). ATPase) proton pumps are the primary pH regulators that The cells were treated with the selection antibiotic (1 mg/ml G418) after 24 h after transfection. Medium containing G418 maintain intravesicular and/or extracellular pH. In normal cells, V-ATPases pump protons from the cytoplasm to the was replenished every 72 h. After confirming the knockdown lumen of the acidic organelles [9]. In cancer cells, plasma by Q-RT PCR, the positive transfectants were cloned and used for further experiments. membrane-associated V-ATPases extrude protons and acid- ify the extracellular matrix [12, 13]. V-ATPase inhibition disrupts tumor pH gradients that alters drug retention and .. Drugs. Anticancer drug cisplatin was obtained from Sigma–Aldrich. Cisplatin 1mM stock was prepared in normal trackin ffi g in tumor cells. Many proton pump/V-ATPase saline (0.9% NaCl) and stored as aliquots at -20 inhibitors are showing efficacy in increasing the sensitivity Cup to 3 of tumor cells to cytotoxic agents [14–16]. Unlike chemi- months. V-ATPase inhibitor balfi omycin A (Sigma–Aldrich, M17931) was dissolved in DMSO at a 100𝜇 M stock solution. cal inhibitors, targeting cancer specific V-ATPase isoforms will modulate autophagy and will potentially decrease the Autophagy modulators rapamycin and chloroquine were procured from Enzo Life Sciences, USA. Rapamycin was associated toxicity to normal cells. Our previous work dissolved in DMSO at a 500𝜇 M stock solution. Chloroquine highlighted that, in OVCA cells, ‘a2’ isoform (V-ATPase- V0a2) is overexpressed in cisplatin resistant cells and is a was dissolved in deionized water for a 60 mM stock solution. For long term storage, all stock solutions were stored at -20 C. component of plasma-membrane V-ATPase and the early Selective MEK inhibitor cobimetinib (10mM in DMSO) was endosomal machinery [17, 18]. Inhibition of V-ATPase-V0a2 acidified the cytosol thereby sensitizing the resistant OVCA obtained from ApexBio and the stock solutions were stored at -20 C. cells to platinum mediated DNA damage [18]. However, it is not known how V-ATPase-V0a2 regulates cisplatin sensitivity through the endosome dependent autophagy .. RNA Isolation and Reverse Transcription-PCR. The OVCA cells were washedwith PBS anddetachedusing process. Here, we investigated the relationship between V-ATPase accutase solution (Sigma–Aldrich, St Louis, MO, USA). For RNA extraction, RNeasy mini kit (Qiagen, Valencia, inhibition, cisplatin sensitization, and the autophagy process. CA) was used according to the manufacturer’s instruction. We provide evidence that, in chemoresistant OVCA cells (cis- R), inhibition of V-ATPase-V0a2 blocks the autophagy u fl x Reverse transcription was performed using the high capacity cDNA kit (Applied Biosystems, Foster City, CA) according and suppresses ERK/MEK pathway that promotes cisplatin- to manufacturer’s protocol. All real-time PCR reactions were mediated cell death. Our n fi dings provide a rationale for the utility of V-ATPase-V0a2 inhibitors in combination with performed in triplicate in 10 𝜇 l volume using Universal fast PCR Master Mix reagent (Applied Biosystems, USA) standard drugs as a novel strategy to improve the treatment according to the manufacturer’s instructions. The results efficacy of the chemoresistant ovarian cancer. were analyzed using theΔΔCt method using GAPDH as the endogenous control. For cell death and autophagy pathway 2. Material and Methods analysis (RT2 proler fi , SA Biosciences, Frederick, MD, USA), PCR array-based expression proli fi ng was performed using .. Cell Lines and Cell Culture. Human ovarian carcinoma SYBR-Green method and the results were analyzed using the cell line A2780 (Sigma–Aldrich), its acquired cisplatin resis- tant counterpart cis-A2780, and TOV-112D cell lines were ΔΔCt method using RT profiler PCR data analysis sow ft are employed in this study as described previously [17, 18]. version 3.5 (SA Biosciences). Journal of Oncology 3 .. Antibodies. The following primary antibodies were incubated at 4 C for 30 min, aer ft which the cells were employed in study: rabbit anti-GAPDH [1:400; Cell Signaling centrifuged at 13,000× rpm at 4 C. The supernatant was then Technology (CST); Catalog number-5174S)], rabbit anti- collected. Protein quantification was performed using the LC3B (1:400; CST; 2775S), rabbit anti-beclin1 (1:350; CST; BCA assay (Pierce Protein Biology, USA). The 30 𝜇 gprotein 5174S), rabbit anti-ATG7 (1:400; CST; 8558S), rabbit anti- lysates were boiled with 4X SDS sample bueff r containing 2- P62 (1:400; CST; 5114S), anti-cleaved caspase 8 (1:200; mercaptoethanol and proteins were separated by SDS-PAGE CST; 9748), anti-phospho BRAF (1:1000; CST; 2696T), on 4–20% gradient acrylamide gels. All primary antibody anti-phospho-MEK (1:400; CST; 9154T), Mouse anti-beta incubations were performed 1h at room temperature followed actin (1:10,000; Abcam; ab184220), rabbit anti-LAMP-1(1:250; bysecondaryantibodyincubation(IRdye,Licor)for1hat RT. Abcam; ab25630), rat anti-LAMP-2 (1:250; Abcam; ab25631), The Blots werescanned using theOdyssey  infrared imaging rabbit anti-Fas L (1:100; Abcam; ab15285), rabbit anti-caspase system (LI-COR Biotechnology Lincoln, NE, USA). Blots 3 (1:400; Thermo Fisher; 4331182), rabbit anti-Fas (1:100; were probed with a𝛽 -actin endogenous control antibody to Bio legend; 305611), and mouse anti-a2V (Covance, Denver, confirm equivalent protein loading (Abcam, USA). USA). For isotype-control antibodies, control mouse IgG (R&D Systems) and rabbit IgG isotype (Invitrogen) were . . Immunofluorescence Analysis. For immunouo fl rescence used. Secondary antibodies were as follows: goat anti-rabbit analysis, the cells were plated in 8-well chamber slides (Nunc, IgG-FITC, donkey anti-mouse IgG AF-594, donkey anti- USA) at 3000 cells/well and were incubated overnight at rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC 37 C, 5% CO2. The cells were then washed thrice with PBS (Abcam), donkey anti-rabbit IRDye-800CW, and donkey (containing 0.5% FBS), fixed with 4% paraformaldehyde anti-mouse IRDye-680 CW (LI-COR Bioscience, Lincoln, for 30 min at room temperature (RT), and permeabilized NE). with 0.1% Triton X-100 in PBS for 12 min, 4 C. Block- ing was performed using 3% FBS in PBS for 1 h at RT. .. Immunohistochemical Staining of Ovarian Cancer Tissue. The cells were then incubated with primary antibodies (in To explore the clinical relevance of V0a2 expression in mod- blocking bueff r) for 1h at RT. The cells were then rinsed ulating cisplatin efficacy, we obtained paraffin-embedded thrice with PBST and incubated with secondary antibodies: tissues from ovarian cancer patients who reported to Advo- Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor cate Lutheran General Hospital (ALGH), Chicago, USA. The 594-conjugated goat anti-rabbit secondary antibody (1:200 study was approved by the Ethics Committee of ALGH. Eight dilution) (Invitrogen) dissolved in 3%FBS in PBS for 1 h samples each from the drug responder and nonresponder at RT. The cells were prepared for viewing using ProLong patient group were selected. 5-𝜇 m serially sectioned slides Gold (Invitrogen) mounting medium containing DAPI and were prepared. For normal control tissues, ovarian tissue allowed to polymerize at room temperature for 24 h. For sections from the normal ovary (n=2) were obtained from confocal microscopy, the stained cells were imaged on an Biochain Institute, Inc. (Newark, CA, USA). The horseradish Olympus Fluoview Fv10i confocal microscope. The analy- peroxidase-labeled polymer (EnVision+Dual Link System- sis was performed using Fv10i Flouview Ver.3.0 software. HRP; DAKO, USA) based staining method was used accord- Experiments were repeated at least twice in duplicate. For ing to the manufacturer’s protocol. For antigen retrieval, the immunou fl orescence microscopy, stained cells were imaged sections were boiled in sodium citrate buffer (pH = 6.0) in Olympus microscope and analyzed using NIS-Elements as described previously [18]. The slides were then cooled, sow ft are (Nikon Inc., NY, USA). blocked with 5% BSA in PBS, and incubated with the primary antibody at 4 C overnight. Concurrently, for negative mouse . . Flow Cytometry Analysis. Sh-V0a2 transfected/untrans- isotype-control antibody (R&D systems, USA) was used. The fected cells (2.5 x10 cells/tube) were washed with HBSS anti-rabbit/mouse secondary antibody was then added for 15 containing 0.1% FBS. For surface staining, the cells were min at 37 C. The sections were counterstained with Mayer’s incubated with mouse monoclonal FasL or Fas antibody hematoxylin and mounted in Faramount aqueous mounting conjugated to A or A (Covance, Denver, PA) in PBS for 647 488 medium (Dako). The immunostaining was evaluated by 40 min at RT. For the intracellular staining, the cells were light photomicroscopy (Leica ICC50 W, USA) using a high- fixed and permeabilized using xa fi tion and permeabilization resolution camera. buffer (BD Biosciences, San Jose, CA, USA) and the cells The IHC scoring was performed using the semiquantita- were stained as described above. For the indirect staining, the tive integration method. In this method, vfi e random fields of cells were incubated with unconjugated antibodies (caspase- view were selected for each specimen at high magnification 8) for 1 h at RT and subsequently washed twice with PBS and (×200). The following criteria were employed to generate then stained with conjugated secondary antibody (Abcam, a score: rfi st, staining area score [SAS] ( ≤1%:0;2–25%: 1; USA) for 30 min at RT. Appropriate isotype and unstained 26–50%: 2; 51–75%: 3 and>75%: 4); second, staining intensity controls were used for the experiments. The stained cells were [SI] (light brown: 1; moderate brown: 2 and tan: 3). The IHC analyzed on a BD LSR II flow cytometer with FlowJo software score was calculated using the formula: IHC score= SAS X SI. (Tree Star). Experiments were performed at least twice in duplicate. . . Western Blot Analysis. The harvested cell pellets were resuspended in NP-40 lysis buffer containing protease and .. Assessment of Autophagosomes. For autophagy analysis, phosphatase inhibitors (Pierce Protein Biology, USA) and V0a2 shRNA transfected/untransfected OVCA cells were 4 Journal of Oncology incubated with 20𝜇 M cisplatin for 24h at 37 Cin5% CO2. clinical relevance of V-ATPase-V0a2 in OVCA with varying To determine the induction of autophagy, we used the Cyto- cisplatin sensitivity, we utilized archival OVCA tissues from ID Autophagy detection kit (Enzo Life Sciences, Raams- cisplatin responder (n=8) and nonresponder patients (n=8). donksveer, The Netherlands). The autophagy detection is The OVCA tissue samples were obtained from patients based on monodansylcadaverine dye that specifically stains that were intrinsically responsive/nonresponsive to cisplatin autophagosomes. For positive autophagy controls, the cells (before any treatment). Normal ovarian tissues (n=2) were were treated with mTOR inhibitor rapamycin (0.5 𝜇 M) employed as the control. Both cisplatin responder and or lysosomal alkalizer chloroquine (60𝜇 M) or V-ATPase nonresponder patient tissues exhibited high V-ATPase-V0a2 inhibitor balo fi mycin (50nM). No treatment control wells staining compared to the normal ovary tissues. The V0a2 were also included in each set of experiment. After 24 h, protein expression was higher in cisplatin nonresponder the cells were washed with assay buffer provided by the (IHC score=8.9±1.06) than cisplatin responder patient tissues manufacturer (supplemented with 5% FBS) and stained with (IHC score= 6.5 ± 1.4, p=0.02) or normal ovarian tissues the Cyto-ID green detection reagent for 30 minutes and (IHC score=2.2±0.28; p<0.01) by IHC analysis [Figures 1(a) subsequently washed twice again with assay buffer. The and 1(b)]. The V-ATPase-V0a2 expression thus correlates u fl orescence signal was immediately captured in LSRII flow with drug unresponsiveness in ovarian cancer patients. Our cytometer measuring the intensity in 10,000 cells. FlowJo previous study showed a very poor expression of V-ATPase- software was used to process the imaging data. V0a2 in normal ovary tissues suggesting that V0a2 expression is selectively upregulated during tumorigenesis [17]. Confocal .. Transferrin Internalization Assay. As a measure of early microscopy analysis showed coexpression of V0a2 with endosomal function, the cellular internalization of A - OVCA antigen marker, CA125, conrfi ming V0a2 expression labeled transferrin (Tfn) was assayed. First, the cells were specifically in OVCA cells in drug nonresponder tissues serum starved by rinsing with 37 CHBSS (Invitrogen, USA) [Figures 1(c)(i) and 1(c)(ii)] as well as drug responder tissues and incubated in serum-free RPMI containing 25mM HEPES [Supplementary Figure S1]. These data suggest that V0a2 is and 1% BSA (RPMI-BSA) for 30 minutes at 37 C, 5% CO . a prominent target in ovarian cancer patients with varying Cells were incubated in ice for 10 minutes and then incubated cisplatin sensitivity. Further studies employing tissues from in RPMI-BSA containing 50 𝜇 g/ml of Tfn-A conjugate relapse/posttreatment patients and including more number to allow internalization for upto 30 minutes. Finally, cells of samples will further improve our understanding of V- were quick rinsed at least 10 times with HBSS to remove ATPase role in cisplatin resistance in OVCA. surface labeling. The slides were fixed in 4% formaldehyde for 15 minutes at room temperature. Immune-uo fl rescence .. Cisplatin Resistant Ovarian Cancer Cells Show Low analysis for early endosome labeling (EEA1) was performed Sphere-Forming Ability upon V-ATPase-Va Inhibition. To as described above. The slides were then processed for investigate the possible association of V0a2 with cisplatin fluorescence microscopy. resistance in OVCA, we next performed in vitro assays employing the sh-V0a2-cisR and sh-scr-cisR control cells. .. Cell Cytotoxicity Assay. OVCA cells were seeded into Our previous study showed that stable V-ATPase-V0a2 96-well plate (10,000 cells/well) overnight. The OVCA cells knockdown cells exhibited 3.8-fold inhibition at mRNA level were exposed to cisplatin (0.5, 1, 2.5, 5, 10, 20, and 50𝜇 M) and a 2.5-fold reduction in protein expression compared to and 10nM cobimetinib (MEK inhibitor) for 48h at 37 Cin sh-scr-cisR control cells [18]. 5% CO . After incubation, in vitro cell viability was measured 3D cancer cell spheroids mimic both in vivo architecture using MTS reagent (Promega, USA). Untreated cells were and low drug penetration properties that represent a relevant used as negative control. All experiments were performed model for studying drug resistance [19]. Here, we found in triplicate. The semilog plots of dose-response curves were that, upon V0a2 inhibition, sh-V0a2-cisR cells exhibited a generated using Microsoft Excel (Microso). ft reduced sphere-forming ability compared to control OVCA cells (sh-scr-cis-R) [Figure 2(a)]. Further, upon cisplatin treatment (20𝜇 M, 48h), we observed dissociation of sh-V0a2- 3. Statistical Analysis cisR spheroids while control spheroids remained unaeff cted The means of two data sets were compared and signif- [Figure 2(b)]. There was no dieff rence in the sphere-forming icance was determined by two-tailed Students t-test or ability between cisplatin sensitive (A2780) and cisplatin Mann–Whitney U test. Differences were considered to be sta- resistant cells (cis-A2780; data not shown). tistically significant where p <0.05. The data were graphically represented as the mean± standard deviation of the mean .. V-ATPase-Va Inhibition Enhances Cisplatin-Mediated (SD). Thedata wereanalyzed using GraphPad Prism (version Cell Death by Elevating Both Intrinsic and Extrinsic Apoptosis. 5) statistical sowa ft re. All experiments were repeated at least Upon therapeutic stress, the fate of a cancer cell is decided twice in duplicate. by its apoptotic capacity. Induction of intrinsic apoptosis is the primary mechanism of cisplatin-mediated cell death [20]. To understand the precise mechanism by which V- 4. Results ATPase inhibition leads to sensitization of cisplatin resistant cells, we performed cell death pathway PCR array using .. V-ATPase Va Is Highly Expressed in Cisplatin Non- responder Human Ovarian Cancer Tissues. To elucidate the cisplatin treated sh-V0a2-cis-R and sh-scr-cis-R cells. The Journal of Oncology 5 (i) Cisplatin Non-responder (ii) Cisplatin Responder (iii) Normal ovary 10X Normal ovary cisplatin cisplatin 40X responder non-responder (a) (b) DAPI CA-125 V-ATPase-V0a2 Merge zoom (i) (ii) (c) Figure 1: V-ATPase-Va is highly expressed in cisplatin nonresponder ovarian cancer tissues. (a) Immunohistochemical analysis of V-ATPase- V0a2 expression in tissues from (i) cisplatin nonresponder and (ii) cisplatin responder ovarian cancer patients compared to (iii) normal human ovary tissue. Original magnification × 100 (upper panel) and X 400 (lower panel). (b) The quantitative IHC data expressed as IHC intensity score revealed higher V0a2 expression in ovarian cancer tissues from cisplatin nonresponder patients compared to responder patients and to normal ovarian tissues. (c) Confocal microscopy analysis of V0a2 (green) in nonresponder OVCA tissues ((i) and (ii)) shows its coexpression with ovarian cancer cell marker CA125 (red). Nuclear DAPI staining in blue. Merged areas are shown in yellow. Original magnification: × 600. Zoomed areas represent white boxes in merged figures. Representative images from three independent experiments are shown. proapoptotic genes (caspase 3, FASL, caspase 8, TNF, and mechanism in cisplatin resistant cancer ovarian cancer cells TNFR1) were signicfi antly upregulated (p < 0.05) in V0a2 [21, 22]. For successful the autophagy process, the proton knockdown cells upon cisplatin treatment [Supplementary pumping activity of V-ATPase is necessary for the acidifi- Figure S2]. Flow cytometry analysis revealed that the pro- cation of the endo-lysosomal vesicles mediated degradative tein levels of active caspase 3 were increased in sh-V0a2- stage [23]. Inhibition of autophagy is known to sensitize the cisR cells (p=0.009) relative to sh-scr-cisR. The intrinsic resistant cells to cisplatin treatment [24–26]. We therefore apoptotic proteins, active caspase-9 (p=0.02) and Bax (p studied the effect of blocking V-ATPase-V0a2 on the modu- =0.03) [Figure 2(c)], were also upregulated in sh-V0a2-cis- lation of autophagy in chemoresistant cells. In our previous R. Further, cell membrane-bound FasL and cleaved caspase study, we showed that the sh-V0a2-cisR growth rate was 8 levels, members of the extrinsic apoptotic pathway, were slower than the sh-scr-cisR cells; however, V0a2 inhibition in also elevated [Figure 2(d)] compared to control cells. This itself did not impose any cytotoxicity to the cisplatin resistant indicates that inhibition of V0a2 expression potentiates the cells. In the context of autophagy, here, we found lower cell death activity of cisplatin by stimulating both intrinsic autophagosome numbers in sh-V0a2-cisR compared to sh- and extrinsic apoptotic pathways. scr-cisR cells by confocal microscopy analysis [Figure 3(a)]. Lower autophagosome accumulation was also conrfi med by .. V-ATPase-Va Inhibition Dampens the Protective Auto- flow cytometry analysis [Figure 3(b)]. Further, a significantly reduced LC3B, Beclin-1 [Figure 3(c)], and ATG7 [Figure 3(d)] phagy Levels in Cisplatin Resistant Ovarian Cancer Cells. An levels were observed in sh-V0a2-cis-R compared to control enhanced autophagy process reflects an enhanced survival V0a2 IHC intensity score 6 Journal of Oncology Caspase 3 Caspase 9 Bax 1000 4000 Sh-scr-cis- R Sh-V0a2-cis-R 400 ∗ 800 300 600 200 400 100 200 0 0 0 (a) Sh-scr-cis-R Sh-V0a2-cis-R Sh-scr-cis-R Sh-V0a2-cis-R Sh-scr-cis-R Sh-V0a2-cis-R UT UT UT cisplatin cisplatin cisplatin (c) Sh-scr-cis- R + cisplatin Sh-V0a2-cis-R + cisplatin Caspase 8 Fas Fas-L 800 400 30000 10X 400 200 200 100 0 0 0 Sh-scr-cis-R Sh-V0a2-cis-R sh-scr-cis-R sh-V0a2-cis-R sh-scr-cis-R sh-V0a2-cis-R 40X UT UT UT cisplatin cisplatin cisplatin (b) (d) Figure 2: Inhibition of V-ATPase-Va in resistant ovarian cancer cells blunts spheroid formation and enhances cisplatin-mediated cell death. (a) Photomicrographs showing the effect of shRNA mediated V-ATPase-V0a2 inhibition on the spheroid formation in cisplatin resistant ovarian cancer cells (sh-V0a2-cisR) compared to control cells (sh-scr-cis-R). eTh sh-V0a2-cisR exhibited decreased tumor spheroid formation while control cells formed large clusters of spheroids. (b) Upon cisplatin treatment (20𝜇 M, 48h), an enhanced spheroid dissociation was observed in sh-V0a2-cisR compared to control spheroids. Original magnification: X100, X400. (c) Geometric mean u fl orescence intensity of effector apoptotic protein (cleaved caspase-3), intrinsic apoptotic (active caspase-9, Bax), and (d) of extrinsic apoptotic proteins (cleaved caspase-8, Fas, and FasL) in cisplatin treated sh-V0a2-cisR compared to cisplatin treated sh-V0a2-cisR cells as quantitated by flow cytometry. Each value represents the mean± SD of three independent experiments,∗P< 0.05. cells as determined by western blot analysis indicating that accumulation and a reduction in the amount of internalized the initial autophagy steps are inhibited by V-ATPase inhibi- Tfn compared to control sh-scr-cis-R cells [Figure 4(a)]. tion. This is in contrast to V-ATPase inhibition using chem- When the cells (tfn internalized, 30 min) were xfi ed and ical inhibitors which are known to acutely induce autophagy stained with EEA1 (early endosome marker), an intense as a protective mechanism. Interestingly, autophagy substrate colocalization of Tfn was observed in control sh-scr-cis-R protein P62 was upregulated in sh-V0a2-cisR, suggesting a cells. In contrast, sh-V0a2-cis-R cells showed low transferrin concomitant block in the autophagy u fl x due to interference signal in early endosomes [Figure 4(a)]. Further, LC3B with endosomal function [Figure 3(d)]. stained autophagosomes and EEA-1 exhibited diminished colocalization in sh-V0a2-cis-R cells compared to control cells (sh-scr-cis-R) [Figure 4(b)]. The autophagy vacuoles .. Inhibition of V-ATPase-Va Disrupts Early Endosome (LC3B) colocalized with the late endosomes/lysosomes in Trafficking in Cisplatin Resistant Ovarian Cancer Cells. V0a2 depleted cisplatin resistant cells similar to the control The isoform-specific V-ATPase inhibition impairs specific organellar functions in contrast to the chemical V-ATPase cells [supplementary Figure S3]. Further in-depth studies are required to understand the precise role of functional early inhibitors that target the predominant subunits on cellu- endosomes in autophagy process. lar V-ATPases. For the formation of autophagolysosomes, autophagic vacuole undergoes maturation through fusion with early/late endosomes and lysosomes [27, 28]. Since V0a2 .. Cisplatin Treatment in V-ATPase-Va Inhibited Chem- is primarily localized on the early endosomal membrane to oresistant Cells Induces Autophagy. In the parental cisplatin regulate the vesicular pH, we rfi st analyzed the effect of V0a2 sensitive OVCA cells, autophagy overactivation is a known knockdown on early endosomal trafficking. To measure the contributor to cisplatin-mediated cell death [22]. In line with function of the early endosome, we examined transferrin the previous reports, we observed an enhanced autophagy (Tfn) uptake, using Alexa -labeled Tfn. In sh-V0a2-cis- response in cisplatin sensitive OVCA cells (Cis-S) upon R cells, a 30-min incubation with Tfn showed a surface cisplatin treatment. There were an increased autophagosome Untreated MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) Journal of Oncology 7 sh-V0a2-cis-R sh-scr-cis-R sh-scr-cisR sh-V0a2-cisR (a) (b) Cis-S sh-scr-cisR sh-V0a2-cisR 75kD ATG7 Fold change 0.8 1 0.6 Normal ovary Cis-S sh-scr-cisR sh-V0a2-cisR 19kD LC3I -Actin 17kD LC3 II Fold change 0.16 0.47 1 0.34 62kD P62 60kD Beclin1 1.9 1 3.5 Fold change 0.2 0.44 1 0.21 Fold change 47kD -Actin -Actin (c) (d) Figure 3: Protective autophagy is dampened upon V-ATPase-Va inhibition in cisplatin resistant ovarian cancer cells. (a) Confocal microscopy analysis of the subcellular distributions of LC3B levels (red) in V-ATPase-V0a2 inhibited cisplatin resistant cells (sh-V0a2-cisR) compared to control (sh-scr-cis-R) cells. eTh sh-V0a2-cisR exhibit low LC3B staining compared to control cells (x600 magnification). (b) Geometric mean u fl orescence intensity (MFU) of LC3B levels in sh-V0a2-cisR compared to the levels in control (sh-scr-cisR) cells as quantified by flow cytometry. (c) Western blot analysis of the autophagy associated proteins LC3 (I and II) and Beclin1 in sh-V0a2-cisR shows decreased expression compared to control cells (sh-scr-cis-R), similar to cisplatin sensitive parental OVCA cells (cis-S). (d) Western blot analysis of ATG-7 shows lower expression upon V-ATPase V0a2 inhibition while P62 (autophagy substrate protein) shows higher expression in sh- V0a2-cisR compared to control cells (sh-scr-cis-R). Fold change in band densities were measured relative to the control (sh-scr-cisR) and the samples were normalized to endogenous beta-actin levels. Representative images from three independent experiments are shown here. number and decreased P62 levels [Supplementary Figure S4] there was a concomitant accumulation of autophagosomes in cisplatin treated cis-S cells. However, the cisplatin resistant with high accumulation of P62 suggesting an overall block cells are known to depict a protective autophagy response that in autophagy ux fl that facilitates cisplatin-mediated cell counteracts apoptotic cell death. Moreover, pharmacological death [Figures 5(c)(i) and 5(c)(ii)]. Flow cytometry analysis inhibition of autophagy either by inhibiting ATGs, beclin, or confirmed upregulated expression of autophagy initiation lysosomal inhibitors enhances cisplatin-mediated apoptosis proteins Beclin-1 and ATG7 [p<0.05; Figures 5(d) and [21, 24–26]. Here, sh-V0a2-cisR when treated with cisplatin 5(e)]. showed induction of several autophagy initiation related genessuch asMAPLC3A, IGFR, and Atg7[Supplementary . . Enhanced Cisplatin Sensitization upon V-ATPase In- Figure S5]. The upregulation in the autophagy initiation hibition Involves Suppression of ERK/MEK Pathway in proteins was conrfi med by ow fl cytometry. The sh-V0a2-cisR Resistant Ovarian Cancer Cells. Upregulation of oncogenic cells exhibited higher autophagosome accumulation upon Ras pathway has been observed in chemoresistant cancer cisplatin treatment compared to sh-scr-cisR as determined cells. The Ras pathway enhances DNA-repair through the by confocal microscopy analysis [Figure 5(a)]. This enhanced Ras/PI3K/Rac1 pathway [29, 30] to protect against cisplatin- autophagosome accumulation was further conrfi med by ow fl induced therapeutic stress. In addition, inhibition of Ras cytometry analysis of LC3B protein in sh-V0a2-cisR and sh- may enhance the cisplatin sensitivity of human glioblas- scr-cisR [Figure 5(b)]. x Therefore, upon cisplatin treatment toma [31]. In the present study, the mRNA prolin fi g of the of sh-V0a2-cis-R, in spite of enhanced autophagy initiation, Ras pathway array of the sh-V0a2-cisR revealed significant LC3B LC3B MFU (Mean ± SD) 8 Journal of Oncology EEA1 Transferrin (30 min) Merge + DAPI (a) Rab5 LC3B Merge (b) Figure 4: Inhibition of V-ATPase-Va disrupts early endosome trafficking in cisplatin resistant ovarian cancer cells. (a) Cisplatin resistant OVCA cells were treated with control shRNA (sh-scr-cis-R) or with shRNA against V-ATPase-V0a2-cisR (sh-V0a2-cis-R). eTh cells were incubated with Tf-Alexa at 37 C for 30 min to label the entire early endosomal compartment. eTh cells were fixed before permeabilization and were stained with anti-EEA1 (green). Merged images of shV0a2-cis-R and sh-scr-cis-R cells. Yellow color indicates colocalization between Tf-Alexa and EEA1. (b) Immunofluorescence analysis of the subcellular distributions of LC3B (red) and Rab5 (early endosome marker; green) in sh-V0a2-cis-R compared to control (sh-scr-cis-R) cells. eTh sh-V0a2-cisR exhibited low poor LC3B/Rab5 colocalization compared to control cells (x600 magnification). downregulation of certain Ras pathway genes (EGFR, Fos, cells (cis-A2780) suggesting a varied activation and role of BRAF, GRB2, ELK, Raf1, and Myc) [Figure 6(a)]. The western ERK/MEK pathway in cisplatin resistance. Taken together, blot analysis conrfi med downregulated phosphorylation of these findings indicate that V-ATPase mediated inhibition MEK1/2 and BRAF, thus confirming the suppression of of autophagy ux fl contributed to the reversal of cisplatin ERK/MEK pathway in these cells compared to sh-scr-cisR resistance in resistant ovarian cancer cells. Knockdown of control cells [Figure 6(b)]. Further, the treatment of cisplatin V0a2 or use of MEK inhibitors suppresses ERK activation and resistant ovarian cancer cells (cis-A2780) with the MEK1/2 blocks the autophagy while increasing cisplatin-induced cell inhibitor cobimetinib sensitized the human ovarian cancer death. cell lines to cisplatin-induced cell death. Cisplatin alone did not elicit significant cell death, whereas enhanced cell death 5. Discussion was seen when cells were treated for 48 h with cisplatin in combination with 10nM cobimetinib [Figure 6(c)]. At the Ovarian cancer is the leading cause of cancer-related deaths same concentration of cobimetinib, an enhanced cell death in women due to high treatment failure rates [32]. To improve was observed more prominently in cisplatin sensitive cell the OVCA patient outcome, it is imperative to understand the lines (A2780 and TOV-S) compared to cisplatin resistant chemoresistance associated pathways to identify the mode Sh-V0a2-cis-R Sh-scr-cis-R Sh-scr-cis-R Sh-V0a2-cis-R Journal of Oncology 9 (i) (ii) Untreated + Cisplatin sh-V0a2-cisR sh-scr-cisR 5000 4000 0 0 (a) (b) Beclin1 1800 ∗ Sh-scr-cisR Sh-V0a2-cisR 1600 (i) (ii) UT +cis,10M +cis,20M UT +cis,10M +cis,20M LC3I LC3I 600 LC3II LC3II Fold change 1 0.42 0.28 1 1.1 1.6 Fold change +cis +cis +cis +cis UT UT P62 P62 10M 20M 10M 20M Sh-scr-cisR Sh-V0a2-cisR Fold change Fold change 1 0.8 1.8 1 0.67 0.43 (d) ATG5 ATG5 ATG7 Fold change Fold change 1 0.5 0.42 1 1.1 1.4 -actin -actin (c) UT +cis +cis UT +cis +cis 10M 20M 10M 20M Sh-scr-cisR Sh-V0a2-cisR (e) Figure 5: Cisplatin induces protective autophagy in V-ATPase-Va inhibited resistant ovarian cancer cells with a concomitant block in autophagy flux leading to drug sensitization. V-ATPase-V0a2 inhibited cisplatin resistant ovarian cancer cells (sh-V0a2-cisR) were treated with cisplatin (20𝜇 g/ml, 48h). (a) Confocal microscopy analysis of the subcellular distributions of LC3B levels (red); nucleus is stained with DAPI (blue). Upon cisplatin treatment, there is a higher accumulation of LC3B in sh-V0a2-cisR compared to untreated cells. eTh uo fl rescence signals of LC3B were sequentially acquired using an Olympus FluoView confocal microscope. Representative confocal micrographs (original magnification: 80X) are shown. Bars, 5 𝜇 m. (b) Geometric mean u fl orescence intensity units (MFU) of LC3B levels in sh-V0a2-cisR compared to the levels in untreated cells as quantified by flow cytometry. (c) Western blot analysis of the autophagy associated proteins LC3, P62, and ATG5 in (i) control sh-scr-cisR cells and (ii) sh-V0a2-cisR cells upon cisplatin treatment. Geometric mean u fl orescence intensity of (d) beclin1 protein levels and (E) ATG7 protein levels in cisplatin treated/untreated sh-V0a2-cisR compared to control cells (sh-scr-cisR) as quantified by flow cytometry. ∗ p<0.05. Experiments were repeated twice in duplicate. for sensitizing cancer cells to therapy [20]. The prominent is therefore highly debatable and reports on the anticancer mechanism of chemoresistance includes resistance to apopto- effects upon autophagy inhibition or its overactivation are sis [33]. Alternative cell survival pathways such as autophagy equally available [37, 38]. In some cases, overactive autophagy have become a critical area of research [34, 35]. The process induces cell death necessary for the cytotoxic effect of the of tumorigenesis exhibits varied dependence on autophagy therapy. Therefore, autophagy inducers such as the mTOR during progression from primary tumor to metastatic stage or inhibitors have also been considered as potential cancer during chemoresistance [36]. The role of autophagy in cancer treatments [39–41]. sh-scr-cis-R sh-V0a2-cis-R MFU (Mean ± SD) UT + Cisplatin + Bafilomycin + Chloroquine MFU (Mean ± SD) MFU (Mean ± SD) MFU (Mean ± SD) UT + Cisplatin + Bafilomycin + Chloroquine 10 Journal of Oncology Normal ovary Cis-S sh-scr-cisR sh-V0a2-cisR P-MEK 1/2 Fold change 0.27 0.27 1 1 0. 0.65 65 EGFR Fos GRB2 ELK1 Raf1 Myc B-Raf −5 -actin −10 −15 s sh-s h-scr cr-ci -cisR sR C Cis-S is-S s sh-V0a2-cisR h-V0a2-cisR −20 P-B-RAF B-RAF −25 −30 Fold ch ha ange nge 1 0.85 0.71 −35 -actin −40 (a) (b) TOV-112D Cis-A2780 A2780 100 100 −10 0 0 0.5 1 2.5 5 10 0 0.5 1 2.5 5 10 Cisplatin concentration (M) Cisplatin concentration (M) 0 0.5 1 2.5 5 10 20 50 cisplatin cisplatin Cisplatin concentration (M) cisplatin + cobimetinib(10 nM) cisplatin + cobimetinib(10 nM) cisplatin cisplatin + cobimetinib(10 nM) (c) Figure 6: V-ATPase-VainhibitionsensitizesthecisplatinresistantovariancancercellsthroughdownregulationoftheRaspathway. The shRNA mediated V-ATPase-V0a2 inhibition was carried out in cisplatin resistant ovarian cancer cells (sh-V0a2-cisR). Cisplatin treated (20𝜇 g/ml, 48h), sh-V0a2-cisR and control cells (sh-scr-cisR) were analyzed for Ras pathway. (a) Transcriptional profiling of the Ras pathway array showed significant downregulation of the Ras pathway associated genes (EGFR, Fos2, GRB2, Raf1, Elk-1 Myc, and B-Raf). (b) Western blot analysis showed downregulation of phosphorylated B-Raf and MEK 1/2. Fold change in band densities was measured relative to the control (sh-scr-cisR) and the samples were normalized to endogenous beta-actin levels. (c) Combination of cisplatin and MEK inhibitor cobimetinib (10nM) showed enhanced cell death in three ovarian cancer cell lines (A2780, TOV-112D, and cis-A2780). The chemoresistant cancer cells, however, exhibit a dis- space, primarily driven by proton pumps V-ATPases [45– tinct relation with the autophagy process. Several studies have 47]. This altered pH gradient interferes with drug uptake revealed that cisplatin resistant ovarian cancer cells express and metabolism in cancer cells [48]. Regulated assembly high levels of autophagy as a survival mechanism [21, 22]. The of the V-ATPase V0 and V1 domain occurs in response fact that anticancer drugs frequently induce cytoprotective to glucose starvation and involves both PI3K and AMPK autophagy has provided the basis for combination therapy tri- pathway, which in turn modulate autophagy [49]. In this als using autophagy-blocking agents with standard antitumor context, it will be interesting to examine the long term drugs [42]. Several clinical trials are investigating the efficacy effect of dysfunctional V-ATPase complex and its effect on of autophagy inhibition with conventional chemotherapy AMPK pathway. In the previous studies, we identified V- in various types of cancers [43]. Given that autophagy is ATPase-V0a2 as a tumor-associated isoform that is distinctly also a basic physiological mechanism in all cells [8, 44], overexpressed in cisplatin resistant ovarian cancer cells, the inhibition of which sensitized the cells to cisplatin treatment directly targeting the autophagy in cancer leads to unwanted consequences in normal cells. It is therefore vital to identify [17, 18]. In continuation with our efforts, in the present indirect autophagy modulators specicfi for cancer cells that study, we demonstrate that the inhibition of V-ATPase-V0a2 can be effectively targeted for anticancer therapy. sensitizes cisplatin resistant OVCA cells by direct modulation Cancer drug resistance is associated with an altered pH of autophagy process. Cisplatin treatment induces extrin- gradient between the cytosol and extracellular/intravesicular sic and intrinsic apoptotic pathways upon V-ATPase-V0a2 % cell survival Fold downregulation to control % cell survival % cell survival Journal of Oncology 11 Sh-scr-cisR Sh-V0a2-cisR +cisplatin Sh-V0a2-cisR (a) (b) (c) Cisplatin treatment V-ATPase Cell death inhibition Cisplatin resistant ovarian cancer cells (1) Dampened protective autophagy, early (1) Cisplatin induced autophagy, high endosomal dysfunction, block in autophagy flux Enhanced autophagy acts as cell survival factor autophagosome accumulation (2) Suppressed ERK/MEK pathway (2) Concomitant block in autophagy flux Cisplatin sensitization due to Inhibition of protective autophagy V-ATPase block in autophagy flux, and block in autophagy flux endosomes enhanced cell death Autophagosome V-ATPase-V0a2 inhibition Block in Autophagy flux cisplatin Figure 7: V-ATPase-Va isoform inhibition mediates cisplatin-associated cell death in resistant ovarian cancer through a block in autophagy flux. (a) Cisplatin resistant ovarian cancer cells exhibit enhanced autophagy that favors cell survival under stress. (b) Upon V-ATPase inhibition, the autophagy initiation proteins are dampened. er Th e is a block in autophagy u fl x reflected by high P62 accumulation. Suppressed ERK/MEK pathway is also observed. (c) Cisplatin treatment of V-ATPase inhibited cells triggers autophagy; however, there is a concomitant block in autophagy u fl x, leading to autophagosome accumulation and enhanced cell death. isoform inhibition in resistant OVCA cells with inhibition in OVCA tissue samples from relapse/posttreatment patients of protective autophagy. This makes the chemoresistant cells to improve our understanding of the acquired chemore- susceptible to any further genotoxic stress. Our data is in sistance. Such tools will greatly improve the prediction of line with the previous reports suggesting that inhibition of sensitivity or resistance to chemotherapy and allow treatment autophagy sensitizes acquired cis-R cells to cisplatin [21, 22]. stratification. Upon cisplatin treatment, sh-V0a2-cisR cells show induc- Cisplatin resistant cancer cells exhibit defective steps in tion of autophagy pathway as a prosurvival mechanism; how- apoptosis with decreased expression of proapoptotic proteins such as BAD, Bid, and caspases 4 and 6 [3, 20, 54]. Our ever, the accumulation of autophagy substrate P62 confirmed a concomitant block in the overall autophagy flux that drives previous study demonstrated that the apoptotic rate of V0a2 the cells towards cell death. V0a2 inhibition suppressed the inhibited cisplatin resistance cells was higher than in the ERK/MEK pathway in cis-R cells. Our findings provide key control group [18]. Here, we confirm that cisplatin treatment evidence that the isoform-specific inhibition of V-ATPase- effectively induces both intrinsic and extrinsic apoptosis in V0a2 inhibits autophagy that contributes to cisplatin sensi- resistant OVCA cells upon V0a2 inhibition by the activation tization in resistant OVCA cells [Figure 7]. of caspase-3, caspase-8, caspase-9, and caspase-7. Previous studies have confirmed that V-ATPase proton However, targeting the apoptotic mechanisms does not pump subunits are inducible by cisplatin treatment [50]. This optimally inhibit chemoresistance [55, 56]. It is therefore prevents cytosolic acidification of cancer cells that is a trigger critical to exploit the alternative pathways, such as autophagy of apoptosis [51]. Further, the acquired cisplatin resistant to counter chemoresistance. Targeting the autophagy for reg- ulation of cancer chemoresistance is a therapeutic strategy yet cells show upregulated expression of V-ATPase subunits [50, 52]. V-ATPase driven proton flux causes acidicfi ation of to be properly designed. With regard to cisplatin resistance, intracellular vesicles as well as the acidification of the extra- several reports suggest that protective autophagy inhibition cellular microenvironment of cancer cells. This interferes is particularly helpful in chemosensitization [22, 57–59]. Our with drug-induced cytotoxicity in addition to promoting study confirms that V-ATPase-V0a2 inhibition significantly cancer invasiveness [53]. inhibits autophagy process with downregulation of beclin-1 In clinical tissues, there are no detailed reports on the and LC3 levels compared to control cells. After cisplatin treat- correlation between V-ATPase expression and drug respon- ment, several early autophagy-related genes were induced siveness in ovarian cancer patients. Our study demonstrates in response to drug-induced stress; however, there was a for the first time increased expression of V-ATPase-V0a2 in concomitant accumulation of autophagosomes and elevated clinical tissues obtained from cisplatin nonresponder patients P62 levels suggesting an overall block in autophagy u fl x that facilitates cisplatin-mediated cell death. compared to the treatment-responder patients. The data pro- vided here include samples showing intrinsic unresponsive- For successful the autophagy process, a fully functional ness to the treatment. Further studies are therefore required endo-lysosomal system is highly critical. For autophagic 12 Journal of Oncology vacuole (AV) maturation, a sequential fusion of AVs with dif- previous observations, we observed suppression of activated ferent populations of early and late endosomes and lysosomes MEK1/2 and B-RAF in V-ATPase-V0a2 inhibited cisplatin [27, 60] is essential which highlights the importance of dif- resistant OVCA cells compared to control. The combination treatment of small molecular MEK1/2 inhibitor, cobimetinib ferent vesicles of the endo-lysosomal pathway in autophagy. Chloroquine (CQ) derivative, hydroxychloroquine (HCQ), along with cisplatin, could enhance cisplatin-mediated cell the only clinically approved autophagy inhibitor, is presently death in OVCA cells. The combination of MEK inhibitor with cisplatin, however, is more effective in sensitive cells under clinical trials as mono- or combination therapy against various types of cancers [61]. HCQ gets sequestered in compared to resistant cells, indicating that there are other the acidic vesicles such as autolysosomes making them significant players in chemoresistance. alkaline, thereby hindering the degradative steps. However, Taken together, these findings indicate that inhibition of CQ derivatives are known to produce harmful side effects autophagy contributes to the reversal of cisplatin resistance in ovarian cancer cells. From the results obtained in this study, specifically on heart and kidney. Similarly, bafilomycin is another known chemical that inhibits V-ATPase and disrupts we propose that targeting V-ATPase-V0a2 is an effective lysosomal acidification and autophagy that contributes to strategy in sensitizing the chemoresistant ovarian cancer cells to cisplatin treatment. tumor cell death; however, it also interferes with Ca2+ pump SERCA [62]. Proton pump inhibitors (PPI) such as omepra- zole and pantoprazole induce the early accumulation of Data Availability autophagosomes, with concomitant inhibition of autophagic ufl x [63, 64].Considering thesideeeff ctsofthe knownchem- The data used to support the findings of this study are ical autophagy and V-ATPase inhibitors, V-ATPase-V0a2 available from the corresponding author upon request. isoform-specific targeting for V-ATPase/autophagy inhibi- tion will provide a safer anticancer alternative. In addition, Conflicts of Interest due to the absence of V0a2 isoform on renal cells (where a4 isoform is predominant), targeted V0a2 based therapy may The authors declare no conflicts of interest. have fewer renal associated side effects. Recently, functional early endosomes were found to be essential for successful autophagy. Defects in early endosome Authors’ Contributions function led to an accumulation of autophagosomes and Arpita Kulshrestha and Gajendra K. Katara contributed to inhibition of autophagy [10]. V0a2 is found in the early conception and design. Arpita Kulshrestha, Gajendra K. endosomes and is also known to regulate protein degradative Katara, and Safaa A. Ibrahim contributed to development of pathway through interaction with Arf6 and ARNO [65]. V- methodology. Arpita Kulshrestha, Gajendra K. Katara, Safaa ATPase inhibition led to dysregulation of Notch signaling due A. Ibrahim, and Kyle W. Meinke contributed to acquisition of to endosomal acidification [66]. Here, V-ATPase inhibition data. Arpita Kulshrestha and Gajendra K. Katara contributed by V0a2 knockdown indicates defective early endosomal to analysis and interpretation of data. Arpita Kulshrestha, function that may contribute to modulation of autophagy Gajendra K. Katara, and Kenneth D. Beaman contributed process. Further in-depth studies are required to decipher to writing and review. Arpita Kulshrestha, Gajendra K. the exact mechanism of V0a2 mediated regulation of early Katara, Safaa A. Ibrahim, M. Sahoo, Valerie Riehl, Kenneth endosomal function. D. Beaman, J. Dolan, and Michael R. Pins contributed to The mechanism of chemoresistance is dependent on the administrative, technical, or material support (i.e., report- balance of the activities of different intracellular signaling ing or organizing data and constructing databases). Arpita systems. There are extensive reports on the involvement Kulshrestha and Kenneth D. Beaman contributed to study of survival signals such as MAP kinase subfamilies in supervision. regulating autophagy [67, 68]. Several studies indicate that ERK (extracellular signal-regulated kinase), one of the six known mammalian MAPK pathways, is aberrantly activated Acknowledgments in cancer cells and promotes cancer cell proliferation, sup- presses apoptosis, and enhances metastasis/drug resistance. The authors thank Dr. Patricia Loomis and Robert Dick- ERK (ERK1 and ERK2) is activated upon phosphorylation inson for technical assistance and the Rosalind Franklin by MEK (MEK1 and MEK2), which is activated upon University of Medicine and Science Confocal Microscopy phosphorylation by Raf (Raf-1, B-Raf, and A-Raf). ERK and Flow Cytometry Core Facility. We thank Advocate pathway activation promotes autophagy, leading to cisplatin Lutheran General Hospital James R. & Helen D. Russell resistance. Inhibition of ERK activation enhances cisplatin- Institute for Research and Innovation, Park Ridge, Illinois, induced growth inhibition [69]. In cisplatin resistant squa- for their assistance in obtaining specimens and obtaining mous cancer cells, both ERK (extracellular signal-regulated IRB approval. This work was partially supported by grants kinase) activation and autophagy induction are observed from the ALGH-RFUMS collaborative research involving [70]. 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