Selective inhibition of tumor cell associated Vacuolar-ATPase 'a2' isoform overcomes cisplatin resistance in ovarian cancer cells: Beyond the Abstract

Ovarian cancer is the deadliest among women related cancers due to problems in early detection and treatment. It is seen that initially, the cancer responds well to therapy; however, in majority of advanced cases, the ovarian tumors relapse with a resistant disease profile. 

The relapsed tumors show unresponsiveness to the mainstay chemotherapy such as platinum compounds (cisplatin/carboplatin) as well as cross-resistance to mechanistically unrelated drugs1. At cellular level, chemo-resistance is a multi-factorial process arising due to an increased pumping out of the drug, drug inactivation, intensified DNA repair, increased anti-apoptotic/ cell death mechanisms or failure to transmit DNA damage response to the cell death signaling machinery 2, 3

Several investigators have tested the combinatorial efficacy of molecular therapies targeted against two or more signaling pathways. The idea of combination therapy relies on the fact that chemo-resistant cancers arise dynamically from diverse changes at genetic and signaling levels. We envision an alternate approach to improve the treatment efficacy by targeting a more centralized mechanism of chemo-resistance i.e., tumor pH. 

In normal cells, the intracellular pH is slightly more acidic than the extracellular pH. In cancer cells however, as a survival mechanism, this pH gradient is reversed. Protons produced by glycolysis are extruded outside the cancer cells to prevent acidosis-induced cell death. As a result, the extracellular pH becomes acidic and the intracellular pH becomes alkaline 4.  This modulates signaling pathways and favors cancer spread as well as tolerance to therapy 5. Many investigators have now focused their studies towards targeting the ‘altered pH gradient’ to overcome the chemotherapy resistance. 

At the heart of regulating the acidity is the vacuolar (V-) ATPase, which is our molecule of interest. V-ATPases are protein complexes that are abundantly expressed in ovarian cancer cells. V-ATPases act just like nano-motors on the cancer cell surface and intracellular vesicles and pump protons out of the cells or into the vesicular lumen, depending on their location6. V-ATPases also increase in response to cisplatin and are over-expressed in drug resistant cells7.

 We reason that the targeted therapies directed against V-ATPases in ovarian cancer will improve the treatment efficacy. However, many V-ATPase subunits are also involved in normal physiological functions and therefore targeting these subunits poses cytotoxicity issues. V-ATPase 'a' subunit is involved in pH sensing and intracellular targeting of the V-ATPase complex8. Our lab is working on one of the ‘a’ subunit isoforms (V-ATPase-V0a2) that is exclusively over-expressed on the surface of cisplatin resistant ovarian cancer cells.  This is particularly important as this suggests that V0a2 regulates the altered pH gradient in these cells. V-ATPase-V0a2 is highly expressed in low and high grade human ovarian cancer tissues. Inhibition of V0a2 in cisplatin resistant cells acidified the cytosolic pH and improved the response to cisplatin therapy. Many investigators have shown that there is reduction in cytosolic pH when the function of the V-ATPase is compromised9.

To understand the concept better, it is important to know how V-ATPase-V0a2 isoform regulates chemotherapy resistance. Many investigators have shown that in sensitive cells, cisplatin binds to DNA and forms double stranded DNA breaks. This recruits damage recognition proteins that transmit signals leading to irreversible cell death program10. In cisplatin resistant cells, there is a reduced cisplatin-DNA binding because the drug is available in neutral form at alkaline intracellular pH. However, in the V0a2 silenced cells, the acidified cytosolic pH favors efficient cisplatin activation and enhanced DNA damage11. Further, at acidic pH, DNA is more prone to damage by cisplatin because of defective DNA damage repair mechanisms12.

Many investigators have shown that proton pump inhibitors (omeprazole, esomeprazole) normalize the intra- and extra-cellular pH in cancer, and when used in combination, these improve the efficacy of the anti-cancer drugs 13, 14. Since proton pump inhibitors target both cancer and normal cells, we reason that isoform specific inhibition of V0a2 is more selective towards cancer cells and will minimize the cytotoxicity to normal cells. 

Our lab has generated a specific monoclonal antibody (2C1) against cell surface associated V0a2. Unlike chemical reagents such as bafilomycin A, concanamycin A, or proton pump inhibitors that also block intracellular V-ATPases, this antibody targets surface V-ATPases that are uniquely present on cancer cells. In our study, combination treatment of cisplatin and 2C1 improved cytotoxicity in resistant cancer cells. We therefore envision that the cell surface form of the V-ATPase is a potentially important target for overcoming cisplatin resistance in ovarian cancer.

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Written By: Arpita Kulshrestha1, Gajendra K. Katara1, Kenneth D. Beaman1
1Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA

References:
1.Tummala MK.  Recurrent ovarian cancer. Clin Adv Hematol Oncol 2005; 3: 723-36.
2.Kartalou M, Essigmann JM. Mechanisms of resistance to cisplatin. Mutat Res 2001; 478: 23–43. 
3.Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003; 22:7265-79.
4.Webb BA, Chimenti M, Jacobson MP, Barber DL. Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer 2011; 11:671-7. 
5.Barar J, Omidi Y. Dysregulated pH in Tumor Microenvironment Checkmates Cancer Therapy. Bioimpacts 2013; 3:149–62. 
6.Nishi T, Forgac M. The vacuolar (H+)-ATPases- nature's most versatile proton pumps. Nat Rev Mol Cell Biol 2002; 3: 94-103. 
7.Murakami T, Shibuya I, Ise T, Chen ZS, Akiyama S, Nakagawa M, Izumi H, Nakamura T, Matsuo K, Yamada Y, Kohno K. Elevated expression of vacuolar proton pump genes and cellular PH in cisplatin resistance. Int J Cancer 2001; 93:869-74.
8.Recchi C, Chavrier P. V-ATPase: a potential pH sensor. Nat Cell Biol 2006; 8:107–9. 
9.Moreno SN, Zhong L, Lu HG, Souza WD, Benchimol M. Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites. Biochem J 1998; 330: 853-60. 
10.Basu A, Krishnamurthy S. Cellular Responses to Cisplatin-Induced DNA Damage. J Nucleic Acids 2010; 2010.
11.Liao C, Hu B, Arno MJ, Panaretou B. Genomic screening in vivo reveals the role played by vacuolar H+ ATPase and cytosolic acidification in sensitivity to DNA-damaging agents such as cisplatin. Mol Pharmacol 2007; 71:416-25.
12.Robinson H, van der Marel GA, van Boom JH, Wang AH. Unusual DNA conformation at low pH revealed by NMR: parallel-stranded DNA duplex with homo base pairs. Biochemistry 1992; 31:10510-7.
13.Spugnini EP, CitroG, Fais S.  Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy. J Exp Clin Cancer Res 2010; 29:44.
14.Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Montinaro A,et al. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst 2004; 96:1702-13.