The ENTPD5/mt-PCPH oncoprotein is a catalytically inactive member of the ectonucleoside triphosphate diphosphohydrolase family

MacCarthy,Caitlin M.; Notario,Vicente

doi:10.3892/ijo.2013.2052

The ENTPD5/mt-PCPH oncoprotein is a catalytically inactive member of the ectonucleoside triphosphate diphosphohydrolase family

Authors:
- Caitlin M. MacCarthy
- Vicente Notario
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Affiliations: Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
Published online on: August 6, 2013 https://doi.org/10.3892/ijo.2013.2052
Pages: 1244-1252

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Abstract

Expression of the ENTPD5/mt-PCPH oncoprotein and overexpression of the normal ENTPD5/PCPH protein contribute to the malignant transformation of diverse mammalian cell types, and PCPH is mutated and/or deregulated in various human tumor types. Expression of PCPH or mt-PCPH caused similar phenotypes, yet the effects promoted by mt-PCPH expression were consistently and substantially greater. ATP depletion and increased stress‑resistance are phenotypes commonly associated with PCPH and mt-PCPH expression. It was suggested that the intrinsic nucleoside triphosphate diphosphohydrolase (NTPDase) activity of PCPH and mt-PCPH may be responsible for these phenotypes, but direct supporting evidence remains to be established. Results from experiments designed to test such hypothesis demonstrate that, as expected, mt-PCPH expression in human colorectal carcinoma (CRC) cells decreased their ATP levels and conferred resistance to oxaliplatin, a colorectal cancer-relevant chemotherapeutic agent. Using a combination of site-directed mutagenesis, immunoprecipitation methods, in vitro enzyme activity assays and in situ enzyme activity determinations in live cells, this report also demonstrates that the mt-PCPH oncoprotein lacks detectable NTPDase activity, indicating that direct ATP cleavage by mt-PCPH did not cause the ATP depletion observed in mt-PCPH-expressing CRC cells. These results strongly suggest that the mt-PCPH oncoprotein may regulate the cellular energy levels and subsequent chemoresistance by an NTPDase-independent mechanism. Understanding possible alternative mechanisms will be essential to devise strategies for the successful treatment of predictably therapeutically resistant tumors expressing either increased PCPH levels or, particularly, the mt-PCPH oncoprotein.

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1.	Cairns RA, Harris IS and Mak TW: Regulation of cancer cell metabolism. Nat Rev Cancer. 11:85–95. 2011. View Article : Google Scholar : PubMed/NCBI
2.	Warburg O: On respiratory impairment in cancer cells. Science. 124:269–270. 1956.PubMed/NCBI
3.	Sattler UG, Hirschhaeuser F and Mueller-Klieser WF: Manipulation of glycolysis in malignant tumors: fantasy or therapy? Curr Med Chem. 17:96–108. 2010. View Article : Google Scholar : PubMed/NCBI
4.	Hardt PD, Mazurek S, Toepler M, et al: Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer. Br J Cancer. 91:980–984. 2004.PubMed/NCBI
5.	Chan EC, Koh PK, Mal M, et al: Metabolic profiling of human colorectal cancer using high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and gas chromatography mass spectrometry (GC/MS). J Proteome Res. 8:352–361. 2009. View Article : Google Scholar
6.	Walenta S, Chau TV, Schroeder T, et al: Metabolic classification of human rectal adenocarcinomas: a novel guideline for clinical oncologists? J Cancer Res Clin Oncol. 129:321–326. 2003. View Article : Google Scholar : PubMed/NCBI
7.	Robson SC, Sevigny J and Zimmermann H: The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance. Purinergic Signal. 2:409–430. 2006. View Article : Google Scholar : PubMed/NCBI
8.	Enjyoji K, Kotani K, Thukral C, et al: Deletion of cd39/entpd1 results in hepatic insulin resistance. Diabetes. 57:2311–2320. 2008. View Article : Google Scholar : PubMed/NCBI
9.	Read R, Hansen G, Kramer J, Finch R, Li L and Vogel P: Ectonucleoside triphosphate diphosphohydrolase type 5 (Entpd5)-deficient mice develop progressive hepatopathy, hepatocellular tumors, and spermatogenic arrest. Vet Pathol. 46:491–504. 2009. View Article : Google Scholar
10.	Notario V, Castro R, Flessate DM, Doniger J and DiPaolo JA: Frequent activation of non-ras transforming sequences in neoplastic Syrian hamster cells initiated with chemical carcinogens. Oncogene. 5:1425–1430. 1990.PubMed/NCBI
11.	Velasco JA, Avila MA and Notario V: The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress. Oncogene. 18:689–701. 1999. View Article : Google Scholar : PubMed/NCBI
12.	Fang M, Shen Z, Huang S, et al: The ER UDPase ENTPD5 promotes protein N-glycosylation, the Warburg effect, and proliferation in the PTEN pathway. Cell. 143:711–724. 2010. View Article : Google Scholar : PubMed/NCBI
13.	Recio JA, Páez JG, Sanders S, Kawakami T and Notario V: Partial depletion of intracellular ATP mediates the stress-survival function of the PCPH oncoprotein. Cancer Res. 62:2690–2694. 2002.PubMed/NCBI
14.	Tirado OM, Mateo-Lozano S, Sanders S, Dettin LE and Notario V: The PCPH oncoprotein antagonizes the proapoptotic role of the mammalian target of rapamycin in the response of normal fibroblasts to ionizing radiation. Cancer Res. 63:6290–6298. 2003.PubMed/NCBI
15.	Villar J, Quadri HS, Song I, Tomita Y, Tirado OM and Notario V: PCPH/ENTPD5 expression confers to prostate cancer cells resistance against cisplatin-induced apoptosis through protein kinase Cα-mediated Bcl-2 stabilization. Cancer Res. 69:102–110. 2009.PubMed/NCBI
16.	Rouzaut A, Recio JA and Notario V: Expression of the protein product of the PCPH proto-oncogene in human tumor cell lines. Radiat Res. 155:181–187. 2001. View Article : Google Scholar : PubMed/NCBI
17.	Blánquez MJ, Arenas MI, Conde I, Tirado OM, Paniagua R and Notario V: Deregulated expression of the PCPH proto-oncogene in human breast cancers. Int J Oncol. 25:821–830. 2004.PubMed/NCBI
18.	Villar J, Arenas MI, MacCarthy CM, Blánquez MJ, Tirado OM and Notario V: PCPH/ENTPD5 expression enhances the invasiveness of human prostate cancer cells by a protein kinase C delta-dependent mechanism. Cancer Res. 67:10859–10868. 2007. View Article : Google Scholar : PubMed/NCBI
19.	Murphy-Piedmonte DM, Crawford PA and Kirley TL: Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase. Biochim Biophys Acta. 1747:251–259. 2005. View Article : Google Scholar : PubMed/NCBI
20.	Recio JA, Páez JG, Maskeri B, Loveland M, Velasco JA and Notario V: Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1. Cancer Res. 60:1720–1728. 2000.
21.	Le Guezennec X, Vermeulen M, Brinkman AB, et al: MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties. Mol Cell Biol. 26:843–851. 2006.PubMed/NCBI
22.	Smith TM and Kirley TL: Site-directed mutagenesis of a human brain ecto-apyrase: evidence that the E-type ATPases are related to the actin/heat shock 70/sugar kinase superfamily. Biochemistry. 38:321–328. 1999. View Article : Google Scholar : PubMed/NCBI
23.	Wachstein M and Meisel E: Histochemistry of hepatic phosphatases of a physiologic pH; with special reference to the demonstration of bile canaliculi. Am J Clin Pathol. 27:13–23. 1957.PubMed/NCBI
24.	Abramoff MD, Magelhaes PJ and Ram SJ: Image processing with Image. J Biophotonics Int. 11:36–42. 2004.
25.	Li Y, Park JS, Deng JH and Bai Y: Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex. J Bioenerg Biomembr. 38:283–291. 2006. View Article : Google Scholar : PubMed/NCBI
26.	Mulero JJ, Yeung G, Nelken ST and Ford JE: CD39-L4 is a secreted human apyrase, specific for the hydrolysis of nucleoside diphosphates. J Biol Chem. 274:20064–20067. 1999. View Article : Google Scholar : PubMed/NCBI
27.	Trombetta ES and Helenius A: Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum. EMBO J. 18:3282–3292. 1999. View Article : Google Scholar
28.	Mulero JJ, Yeung G, Nelken ST, Bright JM, McGowan DW and Ford JE: Biochemical characterization of CD39L4. Biochemistry. 39:12924–12928. 2000. View Article : Google Scholar : PubMed/NCBI
29.	Yang F, Hicks-Berger CA, Smith TM and Kirley TL: Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions. Biochemistry. 40:3943–3950. 2001. View Article : Google Scholar
30.	Smith TM, Lewis Carl SA and Kirley TL: Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase. Biochemistry. 38:5849–5857. 1999. View Article : Google Scholar : PubMed/NCBI
31.	Miura P, Thompson J, Chakkalakal JV, Holcik M and Jasmin BJ: The utrophin A 5′-untranslated region confers internal ribosome entry site-mediated translational control during regeneration of skeletal muscle fibers. J Biol Chem. 280:32997–33005. 2005.
32.	Baykov AA, Evtushenko OA and Avaeva SM: A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem. 171:266–270. 1988. View Article : Google Scholar : PubMed/NCBI
33.	Zhang JT and Ling V: Involvement of cytoplasmic factors regulating the membrane orientation of P-glycoprotein sequences. Biochemistry. 34:9159–9165. 1995. View Article : Google Scholar : PubMed/NCBI
34.	Massé K, Eason R, Bhamra S, Dale N and Jones EA: Comparative genomic and expression analysis of the conserved NTPDase gene family in Xenopus. Genomics. 87:366–381. 2006.PubMed/NCBI
35.	Recio JA, Zambrano N, de la Peña L, et al: cDNA isolation, expression, and chromosomal localization of the mouse pcph proto-oncogene. Mol Carcinog. 26:130–136. 1999. View Article : Google Scholar : PubMed/NCBI
36.	Ivanenkov VV, Murphy-Piedmonte DM and Kirley TL: Bacterial expression, characterization, and disulfide bond determination of soluble human NTPDase6 (CD39L2) nucleotidase: implications for structure and function. Biochemistry. 42:11726–11735. 2003. View Article : Google Scholar
37.	Zebisch M and Strater N: Structural insight into signal conversion and inactivation by NTPDase2 in purinergic signaling. Proc Natl Acad Sci USA. 105:6882–6887. 2008. View Article : Google Scholar : PubMed/NCBI
38.	Crawford PA, Gaddie KJ, Smith TM and Kirley TL: Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): a possible modulator of nucleotidase activity and purinergic signaling. Arch Biochem Biophys. 457:7–15. 2007. View Article : Google Scholar
39.	Riewe D, Grosman L, Fernie AR, Wucke C and Geigenberger P: The potato-specific apyrase is apoplastically localized and has influence on gene expression, growth, and development. Plant Physiol. 147:1092–1109. 2008. View Article : Google Scholar : PubMed/NCBI
40.	Regadera J, Blánquez MJ, González-Peramato P, et al: PCPH expression is an early event in the development of testicular germ cell tumors. Int J Oncol. 28:595–604. 2006.PubMed/NCBI
41.	Mikula M, Rubel T, Karczmarski J, Goryca K, Dadlez M and Ostrowski J: Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors. Funct Integr Genomics. 11:215–224. 2010. View Article : Google Scholar : PubMed/NCBI