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Anti-proliferative and pro-apoptotic actions of a novel human and mouse ovarian tumor-associated gene OTAG-12: downregulation, alternative splicing and drug sensitization

Abstract

In studying the age dependence and chronology of ovarian tumors in follicle stimulating hormone receptor knockout mice, we identified a novel ovarian tumor associated gene-12 (OTAG-12), which is progressively downregulated and maps to Chr. 8B3.3. OTAG-12 protein overexpression in mouse ovarian and mammary tumor cells suggested powerful anti-proliferative effects. In human epithelial ovarian cancers (OCs) and OC cell lines, OTAG-12 mRNA expression is downregulated in comparison with normal ovaries. Cloning and identification revealed that human OTAG-12 mapping to gene-rich Chr. 19p13.12 is expressed in three spliced forms: hOTAG-12a, hOTAG-12b and hOTAG-12c, of which b is predominant in the normal ovary. Functionally active hOTAG-12b is a simple protein with no disulfide bonds and a nuclear localization signal is present in all variants. Transfection of OTAG-12 variants in OC and tumorigenic HEK293 cells confirmed nuclear localization. hOTAG-12b overexpression in OC and HEK293 cells effectively suppressed cell growth, anchorage-dependent and independent colony formation followed by apoptosis, whereas hOTAG-12a and hOTAG-12c had no such effects. Deletion mutants identified the critical importance of carboxyl terminus for hOTAG-12b function. Doxycycline-inducible growth inhibition of HEK293 cells by hOTAG-12a was associated with effects on G2 cell cycle arrest and apoptosis induction. hOTAG-12b expression rendered tumorigenic cells more sensitive to four apoptotic stimuli including etoposide—a topoisomerase-II inhibitor. Doxycycline-induced hOTAG-12b expression blocked xenograft tumor growth in nude mice, whereas hOTAG-12a was ineffective. Although p53-pathway-dependent apoptotic agents could upregulate endogenous hOTAG-12b and p53 in UCI-101/107 OC cells, hOTAG-12b could also induce apoptosis in p53-null and platinum-resistant SKOV3 OC cells and Doxycycline-induced hOTAG-12b did not alter p53. Further study showed that hOTAG-12b increases mRNAs of pro-apoptotic genes such as BAD, GADD45α and CIEDB, while inhibiting anti-apoptotic NAIP and Akt1 expression, suggesting that hOTAG-12b-induced apoptosis might be p53-independent. These results indicate that hOTAG-12b is a putative ovarian tumor suppressor gene warranting further studies.

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Acknowledgements

We are grateful to Dr P Carpenter (UC Irvine) for providing ovarian cancer cell lines UCI101 and UCI107, Dr Roby KF (Univ Kansas) for ID8 cells, Drs Jolicoeur for HC11-26 cells and Seidah at IRCM for the modified HEK293 cell lines. We appreciate the useful suggestions provided by Drs Zhongjun Dong and Hua Lin during this investigation. We also acknowledge the help of Drs Marie-Claude Beauchamp and Amber Yasmeen in this study. This investigation was supported in part by the NCIC and the CIHR.

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Correspondence to M R Sairam.

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Chen, X., Zhang, H., Aravindakshan, J. et al. Anti-proliferative and pro-apoptotic actions of a novel human and mouse ovarian tumor-associated gene OTAG-12: downregulation, alternative splicing and drug sensitization. Oncogene 30, 2874–2887 (2011). https://doi.org/10.1038/onc.2011.11

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