Abstract 5973: GIPC2 is a candidate tumor suppressor gene in sporadic pheochromocytoma

Abstract

Abstract Pheochromocytoma (PCC) is a rare endocrine tumor of the chromaffin cells in adrenal medulla or the paraganglia. Currently about 60% of PCCs can be explained by germline or somatic mutations in well-established susceptibility genes such as RET, VHL and SDHB, while for the remaining, mainly sporadic cases the pathogenesis is still unclear. We explored the molecular basis of these PCCs by analyzing 22 sporadic adrenal PCCs, using SNP6.0 arrays to identify minimal region of the most common chromosomal loss, and expression profiling to identify genes with suppressed expression relative to normal medulla. Here we report the identification of GIPC2, a gene located at 1p31.1 encoding a PDZ domain containing adaptor protein with preferential expression in adrenal, as a putative tumor suppressor gene for sporadic PCC. In a cohort of 55 sporadic PCCs, 38 were found with reduced genomic copy number of GIPC2, with accompanying reduced expression of mRNA. Much reduced or loss of GIPC2 protein expression in tumor tissues was confirmed with Western blot or immunostaining. No GIPC2 mutations were found in the cohort but significant promoter hypermethylation was demonstrated using MassARRAY EpiTYPER assay and methylation-specific PCR. The GIPC2 protein was expressed mostly in the nucleus. With GIPC2 overexpression or siRNA knockdown in both PCC derived cell lines (rat PC12 or hPheo, a cell line derived from human PCC) and primary rat adrenal chromaffin cells (ACC), we showed that GIPC2 suppressed cell proliferation by inducing cyclin kinases such as p27, and by suppressing the activation of MAPK/Erk pathways without affecting pAKT or mTOR pathways. PC12 cells stably overexpressing GIPC2 failed to develop tumor in nude mice. To explore the molecular mechanism of GIPC2 in cell cycle regulation, we searched for its interaction proteins in 293T cells via mass spectrometry and identified NONO, a nucleoprotein with a known role in regulating cell cycle. Reciprocal interaction between GIPC2 and NONO was demonstration in PC12 or hPheo cells in vivo using immune-IP or FRET. We showed that knockdown of NONO blocked GIPC2-mediated p27 up-regulation. Furthermore both GIPC2 and NONO regulated p27 promoter through the same DNA binding site and a GGCC box was significant for GIPC2-regulated p27 promoter activity through NONO. Finally, we found that PCC-causing mutations in RET and SDHB, but not VHL, genes could lead to ACC proliferation and p27 downregulation via down-regulating GIPC2. In summary, our study indicates that GIPC2 acts as a candidate tumor suppressor gene in sporadic pheochromocytomas and regulates cell cycle gene p27 through NONO. Our results provide a new insight into the molecular mechanism of sporadic PCCs. Citation Format: Yeqing Dong, Zhengguang Guo, Chengyan Fan, Zhi Zheng. GIPC2 is a candidate tumor suppressor gene in sporadic pheochromocytoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5973.