Abstract B14: Epigenetic silencing of DAZ-interacting protein 1 in pancreatic ductal adenocarcinoma.

Introduction: The Hedgehog signaling pathway shows aberrant activity in a variety of human cancers including pancreatic cancer. The GLI family of proteins are downstream transcription factors of the hedgehog pathway whose expression is regulated by pathway activity and are commonly altered in pancreatic ductal adenocarcinoma. DAZ-interacting protein 1 (DZIP1) is relatively poorly characterized protein in humans. It is a zinc-finger and coiled-coil domain protein known to regulate the intracellular distribution of GLI family members. Materials and Methods: Oligonucleotide arrays were utilized to examine the DZIP1 levels in 5 pooled microdissected normal pancreatic ducts, 12 intraductal mucinous neoplasms (IPMNs), non-neoplastic ductal epithelial cell lines (HPDE, HPNE) , pancreatic cancer cell lines (A32-1, A38-5, Panc215, Panc2.5, Panc2.8, and Panc3.014) and cancer-associated fibroblast (CAF) cell lines (CAF 11, CAF 12, CAF 13, CAF 15, CAF 16). Methylated CpG Island Amplification and Microarray (MCAM) was additionally performed on pancreatic cancer cell lines and normal pancreata. Further specific methylation analyses included RT-PCR previous to and following epigenetic treatment with 5-AZA and/or TSA, in 10 cancer cell lines (Panc215, A32-1, A38-5, Panc2.5, Panc3.014, MIAPacCa2, AsPC1, Capan1, Panc1, Su86.86) and 2 non-neoplastic pancreatic epithelial cell lines (HPNE, and HPDE); bisulfite sequencing of the DZIP1 gene in cancer cell lines (Panc215, A32-1, A38-5, Panc2.5, Panc2.8, Panc3.014, Su8686, Panc1) and patient-matched non-neoplastic cell lines (215lymph, A32lymph, A38spleen, 2.5ly, 2.8ly, 3.014ly) and non-neoplastic lines (HPNE and HPDE); and methylation-specific PCR analysis in pancreatic cancer xenografts, primary pancreatic tissues and pancreatic cancer cell lines. DZIP1 was transfected into non-expressing cancer cell lines AsPC1, MIAPaCa2, A38-5 and Panc215, as well as DZIP1 expressing cell lines Panc2.5 and HPNE, to analyze the functional impact of re-expression on GLI1 expression. Cell migratory behavior was analyzed in the cancer cell line AsPC1, following re-expression. Results: On oligonucleotide array analysis DZIP1 was under-expressed in 5 out of 6 cancer cell lines, and not in Panc2.5, when compared to CAF and non-neoplastic pancreatic cell lines. IPMNs had lower levels of DZIP1 expression compared to microdissected normal duct, suggestive that the same should be true of pancreatic adenocarcinoma. On MCAM analysis, the DZIP1 gene promoter was shown to be hypermethylated in cancer cell lines. RT-PCR analysis showed DZIP1 expression levels in 8 of 10 cancer cell lines (Panc215, A32-1, A38-5, Panc3.014, MIAPacCa2, AsPC1, Capan1, Su86.86) were lower than non-neoplastic cell lines. All cell lines with reduced DZIP1 expression levels showed re-expression with epigenetic treatment, as did the cell line Panc2.5, despite its normal baseline expression. Bisulfite sequencing of the DZIP1 locus showed greater methylation of the first CpG island in cancer cell lines versus noncancer lines. MSP confirmed this, showed greater methylation in pancreatic cancer cells than non-neoplastic pancreas. Transient expression of DZIP1 in non-expressing lines resulted in modulation of GLI expression consistent with a role of DZIP1 in modulating the hedgehog pathway in pancreatic cancer cells. Re-expression of DZIP1 in the cancer cell line AsPC1 resulted in significant decrease in cell migration. Conclusion: We have identified DZIP1 as a commonly silenced gene in pancreatic cancer cells and pancreatic ductal adenocarcinoma precursor lesions, relative to normal pancreatic duct cells and cancer-associated fibroblasts, and this silencing is associated with promoter methylation. Citation Format: Eileen O’Sullivan, Noriyuki Omura, Michael Goggins. Epigenetic silencing of DAZ-interacting protein 1 in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B14.