Abstract 4528: Chromatin regulation by ING3 leads to tumor suppressive effects in pancreatic cancer through distinct signaling pathways
Authors: Koutsioumpa Marina , Hatziapostolou Maria , Polytarchou Christos , Oikonomopoulos Angelos , Mahurkar-Joshi Swapna , Huerta-Yepez Sara , Tirado-Rodriguez Belen , Karavias Dimitrios , Kourea Helen , Poultsides George A. , Dawson David W. , Donahue Timothy R. , Iliopoulos Dimitrios 
Published: July 2016

Background: Despite our increased knowledge of the molecular events underlying the multi-step development of pancreatic cancer (PanCa), clinically meaningful improvement in survival rates has not yet been achieved. A growing body of evidence supports that pancreatic tumorigenesis is not only led by genetic alterations but also aberrant epigenetic regulation. Identification of epigenetic drivers of the clinical disease is of major importance, as it could potentially offer a means to interfere with novel targets in PanCa therapy.

Methods: Gene expression microarrays were employed in PanCa tissues and adjacent uninvolved tissues. mRNA and protein levels were assessed by qRT-PCR and immunohistochemical analysis of tissue microarrays in extended cohorts of patients. RNAi interference assays or lentiviral expression vector systems were applied for knockdown/overexpression experiments, as evidenced by qRT-PCR and Western Blot Analysis. Molecular analysis, cell proliferation, invasion and colony formation assays were conducted in genetically modified cells. Subcutaneous xenograft mouse models were used to evaluate tumor growth in vivo. Immunoprecipitation followed by mass-spectroscopy analysis was used to evaluate protein-protein interactions. Histone H4 at lysine 16 acetylation (H4K16ac) levels were assessed by Western Blot analysis, while chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) was performed for mapping protein-DNA interactions.

Results: Differential expression analysis of chromatin regulators in PanCa versus normal tissues shows that 27 epigenetic molecules are significantly deregulated (&gt;1.5 fold, P&lt;0.05). The reduced expression of inhibitor of growth family member 3 (ING3) was further validated by qRT-PCR and immunohistochemical analysis in three different cohorts of patients. Importantly, transient or stable depletion of ING3 promotes cell proliferation, anchorage-independent growth, invasiveness and in vivo tumor growth, while its overexpression has opposite effects. Cells lacking or overexpressing ING3 were utilized to reveal ING3-related gene signature. Dual-specificity phosphatase 5 (DUSP5) and c-Met were identified as ING3-downstream targets that mediate different ING3-regulated PanCa cellular properties. Interestingly, genetic manipulation of ING3 leads to profound effects in H4K16ac. Moreover, ING3 expression seems to affect p53 binding to gene promoters, possibly due to the formation of histone H1-ING3 complex that antagonizes for H1-p53 interaction.

Conclusions: Conclusively, we provide evidence that PanCa is characterized by loss of the chromatin regulator ING3 and we elucidated the tumor suppressive role of the latter in PanCa cell growth and invasion in vitro and in vivo.

Citation Format: Marina Koutsioumpa, Maria Hatziapostolou, Christos Polytarchou, Angelos Oikonomopoulos, Swapna Mahurkar-Joshi, Sara Huerta-Yepez, Belen Tirado-Rodriguez, Dimitrios Karavias, Helen Kourea, George A. Poultsides, David W. Dawson, Timothy R. Donahue, Dimitrios Iliopoulos. Chromatin regulation by ING3 leads to tumor suppressive effects in pancreatic cancer through distinct signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4528.