Bilim, Vladimir N.

Abstract Purpose: Enhancer of zeste homologue 2 (EZH2), a histone methyltransferase, plays a key role in transcriptional repression through chromatin remodeling. Our objectives were to determine the expression pattern of EZH2 and to assess the anticancer effect of EZH2 depletion in pancreatic cancer cells. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the expression pattern of EZH2 in normal pancreas and human pancreatic tumors. We used RNA interference, Western blotting, reverse transcription-PCR, and chromatin immunoprecipitation to study the effect of EZH2 depletion on pancreatic cancer cell proliferation and survival. Results: We detected nuclear overexpression of EZH2 in pancreatic cancer cell lines and in 71 of 104 (68%) cases of human pancreatic adenocarcinomas. EZH2 nuclear accumulation was more frequent in poorly differentiated pancreatic adenocarcinomas (31 of 34 cases; P < 0.001). We found that genetic depletion of EZH2 results in reexpression of p27Kip1 and decreased pancreatic cancer cell proliferation. Moreover, we showed that EZH2 depletion sensitized pancreatic cancer cells to doxorubicin and gemcitabine, which leads to a significant induction of apoptosis, suggesting that the combination of EZH2 inhibitors and standard chemotherapy could be a superior potential treatment for pancreatic cancer. Conclusions: Our results show nuclear accumulation of EZH2 as a hallmark of poorly differentiated pancreatic adenocarcinoma; identify the tumor suppressor p27Kip1 as a new target gene of EZH2; show that EZH2 nuclear overexpression contributes to pancreatic cancer cell proliferation; and suggest EZH2 as a potential therapeutic target for the treatment of pancreatic cancer.

Abstract Purpose: We have shown recently that glycogen synthase kinase-3 (GSK-3) β regulates nuclear factor-κB (NF-κB)–mediated pancreatic cancer cell survival and proliferation in vitro. Our objective was to determine the localization of GSK-3β in pancreatic cancer cells and assess the antitumor effect of GSK-3 inhibition in vivo to improve our understanding of the mechanism by which GSK-3β affects NF-κB activity in pancreatic cancer. Experimental Design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the localization of GSK-3β in human pancreatic tumors. We studied the effect of GSK-3 inhibition on tumor growth, cancer cell proliferation, and survival in established CAPAN2 tumor xenografts using a tumor regrowth delay assay, Western blotting, bromodeoxyuridine incorporation, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Results: We found nuclear accumulation of GSK-3β in pancreatic cancer cell lines and in 62 of 122 (51%) human pancreatic adenocarcinomas. GSK-3β nuclear accumulation is significantly correlated with human pancreatic cancer dedifferentiation. We have found that active GSK-3β can accumulate in the nucleus of pancreatic cancer cells and that inhibition of GSK-3 kinase activity represses its nuclear accumulation via proteasomal degradation within the nucleus. Lastly, we have found that inhibition of GSK-3 arrests pancreatic tumor growth in vivo and decreases NF-κB-mediated pancreatic cancer cell survival and proliferation in established tumor xenografts. Conclusions: Our results show the antitumor effect of GSK-3 inhibition in vivo, identify GSK-3β nuclear accumulation as a hallmark of poorly differentiated pancreatic adenocarcinoma, and provide new insight into the mechanism by which GSK-3β regulates NF-κB activity in pancreatic cancer.