Vladimir Bilim

Regulation of Pancreatic Tumor Cell Proliferation and Chemoresistance by the Histone Methyltransferase Enhancer of Zeste Homologue 2

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.

Aberrant Nuclear Accumulation of Glycogen Synthase Kinase-3β in Human Pancreatic Cancer: Association with Kinase Activity and Tumor Dedifferentiation

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.

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma

Background:Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3β positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-κB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3β and to assess the anti-cancer effect of GSK-3β inhibition in RCC.Methods:Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3β in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT–PCR, BrDU incorporation and MTS assays to study the effect of GSK-3β inactivation on renal cancer cell proliferation and survival.Results:We detected aberrant nuclear accumulation of GSK-3β in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-κB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells.Conclusions:Our results show nuclear accumulation of GSK-3β as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Re-expression of miR-199a suppresses renal cancer cell proliferation and survival by targeting GSK-3β

Recently, we have identified GSK-3 as a new therapeutic target in renal cell cancer (RCC). miR-199a could potentially downregulate GSK-3β expression. Here, we found a decreased miR-199a expression in 59% (32 of 54) of RCCs and it was correlated with higher tumor stage (p < 0.05) and nuclear overexpression of GSK-3β (p < 0.05). We show that re-expression of miR-199a downregulates GSK-3β and suppresses cancer cell growth. Our results demonstrate low miR-199a expression as a feature of advanced RCCs, identify miR-199a as a negative regulator of GSK-3β, and suggest re-expression of pre-miR-199a as a new potential treatment of RCC.

The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells.

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.

Glycogen Synthase Kinase-3β: A Prognostic Marker and a Potential Therapeutic Target in Human Bladder Cancer

Purpose: Although recent studies have shown glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, as a positive regulator of pancreatic, colon, and kidney cancer cell survival and proliferation, the role of GSK-3 in bladder cancer remains unknown. Our objectives were to determine the subcellular localization of GSK-3β and to evaluate the effect of GSK-3 inhibition in bladder cancer. Experimental Design: We used immunohistochemical staining and nuclear/cytosolic fractionation to determine the expression pattern of GSK-3β in human urothelial carcinomas. To study the effect of GSK-3 inhibition on bladder cancer cell proliferation and survival, we used pharmacologic inhibitors of GSK-3, RNA interference, MTS assay, bromodeoxyuridine incorporation assay, quantitative reverse transcriptase-PCR, and Western blotting. Results: We found aberrant nuclear accumulation of GSK-3β in 62% (43 of 69) and 91% (21 of 23) of noninvasive and invasive human urothelial carcinomas, respectively. GSK-3β nuclear staining was significantly associated with high-grade tumors (P < 0.001), advanced stage of bladder cancer (P < 0.05), metastasis (P < 0.05), and worse cause-specific survival (P < 0.05) in bladder cancer patients. Moreover, we found that pharmacologic inhibition or genetic depletion of GSK-3β resulted in decreased viability of bladder cancer cells. Conclusions: Our results suggest nuclear accumulation of GSK-3β as a novel prognostic marker in bladder cancer, show that GSK-3 contributes to urothelial cancer cell proliferation and survival, and identify GSK-3 as a potential therapeutic target in human bladder cancer. Clin Cancer Res; 16(21); 5124–32. ©2010 AACR.

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis

Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatins effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.

Proteomics identified nuclear N-myc downstream-regulated gene 1 as a prognostic tissue biomarker candidate in renal cell carcinoma.

The aim of this study was to identify proteins with aberrant expression in clear cell renal cell carcinoma (ccRCC), and elucidate their clinical utilities. The protein expression profiles of primary ccRCC tumor tissues and neighboring non-tumor tissues were obtained from 9 patients by two-dimensional difference gel electrophoresis and mass spectrometry. Comparative analysis of 3771 protein spots led to the identification of 73 proteins that were expressed at aberrant levels in tumor tissues compared with non-tumor tissues. Among these 73 proteins, we further focused on N-myc downstream-regulated gene 1 protein (NDRG1). NDRG1 expression is regulated by members of myc family as well as by p53, HIF1A, and SGK1. The biological and clinical significance of NDRG1 is controversial for various malignancies and no detailed studies on NDRG1 have been reported in ccRCC until our study. For the 82 newly enrolled ccRCC patients, immunohistochemical analysis revealed a significant association between nuclear NDRG1 and favorable prognosis (p<0.05). Multivariate analysis demonstrated the role of NDRG1 as an independent factor of progression-free survival (p=0.01). Subsequent in vitro gene suppression assay demonstrated that NDRG1 silencing significantly enhanced cell proliferation and invasion of RCC cells. The cytotoxic effects of NDRG1 up-regulation induced by an iron chelator were also confirmed. These findings suggest that nuclear NDRG1 has tumor suppressive effects, and the NDRG1 expression may have clinical values in ccRCC. Nuclear NDRG1 may provide additional insights on molecular backgrounds of ccRCC progression, and contribute to the development of novel therapeutic strategy.

GSK-3 inhibition in vitro and in vivo enhances antitumor effect of sorafenib in renal cell carcinoma (RCC).

Sorafenib is a multikinase inhibitor approved for the systemic treatment of renal cell carcinoma (RCC). However, sorafenib treatment has a limited effect due to acquired chemoresistance of RCC. Previously, we identified glycogen synthase kinase-3 (GSK-3) as a new therapeutic target in RCC. Here, we observed that sorafenib inhibits proliferation and survival of RCC cells. Significantly, we revealed that sorafenib enhances GSK-3 activity in RCC cells, which could be a potential mechanism of acquired chemoresistance. We found that pharmacological inhibition of GSK-3 potentiates sorafenib antitumor effect in vitro and in vivo. Our results suggest that combining GSK-3 inhibitor and sorafenib might be a potential new therapeutic approach for RCC treatment.

A case of myxoid adrenocortical neoplasm: computed tomography and magnetic resonance imaging characteristics

Myxoid adrenocortical neoplasms are rare; to our knowledge, only 56 cases have been reported in the literature. Therefore, distinguishing benign from malignant cases is challenging. Although the histopathological features of myxoid adrenocortical neoplasia have been amply demonstrated, their imaging characteristics are yet to be reported. We describe here these characteristics for such a neoplasm. Our patient, a 70-year-old male, was found to have a 3-cm left adrenal incidentaloma through a non-enhanced computed tomography. Attenuation measurements were 22 Hounsfield units on precontrast imagery, and percentage enhancement washout was 92%. Magnetic resonance imaging showed no loss of signal intensity in T1-weighted out-of-phase images, but high signal intensity on T2-weighted and diffusion-weighted images. Left adrenalectomy was performed and the pathological diagnosis was confirmed as myxoid adrenocortical neoplasm. The imaging characteristics reported here will be beneficial to the differential diagnosis of myxoid adrenocortical neoplasms based upon image analysis and will help distinguish benign from malignant neoplasms.