Citations

AbstractMicroRNA‐200 (miR‐200) has emerged as a regulator of the PI3K/AKT pathway and cancer cell growth. It was reported that miR‐200 can activate PI3K/AKT by targeting FOG2 (friend of GATA 2), which directly binds to the p85α regulatory subunit of PI3K. We found that miR‐200 was elevated in early stage lung adenocarcinomas when compared with normal lung tissues, and the expression of miR‐200 promoted the tumor spheroid growth of lung adenocarcinoma cells. We show that AKT activation was essential for such oncogenic action of miR‐200. However, depletion of FOG2 had little effect on AKT activation. By performing a reverse‐phase protein array, we found that miR‐200 not only activated AKT but also concomitantly inactivated S6K and increased IRS‐1, an S6K substrate that is increased on S6K inactivation. Depletion of IRS‐1 partially inhibited the miR‐200‐dependent AKT activation. Taken together, our results suggest that miR‐200 may activate AKT in lung adenocarcinoma cells through a FOG2‐independent mechanism involving IRS‐1. Our findings also provide evidence that increased miR‐200 expression may contribute to early lung tumorigenesis and that AKT inhibitors may be useful for the treatment of miR‐200‐dependent tumor cell growth. © 2015 IUBMB Life, 67(9):720–725, 2015

Background Preoperative assessment of anterior communicating artery (AcoA) aneurysms with cerebral angiography is common, but not without risk. Computed tomography angiography (CTA) is a widely available imaging modality that provides quick acquisition, low morbidity, and low cost. One disadvantage is that it does not provide dynamic information. In this study, the authors sought to determine whether CTA alone can reliably predict the inflow dominance to an AcoA aneurysm. Methods Eighty-three patients with ruptured AcoA aneurysms were reviewed retrospectively. Only those patients with both preoperative CTA and cerebral angiogram were included, thus excluding six patients. Four independent observers reviewed the CTAs and attempted to identify the dominant A1. Additionally, three mathematical models were created to identify the dominant A1. These responses were compared to cerebral angiograms. Results Four observers were correct in judging the dominant A1 an average of 93% of the time. Seventeen cases were read incorrectly by only one of four observers, and three cases were read incorrectly by two observers. For cases with incorrect readings, the average percentage difference in A1 sizes was 19.6%. For cases read unanimously correct, the average percentage difference in A1 sizes was 42.7%. Mathematical model #3 correctly evaluated the dominant A1 in 97% of the cases. Conclusions This study found CT angiograms can be reliable in predicting the inflow dominance to the majority of AcoA aneurysms.

Abstract FoxM1, a member of the Forkhead transfection factors, has been found to be overexpressed in many human cancers including ovarian cancer. Although FoxM1 is known to be downregulated by p53, the molecular mechanisms are not well characterized. In this study, we used the MDM2 inhibitor Nutlin-3 as a tool to modulate p53 expression and investigated the mechanism by which p53 regulates FoxM1 expression in ovarian cancer cells. We observed that Nutlin-3 upregulated p53 protein and downregulated FoxM1 mRNA and protein levels in ovarian cancer cell lines with wild type TP53, but not in cell lines with mutant TP53. This effect was at least partially blocked by cycloheximide or actinomycin D, indicating a transcription- and translation-dependent mechanism. Pulse-chase studies revealed that downregulation of FoxM1 by Nutlin-3 is due to enhanced FoxM1 mRNA decay. Knockdown of p53 using shRNAs abrogated the suppression of FoxM1 expression by Nutlin-3, indicating a p53-dependent mechanism. Furthermore, silencing of FoxM1 by shRNAs in ovarian cancer cells with or without wild-type TP53 led to a significant decrease in cell proliferation. The FoxM1 inhibitor, thiostrepton, induces cytotoxicity in ovarian cancer cell lines and enhances sensitivity to cisplatin in these cells. Thiostrepton downregulates FoxM1 expression in several ovarian cancer cell lines and enhances sensitivity to carboplatin in vivo. Finally, FoxM1 expression is elevated in nearly all (47/48) ovarian tumors. In summary, the present study provides novel evidence that p53 downregulates FoxM1 in ovarian cancer cells at least in part by destabilizing FoxM1 mRNA and that FoxM1 may be targeted for therapeutic benefits in ovarian cancer. Citation Format: Xuan Zhang, Lihua Cheng, Kay Minn, Magdalena Waggoner, Nikhil K. Parelkar, Iman Jokar, George A. Vielhauer, Rashna Madan, Andrew K. Godwin, Viji Shridhar,, Jeremy Chien. Targeting p53-FoxM1 axis in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1409.

AbstractGenetic risk factors for cholangiocarcinoma (CCA) remain poorly understood. We assessed the effect of single‐nucleotide polymorphisms (SNPs) of genes modulating inflammation or carcinogenesis on CCA risk and survival. We conducted a case‐control, candidate gene association study of 370 CCA patients and 740 age‐, sex‐, and residential area‐matched healthy controls. Eighteen functional or putatively functional SNPs in nine genes were genotyped. The log‐additive genotype effects of SNPs on CCA risk and survival were determined using logistic regression and the log‐rank test, respectively. Initial analysis identified significant associations between SNP rs2143417 and rs689466 in cyclooxygenase 2 (COX‐2) and CCA risk, after adjusting for multiple comparisons (cutoff of P = 0.0028). However, these findings were not replicated in another independent cohort of 212 CCA cases and 424 matched controls. No significant association was found between any SNP and survival of CCA patients. Although COX‐2 has been shown to contribute to cholangiocarcinogenesis, the COX‐2 SNPs tested were not associated with risk of CCA. This study shows a lack of association between variants of genes related to inflammation and carcinogenesis and CCA risk and survival. Other factors than these genetic variants may play more important roles in CCA risk and survival.

Abstract Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) and is the most common NHL subtype diagnosed worldwide. Some epidemiologic data have indicated differences in DLBCL incidence rates and subtype distributions between Eastern Asian and Western countries. The first large-scale genome-wide association study (GWAS) of DLBCL with over 4000 cases conducted among individuals of European ancestry recently identified five independent SNPs that achieved genome-wide significance, and two SNPs that showed a suggestive association with DLBCL risk. In order to evaluate whether Eastern Asians and individuals of European ancestry share similar genetic risk factors for this disease, we attempted to replicate these GWAS findings in a pooled series of 1124 DLBCL cases and 3596 controls from Hong Kong, South Korea, and Thailand. Three of the five genome-wide significant SNPs from the DLBCL GWAS were significantly associated with DLBCL in our study population, including the top finding from the GWAS, EXOC2 rs116446171, which achieved genome-wide significance in our data (per allele OR = 2.04, 95% CI = 1.63-2.56; ptrend = 3.9 × 10-10). Additionally, we observed a significant association with PVT1 rs13255292 (per allele OR = 1.34, 95% CI = 1.19-1.52; ptrend = 2.1 × 10-6), which was the second strongest finding in the GWAS, and with HLA-B rs2523607 (per allele OR = 3.05, 95% CI = 1.32-7.05; ptrend = 0.009). Our study, which provides the first evaluation in Eastern Asians of SNPs definitively associated with DLBCL risk in individuals of European ancestry, indicates that at least some of the genetic factors associated with risk of DLBCL are similar between these populations. Citation Format: Nathaniel Rothman, Bryan A. Bassig, James R. Cerhan, Wing-Yan Au, Hee Nam Kim, Suleeporn Sangrajrang, Wei Hu, Jovic Tse, Sonja Berndt, Tongzhang Zheng, Heping Zhang, Pattarapong Pornsopone, Je-Jung Lee, Hyeoung-Joon Kim, Christine F. Skibola, Joseph Vijai, Laurie Burdette, Meredith Yeager, Paul Brennan, Min-Ho Shin, Raymond Liang, Stephen Chanock, Qing Lan. Genetic susceptibility to diffuse large B-cell lymphoma in a pooled study of three Eastern Asian populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4626. doi:10.1158/1538-7445.AM2015-4626

Abstract Introduction: Prognosis in CLL is heterogeneous with the elderly, males and those with multiple co-morbidities having the highest risk of poorer outcomes. Whether this reflects differences in the biology of CLL or the result of altered immunosuppression is unknown. In normal individuals, the telomeres in somatic cells shorten with age, predisposing those with shorter telomeres to death from infections, cancer and cardiovascular disease. In chronic lymphocytic leukemia (CLL), short telomere length in the leukemia cells predicts poor prognosis. The contribution of un-modifiable factors of age, sex or comorbidities to telomere shortening in CLL has not been elucidated. In the present study, we evaluated buccal cell (BC) telomere length in patients with CLL, to determine if this is predictive of survival, and can enhance the predictive value of leukemia cell telomere length. Methods: The Manitoba CLL tumor bank contains 168 samples from newly diagnosed CLL patients between 2007-2011; with a median follow-up of 4 years. Half of these patients required chemotherapy and one-tenth have died. Genomic DNA was extracted from purified CLL cells and buccal cells (BC) collected at diagnosis. Telomere length was established by multiplex quantitative real-time PCR. Telomere/standard (t/s) ratio was calculated using the beta-globulin gene as the standard. Statistical analysis was performed using Statistical Analysis Software (SAS) and Prism software. Results: The median adjusted telomere length was shorter in CLL cells than in BCs being 0.53 and 2.01, respectively. In BCs, telomere length significantly shortened with increasing age (p = 0.01) but was not reflective of the number of comorbidities or survival. In contrast, telomere length in CLL cells was independent of age (p = 0.44) and sex (p = 0.75). Short CLL telomere length correlated with other biological and clinical markers of poorer prognosis including un-mutated IGHV status (p<0.0001), Zap70 positivity (p = 0.05), CD38 positivity (p = 0.003), short lymphocyte doubling time (p = 0.004), higher Rai stage (p = 0.02) and an earlier time to treatment (p<0.0001). Patients with short CLL telomeres had also decreased survival at four years (p = 0.05); living 22 months less than those with normal telomeres. Adjusting for BC telomere length improved the prognostic value of CLL telomere length as an independent predictor of survival. Conclusions: These results demonstrate that BC telomere length in CLL patients shorten with age independent of co-morbidities, secondary malignancies and the respective CLL telomere length. CLL telomere shortening is strongly associated with biochemical and clinical markers of disease progression and survival. The predictive value for overall survival is enhanced by correcting for changes in buccal cell telomere length. Note: This abstract was not presented at the meeting. Citation Format: Lin Yang, Sara Beiggi, Yunli Zhang, Robert Schmidt, Spencer B. Gibson, James B. Johnston. Clinical impact of telomere shortening in normal and leukemia cells in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2015-4624

Abstract Genome wide association studies (GWAS) have mapped multiple independent cancer risk loci (n = 6) to a small region on chr5p15.33 for at least ten distinct cancers, including bladder, breast, glioma, lung, melanoma, non-melanoma skin, ovarian, pancreas, prostate, and testicular germ cell cancer. This region harbors two plausible target genes, TERT which encodes the catalytic subunit of telomerase reverse transcriptase which maintains chromosome ends by adding telomeres repeats, and CLPTM1L which encodes the cleft lip and palate transmembrane protein 1-like protein which promotes cancer cell growth, protect cells from apoptosis, and can induce abnormal cytokinesis. The most significant pancreatic cancer GWAS SNP on chr5p15.33 was rs401681 (P = 3.7×10−7, OR = 1.19), located in the 13th intron of the CLPTM1L gene and within the independent multi-cancer risk locus in this region most distant from TERT. Imputation and fine mapping in pancreatic cancer resolved this signal by three orders of magnitude (P = 1.4×10−10, OR = 1.30) to a set of seven highly correlated SNPs. Electrophoretic mobility shift assays (EMSA) and luciferase assays across these SNPs led us to a single SNP, rs36115365, that showed allele-specific effects in both assays and overlapped with prominent ENCODE marks indicating regulatory potential. Examining this SNP across cell lines derived from multiple types of cancer linked to this locus, specifically pancreatic, testicular, lung, and melanoma, established a consistent pattern of a specific protein binding and enhanced regulatory activity for the risk allele across eight cancer cell lines (two cell lines per cancer type). siRNA-based targeting of the putative gene regulatory-region harboring rs36115365 resulted in a strong inhibition of TERT expression (average 60%, range 45-85%) but no effect on CLPTM1L expression. This inhibition of TERT expression was stronger from the pancreatic cancer risk as compared to the protective allele. Quantitative mass spectrometry identified Zinc finger protein 148 (ZNF148) as the protein specifically binding the risk allele, further supported by direct and allele-specific binding of purified recombinant ZNF148 in EMSA assays. Lastly, siRNA-mediated knockdown of ZNF148 resulted in significant reduction of TERT but not CLPTM1L expression across multiple cancer cell lines. Our results indicate that the pancreatic cancer GWAS signal at the single independent chr5p15.33 multi-cancer risk locus most distant from TERT may be explained by a single SNP that influences TERT expression via ZNF148. Citation Format: Kevin M. Brown, Jun Fang, Jinping Jia, Zhaoming Wang, Matthew Makowski, Tongwu Zhang, Jason Hoskins, Jiyeon Choi, Younghun Han, Mingfeng Zhang, Mai Xu, Peter Kanetsky, Andresson Thorkell, Gloria M. Petersen, Katherine L. Nathanson, Christopher I. Amos, Maria T. Landi, Stephen J. Chanock, Michiel Vermeulen, Laufey T. Amundadottir. Functional characterization of a multicancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2015-4610

Abstract Background: The Tumor Growth Factor-β (TGF-β) pathway is a potent tumor suppressor pathway. Its inactivation has been implicated in hepatocarcinogenesis. However, in patients with advanced stage Hepatocellular Carcinoma (HCC), TGF-β1 serum concentrations are elevated and correlated to tumor size, recurrence and shortened survival. The mechanisms mediating TGF-β1 ligand overexpression in HCC are unknown. Material and Methods: Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Array. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analysis. shRNA was used for gene silencing. Transcription factor DNA binding was evaluated by chromatin immunoprecipitation. For promoter analysis, transcription factor binding site containing DNA fragments were cloned into the enhancer or promoter position of luciferase expression vectors and subsequent transfected. Results: In up to 68% of advanced stage HCC tumors Stem Cell Factor (SCF) is overexpressed, and SMAD2 and STAT3 are constitutively activated. SCF expression in human HCC tumors is intratumorally and intracellularly correlated to expression of pSer465/467SMAD2 and pTyr705STAT3. In HCC cell lines, SCF stimulation induces TGF-β1 transcription, expression and secretion, all of which are abrogated by genetic silencing of STAT3. TGF-β1 stimulation of human HCC cells induces TGF-β1 transcription, and genetic silencing of SCF prevents TGF-β1 autoregulation. We identified two STAT3 recognition sites within the TGF-β1 gene. In human HCC cells, TGF-β1 stimulation results in STAT3 binding to one of the identified recognition sites. TGF-β1 activates luciferase expression vectors under control of the corresponding STAT3 binding site but not in its absence. TGF-β1-induced STAT3 binding and expression vector activation is abrogated in HCC cells with genetically silenced SCF. TGF-β1 induces SCF expression but SMAD2 knockdown inhibits TGF-β1-induced SCF transcription and expression. We identified 7 putative SMAD2-binding elements (SBE) within the SCF promoter. TGF-β1 stimulation results in SMAD2 binding to the SCF promoter and activation of luciferase expression vectors regulated by the SCF promoter; conversely, genetic silencing of SMAD2 abrogates TGF-β1-induced SCF promoter activation. Importantly, knockdown of SCF restored the antiproliferative functions of TGF-β, and prevented TGF-β-induced tumor cell migration and invasion. Conclusion: In advanced stage HCC, SCF drives TGF-β1 autoregulation through STAT3-mediated regulation of the TGF-β1 promoter. Thereby, SCF and TGF-β1 form a positive feedback loop that functionally switches the TGF-β pathway from a tumor suppressor to a tumor promoter in advanced stage HCC. Citation Format: Pingyu Zhang, Andres Rojas, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. A positive feedback loop between TGF-β and SCF mediates TGF-β1 ligand overexpression in advanced hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2015-3911

Abstract Background: Current systemic agents for advanced Hepatocellular carcinoma (HCC) achieve minimal survival benefits. Tumor Growth Factor-β (TGF-β) is a potent tumor suppressor via its SMAD2/3 signaling axis; its inactivation has been implicated in hepatocarcinogenesis. Paradoxically, TGF-β serum concentrations are elevated in patients with advanced HCC. The TGF-β receptor is currently explored in clinical trials as a therapeutic target. However, the mechanisms mediating the functional switch of the TGF-β pathway to an oncogenic role in advanced HCC are unknown. Material and Methods: Gene expression profiling of human HCC was performed using the Oncomine database. Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Arrays. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analyses. shRNA was used for gene silencing. Cell cycle analysis was performed by FACS. Cell proliferation was assessed by microscopic quantification, MTS- and BrdU-assay. Apoptosis was analyzed by TUNEL assay and DAPI. Migration was evaluated by wound assay, and invasion by Boyden Chamber assay. Results: In human HCC, SMAD2 overexpression and decreased expression of the negative TGF-β/SMAD regulator SKI was correlated to shortened survival. Intranuclear pSer465/467-SMAD2 was identified in up to 65% of human HCC tumors, and correlated with TNM-stage, survival and pTyr705STAT3. In 50% of human HCC cell lines, the TGF-β/SMAD2 axis was functional. Exclusively in HCC cells with intact SMAD-signaling, TGF-β induced STAT3 Tyr705-phosporylation and nuclear translocation. We show that TGF-β1 transcriptionally regulates expression of the c-KIT ligand Stem Cell Factor (SCF). SCF is overexpressed in advanced HCC tumors, and correlated to pSMAD2, metastases and decreased survival. In human HCC cells, SCF activates STAT3 via auto- and paracrine stimulation of its cognate receptor c-KIT. TGF-β1-induced activation of STAT3 is inhibited by SCF-knockdown, inhibition of c-KIT or JAK1. The TGF-β1/STAT3 axis neutralizes the cell cycle inhibitory function of TGF-β in human HCC cell lines, and instead promotes EMT, tumor cell migration and invasion. Disruption of the TGF-β/STAT3 signaling axis inhibits its tumor promoting effects and restores TGF-β antiproliferative effects. Conclusion: In advanced HCC, TGF-β/SMAD2-signaling is constitutively activated and correlated to worse outcomes. The functional switch of TGF-β is mediated through aberrant expression of SCF with subsequent stimulation of the c-KIT/STAT3 signaling axis. Disruption of this axis restores TGF-β tumor suppressor function. Disruption of the molecular network between TGF-β1/SMAD2 and SCF/STAT3 constitutes a novel therapeutic strategy aiming at restoration of a dysregulated tumor suppressor. Citation Format: Andres Rojas, Pingyu Zhang, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. TGF-β/SMAD2 signaling drives tumor-progression via c-KIT/STAT3-signaling in advanced HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2015-1962