Zhixing Yao

Transcriptional regulation of STAT3 by SPTBN1 and SMAD3 in HCC through cAMP-response element-binding proteins ATF3 and CREB2

The cytoskeletal protein Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein to SMAD3 in TGF-β signaling, may prevent hepatocellular carcinoma (HCC) development by downregulating the expression of signal transducer and activator of transcription 3 (STAT3). To elucidate the as yet undefined mechanisms that regulate this process, we demonstrate that higher levels of STAT3 transcription are found in livers of heterozygous SPTBN1(+/-) mice as compared to that of wild type mice. We also found increased levels of STAT3 mRNA, STAT3 protein, and p-STAT3 in human HCC cell-lines after knockdown of SPTBN1 or SMAD3, which promoted cell colony formation. Inhibition of STAT3 overrode the increase in cell colony formation due to knockdown of SPTBN1 or SMAD3. We also found that inhibition of SPTBN1 or SMAD3 upregulated STAT3 promoter activity in HCC cell-lines, which is dependent upon the cAMP-response element (CRE) and STAT-binding element (SBE) sites of the STAT3 promoter. Mechanistically, suppression of SPTBN1 and SMAD3 augmented the transcription of STAT3 by upregulating the CRE-binding proteins ATF3 and CREB2 and augmented the binding of those proteins to the regions within or upstream of the CRE site of the STAT3 promoter. Finally, in human HCC tissues, SPTBN1 expression correlated negatively with expression levels of STAT3, ATF3, and CREB2; SMAD3 expression correlated negatively with STAT3 expression; and the level of phosphorylated SMAD3 (p-SMAD3) correlated negatively with ATF3 and CREB2 protein levels. SPTBN1 and SMAD3 collaborate with CRE-binding transcription factors to inhibit STAT3, thereby preventing HCC development.

Abstract 115: Telomerase modulation in a human cancer stem cell syndrome with loss of TGF-β signaling is a promising treatment strategy in liver and gastrointestinal cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome with an 800 fold risk of multiple cancers, is currently linked to deregulated imprinting at chromosome 11p15, IGF2, CDKN1C and others. Yet, causal molecular defects and genetic models of this overgrowth syndrome have remained elusive to date in the majority of cases. CCCTC-Binding Factor CTCF is a highly conserved zinc finger protein that has diverse regulatory functions, including transcriptional ctivation/repression/imprinting of molecules such as TERT, IGF-2 and c-Myc. Recent studies demonstrate a high frequency of TERT promoter mutations in early stage of multiple cancers, suggesting that these promoter mutations may function as driver events that contribute to oncogenesis through TERT deregulation. However, telomerase remains a challenge to target effectively. We have recently discovered identified a mouse model for BWS with loss of TGF-β signaling with multiple liver and GI cancers. CTCF expression is markedly decreased, with concomitantly high TERT levels in these cancers. Therefore, we hypothesize that TGF-β/β2SP/Smad3/ signaling regulates CTCF and TERT regulation. Rescuing TGF-β signaling may successfully reduce tumor burden with inhibition of telomerase. Materials & Methods: Generation of β2SP+/-/Smad3+/- mice and genotype analysis. Database of Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. BWS tumors and mice liver, pancreas and stomach tissue specimens were immunostained with anti-TERT antibody. ChIP assays were to demonstrate the recruitment of β2SP/Smad3/CTCF at the promoter of TERT. Results: (1) β2SP+/-/Smad3+/- heterozygote mice spontaneously develop visceromegaly, multiple cancers, phenocopy a hereditary human cancer stem cell syndrome BWS. (2) Genomics and transcriptomics analyses revealed aberrant TGF-β signaling in β2SP+/-/Smad3+/- mice and human BWS. (3) Increased TERT expression in β2SP+/-/Smad3+/- mice is similar to that observed in human BWS. (4) CTCF levels are markedly decreased in β2SP+/-/Smad3+/- mice tissues. (5) CTCF interacts with β2SP/Smad3 in cell nucleus in a TGF-β-dependent manner. (6) TGF-β promotes the complex of β2SP/Smad3/CTCF at TERT promoter region. Conclusions: Loss of TGF-β signaling pathway leads to dysfunctional chromosome modulation through TERT deregulation. Smad3/β2SP/CTCF complex regulates TERT transcriptional activity. Our results provide a better mechanistic understanding of this dysfunctional stem cells cancer syndrome. Importantly, this study may lead to identify new treatment strategies and functional markers for the early detecting lethal cancers. Citation Format: Jian Chen, Jiun-Sheng Chen, Zhixing Yao, Wilma Jogunoori, Bibhuti Mishra, Lopa Mishra. Telomerase modulation in a human cancer stem cell syndrome with loss of TGF-β signaling is a promising treatment strategy in liver and gastrointestinal cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 115. doi:10.1158/1538-7445.AM2014-115

614 The Oncogenic Role of Praja1 on Disrupting TGF-β Signaling in Hepatocellular Cancer

BACKGROUND AND AIMS: In addition to hepatitis B virus, HCV is also an important cause of HCC in Asians; however, it is often overlooked. This study aims to examine baseline characteristics, timing of HCV diagnosis and long-term survival of HCV-related HCC in Asians compared to non-Asian patients. METHODS: We conducted a retrospective cohort study of 798 consecutive Asian (n=220) and non-Asian (n=572) patients with HCV-related HCC who were identified via computer query using ICD-9 diagnosis at a U.S. university medical center between 7/1996 and 6/2012. Individual records were reviewed. RESULTS: Asians were much older (66 [38-88] vs. 56 [31-87] years, P ,0.0001) and more likely to be female (33% vs. 19%, P,0.0001). A significantly larger proportion of Asians were also diagnosed with HCC within only 2 years of HCV diagnosis compared to non-Asians (35% vs. 20%, p=0.001). Non-Asians were more likely to have decompensated liver disease and had higher median Child-Turcotte-Pugh score (6 [5-11] vs. 7 [5-13], P ,0.0001). Asian patients were more likely to undergo liver-directed palliative therapy (46% vs. 28%) and much less likely to be listed for liver transplantation(20% vs. 48%) (P ,0.001), despite similar rates of meeting Milan criteria for liver transplantation (52 vs.58%, P=0.16). Overall, there was a trend for higher median survival rates in Asians compared to non-Asians (30 vs. 21 months, P=0.091). Among those who were listed for liver transplantation, there was no statistically significant difference in survival between Asians and non-Asians in the first 2 years (72% vs. 68%) but there was divergence with Asians having higher survival afterwards (61% vs. 51%) (Figure 1). However, among those undergoing liver-directed palliative therapy, Asians had significantly higher long-term survival compared to non-Asians (5-year survival: 28% vs. 10%, P,0.0001) (Figure 2). On multivariate Cox proportional hazards model also inclusive of age, sex, BCLC staging, Child-Pugh score, meeting Milan criteria, liver transplantation listing, non-Asian ethnicity was an independent predictor for lower overall survival (HR=0.70 [0.52-0.86]). CONCLUSIONS: Despite being older and having a lower rate of liver transplantation listing, Asian HCV/HCC patients had higher median survival compared to non-Asian patients, particularly among those undergoing palliative therapy. Non-Asians were more likely to have decompensated liver disease and non-Asian ethnicity was an independent predictor for poorer long-term survival. Compared to non-Asians, Asians with HCV-related HCCwere also muchmore likely to have delayed HCV diagnosis. Improved strategies in HCV screening in Asians are needed as it may lead to earlier diagnosis and treatment of HCV infection and possible prevention of HCC development in this ethnic population with a disproportionate HCC disease burden.

Tu1175 The Protective Influence of Vitamin D in Hepatocellular Carcinoma

Backgrounds: Green tea catechins (GTCs) have been shown to provide anti-carcinogenic effects in several laboratory studies. Although the effect of green tea consumption on the risk of gastric cancer has been studied in epidemiological studies, the results remain controversial. The anti-carcinogenic mechanisms of GTCs have not been fully evaluated in rodent models of gastric cancer. Previous reports indicate that transgenic INS-GASmice are hypergastrinemic and induced corpus gastritis and eventually developed gastric cancer at over 20 months old. Aim: The aim of our study was to investigate the molecular effects of GTCs on gastritis and premalignant lesions in INS-GAS mice. Methods: Thirty eight, male INSGAS mice on a FVB background were used in this study. Mice were divided into two groups: A GTCs group and control. Mice in the GTCs group were allowed free access to drinking water containing 2000ppm of GTCs from the age of 9 weeks. Mice were euthanized at the age of 13 or 37 weeks. Histological scores (inflammation and dysplasia) were graded on a blinded basis by pathologist on a scale from 0 to 4. The mRNA expressions of IFN-gamma and TNF-alpha in the corpus of stomachs were evaluated using quantitative PCR. Statistical analysis was performed using a Mann-Whitney U test. Results: Body weights in mice supplemented with GTCs were significantly lower than that of controls at both 13 and 37 weeks of age (p < 0.05). Treated and control mice developed minimal to mild corpus gastritis, although dysplasia was evident. The histological score of dysplasia in mice supplemented with GTCs was significantly lower than that in controls at 37 weeks of age (p < 0.05). GTCs supplementation did not affect the inflammation score. The mRNA level of IFN-gamma in mice administrated GTCs was significantly lower than that of controls at 13 weeks of age (p < 0.05), although it did not reach statistical significance at 34 weeks of age (p = 0.056). GTCs supplementation did not alter TNF-alpha mRNA expression levels between both groups. Conclusions: Administration of GTCs attenuated progression of gastric dysplasia in INS-GAS male mice. These results demonstrated that IFN-gamma dependent mechanisms may be involved in the protective role of GTCs in development of gastritis and premalignant lesions in this rodent model.

Abstract 1619: Preventive effects of vitamin D in liver cancer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer related mortality worldwide. In view of its limited therapeutic options, strategies for HCC prevention are urgently needed. Vitamin D (VD) and calcitriol have anti-proliferative and other anti neoplastic activities. Low levels of VD are associated with increased risk for cancer, and calcitriol can be active agent in cancer treatment. Therefore, VD might be a strong candidate for HCC prevention. We show that vitamin D can prevent the development of HCC in cell lines, reduce proliferation in animal models and modify cell cycle protein levels in human livers. Methods: 1) Hep3B HCC cells were treated with either calcitriol, or two synthetic VD analogs - EB1089 and QW-1624F. Growth was measured using an MTS assay. 2) Livers were harvested from 12 months-old wild-type (WT) mice fed standard Purina chow, and age-matched β2spectrin (≥2SP) +/− mice after 6 weeks of feeding with a semi-purified AIN-93 diet containing 1000 IU VD/kg or 10,000 IU VD/kg. Hepatocyte proliferation (Ki67 staining) and fat content (Oil red staining) were assessed as well as cell cycle proteins (by Western blot). 3) Human liver tissue from healthy and cirrhotic livers as well as from HCC patients were stained for TGF ≤ Receptor II (TBRII) and for β2SP with and without treatment with VD. Results: 1) HCC cell lines - Doses of 10-1000nM of calcitriol or vitamin D analogs suppress the growth of HCC cells. 2) Serum levels of VD in WT and β2SP +/− mice on regular diet were 25.6+/− 2.9 ng/mL and 21.7+/− 8 ng/mL, respectively (p=NS). High dose vitamin D in the diet increased significantly serum levels of vitamin D in β2SP +/− mice to 49.6+/− 12.7 (p=0.0036). Proliferation and steatosis were higher in hepatocytes from β2SP +/− mice fed low dose VD as compared to the wild-type mice fed standard diet. However, high dose VD reduced hepatocyte proliferation and steatosis in β2SP +/− mice. Levels of cyclin D1 were increased in the β2SP +/− mice fed with regular diet as compared to the wild type mice. However, high-dose VD restored the levels of cyclin D1 almost to that of the WT (p=0.069). 3) Normal human hepatocytes have active TGF-β signaling as demonstrated by high levels of β2SP and TBRII staining, while cirrhotic liver cells and HCC cells have reduced β2SP and TBRII staining. VD treatment partially restores TGF-β signaling with similar β2SP and TBRII staining to that in normal hepatocytes. A potential mechanism through KLF-4 modulation may contribute to it. A disappearance of an MRI detected nodule was observed in one of the patients treated with Vitamin D with restoration of TGF-β markers. Conclusions: 1) Calcitriol and its analogs suppress proliferation of HCC cell lines. 2+3) High doses of VD in the diet suppress hepatocyte proliferation and hepatic steatosis in susceptible mice. Possible mechanism can be the effect of VD on cell cycle proteins through the TGF-β pathway as seen in humans. These findings emphasize the use of vitamin D as a novel preventive treatment for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1619. doi:1538-7445.AM2012-1619

S2034 CEA Increases Metastatic Potential of CRC Cells By Targeting Multiple Signaling Pathways

BCL6 and nuclear translocation of HB-EGF-CTF in gastric cancers. Method: We examined BCL6 and cyclinD2 expression level under the 12-0-tetradecanoylphorbor-13-acetate (TPA) stimulation. KB-R7785 was used to suppress HB-EGF-CTF nuclear translocation. We also investigated the interaction and translocation of BCL6 with HB-EGF-CTF by using immunofluorescence microscopy and immunoprecipitation. Furthermore, we immunohistochemically studied 100 surgical specimens of advanced gastric cancers to analyze the expression of HB-EGF, BCL6, and cyclin D2. Results: TPA treatment resulted in BCL6 degradation and cyclin D2 up-regulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF. The HB-EGF-CTF nuclear translocation lead to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and the ubiquitin/proteasome pathway mediated the BCL6 degradation. siRNA targeting BCL6 revealed that the upregulation of cyclin D2 depends on BCL6 degradation. BCL6 interacts with nuclear translocated HB-EGF-CTF and the nuclear export and degradation of BCL6 (caused by this interaction and the ubiquitin/proteasome pathway) induces cyclin D2 up-regulation. In human gastric cancer tissues, BCL6 expression is associated with differentiated gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also induced cyclin D2 expression In Vivo (human cancer tissues). Conclusion: Inhibition of HB-EGF-CTF nuclear translocation and the resulting maintenance of BCL6 function are important for the regulation of gastric cancer progression.

800 TGF-β Pathway Members ELF and SMAD3 Are Key Tumor Suppressors in Human Beckwith-Wiedemann Syndrome

The Smad3/4 adaptor protein ELF is emerging as a potent regulator of tumorigenesis by its ability to affect TGF-β tumor suppressor function (Science, 2003, 299:574-577, Science, 2005, 310:68-71). However, to date, the role of the TGF-β pathway at specific stages in gastrointestinal (GI) tumor development such as metaplasia, dysplasia and carcinoma remains unclear. A surprising and dramatic discovery by us is that elf+/and elf+/-/Smad3+/mice develop multiple GI cancers, with a phenotype strongly suggestive of Beckwith Wiedemann syndrome (BWS), a hereditary human cancer syndrome with no known causal mutations, and associated with loss of imprinting (LOI) at multiple loci on chromosome 11p15 including IGF2. Methods and Results: 1. Elf+/and Elf+/-/Smad3+/mice develop visceromegaly, multiple liver lobes, abnormal ear folds, and 70% of the mice develop multiple tumors that include metastatic pancreatic carcinoma, hepatocellular carcinoma, and small bowl adenocarcinoma. 2. A dramatic decrease in elf mRNA (200-2000 fold) was observed in elf+/-/Smad3+/tumor tissues without significant decrease of Smad3 or p53 compared to normal tissues. 3.We observed a loss of the 200 Kda ELF protein expression 4 out of 11 human cancer cell lines (HCC, gastric and pancreatic) by Western Blotting and in 19/20 human HCC and gastric cancer tissues by IHC labeling using a N-terminal peptide specific anti-ELF Ab. 4.We identified a missense mutation Arg 928 to Ile (G→A) in elf exon15 in 2 of the human GI cancer cell lines with loss of ELF expression. 5. Importantly we observed a loss of “C” to “U” RNA editing at elf Exon 15 in GI cancer cell lines with loss of ELF expression. 6. All 4/4 Human BWS patient tissues displayed a marked decrease in ELF RNA (by 50%), and a complete loss of ELF protein expression (western blot and IHC). Smad3 RNA and protein expression were not significantly altered. 7. In all 4 BWS tissues, we observed a loss of “C” to “U” RNA editing at elf Exon 15 with loss of ELF expression. 8 Array, proteomic analyses and IHC revealed a markedly high expression (about 4-6 fold) of IGF2, β-catenin, CDK4, and hTERT in Elf+/and Elf+/-/Smad3+/tissues. Conclusions: 1.The TGF-β pathway through elf+/and elf+/-/Smad3+/phenotype provides a novel and strongly compelling model for Beckwith-Wiedemann (BWS) a human cancer overgrowth syndrome. 2. Alterations of ELF/ SMAD3 expression at the mRNA level are the major genetic disposition of human BWS. 3. In BWS patient samples, aberrant elf RNA editing may be a predominant mechanism for loss of ELF protein expression observed in BWS.

W1964 Telomerase Reverse Transcriptase Regulation By TGF-β Signaling Through Adaptor ELF and SMAD3 That Is Independent of C-MYC

3442 Transforming Growth Factor-β (TGF-β) induced antiproliferative responses is a hallmark of many cancer cells. Smad proteins as intracellular mediators for TGF-β signaling and regulate target gene expression by activating or repressing gene transcription. Smads 2, 3 and 4 inhibit G1/S cell cycle progression mostly by suppression of c-Myc and cyclin-dependent kinases (cdks). Myc amplification, telomere maintenance, and telomerase reverse transcriptase (TERT) reactivation are common features of human foregut cancers, such as hepatocellular carcinoma (HCC). Emerging evidence indicates that ELF, a Smad3/Smad4 adaptor protein required for TGF-β signaling, is a powerful tumor suppressor [Science, 2005, 310(5745):68-71, Oncogene, 2007, 26(50):7103-10]. Elf+/-, elf+/-/Smad3+/- and elf+/-/Smad4+/-, (but not Smad3-/-) mice dramatically develop foregut cancers, including hepatocellular, pancreatic and gastric cancers. However, the specific role(s) of h-TERT, c-MYC and Elf, and their relation to the TGF-β pathway, in foregut cancer formation are poorly understood. Deletion of ELF results in a dramatic and spontaneous formation of liver and gastrointestinal cancers, with exon 15 mutations in 11% of human HCC and gastric cancer cell lines tested. Elf+/-andelf+/-/Smad3+/- mice develop visceromegaly and multiple GI cancers (70% of mice), spontaneously. This phenotype provides compelling evidence that elf+/-and elf+/-/Smad3+/- mice are a model of the hereditary human cancer syndrome Beckwith-Wiedemann (BWS). Aims: In this study, we investigated the mechanism of role of Elf, Smad3 and c-Myc in regulating human TERT gene expression by TGF-β in HepG2, PLC/PRF/5, SNU 298 cell lines and elf+/-/Smad3+/- mice tumor tissues in vitro and in vivo. Results show that: 1) TheElf+/-/Smad3+/-mice develop visceromegaly and multiple cancers, including metastatic pancreatic, hepatocellular, small bowel lymphomas, adrenocortical carcinomas, renal carcinomas and tumor tissues from elf+/-/Smad3+/- mice show a dramatic decrease of ELF mRNA; 2) TERT and c-Myc are markedly elevated in elf+/-andelf+/-/Smad3+/- mice; Interestingly, TERT levels are far higher than can be accounted for by c-Myc levels in elf+/-andelf+/-/Smad3+/- tumors; 3)Ectopic ELF and Smad3 suppress TERT greater than c-Myc in the absence of TGF-β. 4) Both ELF and Smad3 associate with c-Myc in TGF-β stimulated hepatocytes and suppress TERT. 5)Overexpression of ELF and/or Smad3 decreases hTERT RNA levels in PLC/PRF/5 and SNU 298 human cell lines. Conclusions: Taken together our preliminary data suggest that divergent pathways converge on ELF and Smad3 that then regulate TERT. Inactivation of the TGF-β signaling pathway with TERT activation provides a strong strategy for generating targeted therapeutics at TERT in these lethal human cancers.