Shoujun Gu

Vitamin D Deficiency Promotes Liver Tumor Growth in Transforming Growth Factor-β/Smad3-Deficient Mice Through Wnt and Toll-like Receptor 7 Pathway Modulation

Disruption of the TGF-β pathway is associated with liver fibrosis and suppression of liver tumorigenesis, conditions associated with low Vitamin D (VD) levels. However, potential contributions of VD to liver tumor progression in the context of TGF-β signaling remain unexplored. Our analyses of VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold increases in tumor burden in Smad3+/− mice, with a three-fold increase in TLR7 expression compared to controls. ChIP and transcriptional assays confirm Smad3 binding at two TLR7 promoter SBE sites. Molecular interactions between TGF-β pathway and VDD were validated clinically, where an absence of VD supplementation was associated with low TGF-β pathway member expression levels and β-catenin activation in fibrotic/cirrhotic human liver tissues. Subsequent supplementing VD led to restoration of TGF-β member expression with lower β-catenin levels. Bioinformatics analysis provides positive supportive correlation between somatic mutations for VD-related genes and the TGF-β pathway. We conclude that VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and β-catenin activation. VD could therefore be a strong candidate for liver cancer prevention in the context of aberrant Smad3 signaling.

Mutational Profiles Reveal an Aberrant TGF-β-CEA Regulated Pathway in Colon Adenomas

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Alcohol, stem cells and cancer

Dosage, gender, and genetic susceptibility to the effects of alcohol remained only partially elucidated. In this review, we summarize the current knowledge of the mechanisms underlying the role of alcohol in liver and gastrointestinal cancers. In addition, two recent pathways- DNA repair and TGF-β signaling which provide new insights into alcohol in the regulation of cancers and stem cells are also discussed here.

Abstract 5330: Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases

Hepatocellular carcinoma (HCC) is the 3 rd leading cause of cancer deaths worldwide, and rising in the United States at an alarming rate. Multiple E3 ubiquitin ligases such as the SMURFs and RINGH2 proteins have been identified as negative regulators of the TGF-β pathway. However, to our knowledge, there remains a gap in the integration between genomics, underlying mechanisms and the development of targeted therapeutics harnessing these TGF-β-associated E3 ligases for HCC. The aim of this study is to elucidate the role of E3 ligases in HCC, through TCGA analyses and provide mechanistic insight into these as therapeutic targets for HCC. We first analyzed the 488 hepatocellular cancers and screened for alterations in The Cancer Genome Atlas (TCGA). Immunohistochemistry (IHC), Q-PCR, Western blot analysis were used to validate the expression levels of two of the most highly altered E3 ligases, PRAJA and Keap1 in hepatocellular cancer tissues and cell lines in human and in TGF-β-deficient β2SP +/- mouse models. Inhibition studies of PRAJA and Keap1 were performed by lentivirus shRNA in HCC cell lines, and xenograft studies. From the TCGA data, we observe two different signatures (activated and inactivated) for 18 TGF-β pathway genes. While increased levels of TGF-β-related transcripts were associated with activation of hepatic fibrosis/immune microenvironment pathways, decreased levels of TGF-β members were associated with loss of TGF-β tumor suppressor function. HCCs characterized by the “inactivated” TGF-β signature were associated with a significantly poorer survival, compared to HCCs with the “activated” TGF-β signature (p=0.0027). We next analyzed 29 TGF-β-related E3 ligases, and observed raised expression of the following: PRAJA1 (12.7% of HCCs), KEAP1 (6.4%), UCHL5 (16.4%), WWP2 (11.8%), WWP1 (10%), Smurf2 (9.1%), Skp2 (9.1%), and Smurf1 (8.2%). Interestingly, expression patterns corresponded with a few TGF-β signaling members regulated by some of these E3 ligases, namely Smad3 (altered in 54%) and β2SP (27%). We identified that PRAJA1 targets Smad3 and β2SP for ubiquitination and degradation. We further observe raised levels of PRAJA (25%) and KEAP1 (70%) in 176 human liver cancers, by IHC, compared to normal controls. Depletion of PRAJA and KEAP1 with either shRNAs or E3 ligase inhibitors, substantially inhibited growth and induced apoptosis through PRAJA/Smad3/β2SP and KEAP1/Nrf signaling in HCC cell lines and xenografts. These results suggest that E3 ligases such as PRAJA1 and KEAP1 may be valuable therapeutic targets for liver cancer in the context of TGF-β signaling, an important approach given that few effective targeted therapeutics are available for this cancer with poor prognosis. Citation Format: Shuyun Rao, Heather Levin, Jian Chen, Rehan Akbani, Jon White, Wilma Jogunoori, Shoujun Gu, Kazufumi Ohshiro, Sobia Zaidi, Bibhuti Mishra, Asif Rashid, Shulin Li, Lopa Mishra. Targeting hepatocellular carcinoma through TGF-β pathway E3 ligases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2017-5330