Ji-Hyun Shin

Glutamine synthetase mediates sorafenib sensitivity in β-catenin-active hepatocellular carcinoma cells

The gene encoding β-catenin is frequently mutated in hepatocellular carcinoma cells. While the oncogenicity of β-catenin has been extensively studied, β-catenin’s role in hepatocellular carcinoma tumor metabolism is currently less well understood. In this study, we found that β-catenin regulates the expression of glutamine synthetase and triggers a series of metabolic changes leading to induction of autophagy in hepatocellular carcinoma cells. β-Catenin-active Hep3B and HepG2 cells exhibited higher basal levels of autophagic activity than did β-catenin wild-type cells. We also found that autophagy in β-catenin-active cells was mediated by glutamine synthetase, as silencing of glutamine synthetase significantly reduced autophagic activity. We also showed that β-catenin-active hepatocellular carcinoma cells were more sensitive to sorafenib than were β-catenin wild-type cells. Our results demonstrated that glutamine synthetase-mediated autophagy explains the high sensitivity of β-catenin-active hepatocellular carcinoma cells to sorafenib. Our results highlight the importance of glutamine metabolism in the regulation of autophagy in hepatocellular carcinoma cells. More importantly, our study unravels the molecular mechanisms leading to sorafenib sensitivity in hepatocellular carcinoma.

Abstract 3594: The TGF-β effector β2SP depletion abrogates DNA damage repair

Objective: Exposure to genotoxins, such as ethanol-derived acetaldehyde, leads to DNA damage, liver injury, and promotes the development of cancer. Alcohol-related genotoxicity, arising from DNA damage by metabolically generated reactive aldehydes, has recently been observed in models with genetic inactivation of members of the Fanconi anemia pathway. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Transforming growth factor β (TGF-β) is a critical protein in the regulation of several cancer phenotypes and also functions as an extracellular sensor of ionizing radiation-induced cell damage. Yet, how the TGF-β pathway contributes to toxin-induced DNA damage repair remains unclear. We utilized the TGF-β/β2SP mutant mouse model to investigate the mechanisms in relation to β2SP-mediated TGF-β modulation of the Fanconi anemia pathway for DNA damage repair, alcohol sensitivity, and liver tumorigenesis. Methods: (1) β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. (2) Genomic instability and sensitivity to DNA damaging agents in primary β2SP+/+, β2SP-/- MEFs were determined by clonogenic survival and metaphase chromosome aberrations analysis. (3) Defective S-phase specific DNA repair in β2SP-/- MEFs were determined by DNA replication restart assays. (4) ChIP assays were performed to determine the recruitment of β2SP/Smad3 at FancD2 promoter. (5) We investigated the clinical relevance of altered β2SP and FancD2 function using immunohistochemical analyses of 20 human liver specimens from alcoholic hepatitis (n = 5), alcoholic cirrhosis (n = 5), and alcohol-associated liver cancer (n = 5), as well as normal controls (n = 5). Results: (1) Sptbn1-deficient mice exhibit a phenotype similar to human fetal alcohol syndrome and are sensitive to ethanol exposure. (2) Sptbn1-deficient cells exhibit genomic instability and hypersensitivity to DNA damage (3) Sptbn1-deficiency delays DNA damage repair. (4) Furthermore, Sptbn1-deficient cells are defective in stalled DNA replication fork resolution and homologous recombination. (5) FancD2 ectopic expression rescues the DNA repair defect in Sptbn1 null cells. (6) β2SP and FancD2 are clinically correlated in alcoholic hepatitis and HCCs. Conclusions: Our model proposes that in response to liver toxins such as alcohol, the TGF-β/β2SP/Smad3 pathway prevents liver injury and cancer through its direct effects on DNA repair and genomic stability. Thus, characterizing the role of TGF-β in alcohol-induced injury could potentially enhance our mechanistic insight into the basis for therapeutics targeting toxin-induced DNA damage and tumorigenesis. Citation Format: Jian Chen, Vivek Shukla, Jiun-Sheng Chen, Raj K. Panditab, Yun Seong Jeong, Lior H Katz, Ji-Hyun Shin, YoungJin Gi, Lawrence N. Kwongc, Clayton R. Huntb, Patrizia Farci, Xiaoping Su, Jon White, Bibhuti Mishra, Asif Rashid, Milind Javle, Lei Li, Junjie Chen, John R, Stroehlein, Marta Davila, Rehan Akbani, Keigo Machidao, Hidekazu Tsukamoto, Tej K. Pandita, Lopa Mishra. The TGF-β effector β2SP depletion abrogates DNA damage repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3594.

Sa1851 Prognostic Significance of TGF-β Signaling in Human Hepatocellular Carcinoma

A S L D A b st ra ct s (LCD) including the variants small cell change (SCC), and large cell change (LCC), HCC, and NLC control tissues using Vectra® automated multispectral imaging system, and to evaluate any differential staining features between groups.Design: FOXM1 immunohistochemistry was performed on a liver cancer progression tissue array consisting of 85 paired liver tissue cores representing 20 NDC, 40 LCD consisting of 20 SCC and 20 LCC, 20 HCC, and 5 NLC control tissues in optical density by Vectra® automated multispectral imaging system. Staining was statistically compared between sample groups using a 2 tailed p-value derived from Welchs T Test. Results: The mean FOXM1 nuclear staining in OD was NLC 0.0318 ±0.0107, SCC 0.0741 ±0.0239, NDC 0.0717 ±0.0216, LCC 0.1103 ±0.1210, and HCC 0.1134 ±0.0378. Nuclear staining features by FOXM1 had three distinct groups with the highest staining seen in HCC and LCC, followed by SCC and NDC, and the least in NLC. The nuclear staining features of FOXM1 in HCC and LCC were similar (p=0.9158), as were those seen in SCC and NDC (p=0.7437). NLC showed markedly less staining (p <0.0088) than all other sample groups. SCC and NDC had statistically less staining than HCC (p<0.0003). SCC and NDC did not have statistically different nuclear staining from LCC (p=0.1961 for SCC, and p=0.1677 for NDC). Conclusions: Here, we provide evidence that the hepatocyte nuclear expression of the protein product of human proto-oncogene FOXM1 shows a stepwise increase in NLC and paired liver tissue samples of NDC and SCC, and LCC and HCC by Vectra®automated multispectral imaging system. Further, FOXM1 nuclear expression rises in LCC, before the development of HCCduring hepatocarcinogenesis. Additionally, the FOXM1 nuclear immunolevels did not differ in LCC and HCC.

CSN6 positively regulates c-Jun in a MEKK1-dependent manner

c-Jun is a proto-oncoprotein that is commonly overexpressed in many types of cancer and is believed to regulate cell proliferation, the cell cycle, and apoptosis by controlling AP-1 activity. Understanding the c-Jun regulation is important to develop treatment strategy for cancer. The COP9 signalosome subunit 6 (CSN6) plays a critical role in ubiquitin-mediated protein degradation. MEKK1 is a serine/threonine kinase and E3 ligase containing PHD/RING domain involved in c-Jun ubiquitination. Here, we show that CSN6 associates with MEKK1 and reduces MEKK1 expression level by facilitating the ubiquitin-mediated degradation of MEKK1. Also we show that CSN6 overexpression diminishes MEKK1-mediated c-Jun ubiquitination, which is manifested in mitigating osmotic stress-mediated c-Jun downregulation. Thus, CSN6 is involved in positively regulating the stability of c-Jun. Overexpression of CSN6 correlates with the upregulation of c-Jun target gene expression in cancer. These findings provide new insight into CSN6-MEKK1-c-Jun axis in tumorigenesis.

Abstract 892: Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Hepatocellular carcinoma (HCC) is the fifth most common tumor and the third leading cause of cancer-related deaths. Loss of TGF-β signaling has been associated with development of HCC. Cancer preventive effects of Vitamin D (VD) have been implicated in multiple cancers, however a clear role for Vitamin D in specific high risk populations remains undefined for HCC. Therefore, we examined for a potential chemopreventive role of VD in HCC in the context of TGF-β inactivation. Methods: (1) We screened for somatic mutation of the TGF-β pathway and VD related genes in 202 HCCs from The Cancer Genome Atlas (TCGA). (2) Wild type, Sptbn1+/- and Smad3+/- mice were fed with low VD (200 IU VD/kg) or high VD diet (10,000 IU VD/kg) for 9 weeks. Liver tissues were subjected to microarray analyses and further evaluation by quantitative PCR. (3) Wild type, Sptbn1+/- and Smad3+/- mice were injected Diethylnitrosamine (DEN) and at 8 month, 26 mice receiving low VD (200 IU VD/kg) and 26 mice receiving high VD (10,000 IU VD/kg) chow. HCC development was assessed at 4 months after VD treatment. (4) Reverse Phase Protein Array (RPPA) was performed to analyze expression profiles of 164 proteins of mouse liver tumors. (5) Liver samples from patients with HCV cirrhosis receiving VD supplements were examined to evaluate expression of TGF-β, Wnt and VD pathway molecules by immunohistochemistry. Results: (1) We observed a high rate of somatic mutation in TGF-β and VD pathway related genes in the TCGA genomic analysis. (2) None of the VD treated mice developed HCC but high VD treatment increased TLR7 mRNA expression about 3-fold in liver from Smad3+/- mice compared with livers from WT mice. (3) Smad3+/- mice with low VD showed 3-fold larger HCC formation, compared to the high VD group (Smad3+/-) that did not develop significant tumors. However, correction of VD in the Chow after 10 months did not reverse HCC formation. (4) RPPA data revealed that the tumor suppressor protein PDCD4 was reduced in Smad3+/- mice with low VD treatment. However, oncoproteins such as β-catenin, Stat5A and Bcl2-XL were induced in the same sample. (5) Expression of β2SP and TβRII were higher in HCV cirrhosis patients receiving VD supplementation compared to non-treated group. Conclusions: Loss of TGF-β signaling pathway developed HCC and VD deficiency promotes tumor growth in the context of Smad3 disruption potentially through regulation of TLR7 expression. However, after 10 months restoring VD does not have any significant effect on altering tumors. Therefore VD could be a potential candidate for prevention in early identified HCC high risk individuals who has inactivation of TGF-β/Smad3 signaling. Citation Format: Ji-hyun Shin, Lior H. Katz, Nina M. Munoz, Andrea Cortes, Vivek Shukla, Sang-Bae Kim, Franklin Herlong, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Jian Chen, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice. [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 892. doi:10.1158/1538-7445.AM2015-892