Vivek Shukla

Loss of transforming growth factor β adaptor protein β-2 spectrin leads to delayed liver regeneration in mice

Liver regeneration, following partial hepatectomy (PHx), occurs through precisely controlled and synchronized cell proliferation, in which quiescent hepatocytes undergo one to two rounds of replication, with restoration of liver mass and function. We previously demonstrated that loss of the Smad3/4 adaptor protein β‐2 spectrin (β2SP) is associated with faster entry into S phase, and hepatocellular cancer formation. These observations led us to further pursue the role of β2SP in cell cycle progression in vivo. Liver regeneration studies with PHx in β2SP+/− mice reveal a surprising and significant decrease in liver/body weight ratio at 48 hours after PHx in β2SP+/− mice in comparison to wildtype mice. At 48 hours after PHx we also observe decreased levels of cyclin E (2.4‐fold, P < 0.05), Cdk1 (7.2‐fold, P < 0.05), cyclin A, pRb (Ser249/Thr252), proliferative cell nuclear antigen (PCNA), cyclin D1 with elevated levels of pCdk1 (Thr14) (3.6‐fold, P < 0.05). Strikingly, at 24 hours elevated levels of p53 (4‐fold, P < 0.05), phospho‐p53 (ser15 and ser20), and p21 (200‐fold, P < 0.05) persisting to 48 hours after PHx further correlated with raised expression of the DNA damage markers pChk2 (Thr68) and γH2AX (S139). However, compromised cell cycle progression with loss of β2SP is not rescued by inhibiting p53 function, and that G2/M phase arrest observed is independent and upstream of p53. Conclusion: β2SP deficiency results in dysfunctional hepatocyte cell cycle progression and delayed liver regeneration at 48 hours after PHx, which is p53‐independent. β2SP loss may increase susceptibility to DNA damage, impair cell cycle progression, and ultimately lead to hepatocellular cancer. (HEPATOLOGY 2011;)

Loss of histone acetyltransferase cofactor transformation/transcription domain‐associated protein impairs liver regeneration after toxic injury

Organ regeneration after toxin challenge or physical injury requires a prompt and balanced cell‐proliferative response; a well‐orchestrated cascade of gene expression is needed to regulate transcription factors and proteins involved in cell cycle progression and cell proliferation. After liver injury, cell cycle entry and progression of hepatocytes are believed to require concerted efforts of transcription factors and histone‐modifying activities; however, the actual underlying mechanisms remain largely unknown. The purpose of our study was to investigate the role of the histone acetyltransferase (HAT) cofactor transformation/transcription domain‐associated protein (TRRAP) and histone acetylation in the regulation of cell cycle and liver regeneration. To accomplish our purpose, we used a TRRAP conditional knockout mouse model combined with toxin‐induced hepatic injury. After we treated the mice with a carbon tetrachloride toxin, conditional ablation of the TRRAP gene in those mice severely impaired liver regeneration and compromised cell cycle entry and progression of hepatocytes. Furthermore, loss of TRRAP impaired the induction of early and late cyclins in regenerating livers by compromising histone acetylation and transcription factor binding at the promoters of the cyclin genes. Our results demonstrate that TRRAP and TRRAP/HAT‐mediated acetylation play an important role in liver regeneration after toxic injury and provide insight into the mechanism by which TRRAP/HATs orchestrate the expression of the cyclin genes during cell cycle entry and progression. (HEPATOLOGY 2011)

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.

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

Abstract 4111: Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway

Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with HCC. We analyzed whole genome data for TGF-β signaling and examined the role of VD in the context of TGF-β inactivation in HCC. Methods: Databases of HCC Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. The effect of calcitriol on cell proliferation was measured in HCC cell lines; Hep-G2 and MEF cells, with knocked down TGF-β/β2-spectrin (β2SP). Wild-type (WT), Sptbn1+/- and Smad3+/- mice were fed with diets containing 200 IU VD/kg or 10,000 IU VD/kg for 9 weeks. Hepatocyte proliferation was analyzed through Ki67 staining. The expression of cyclin D1 was evaluated by Western blotting. A whole genome gene expression profiling array was performed from liver samples of those mice using Illumina MouseWG-6 v2.0 gene expression arrays®. Lastly, liver samples from patients with HCC who had received VD supplementation were evaluated by immunohistochemistry. Results: Whole genome data revealed aberrant TGF-β signaling in ∼70% of HCCs. Treatment with calcitriol suppressed the growth of HCC cell lines, regardless of their TGF-β pathway status. Inhibition of proliferation was noted in WT MEFs as well as in the Sptbn1+/- and Sptbn1-/- cells. High dose VD reduced hepatic proliferation and cyclin D1 levels in Sptbn1+/- and Smad3+/- mutants, and in WT mice. The microarray data demonstrated the most significant changes in gene expression were associated with acute phase inflammatory response (p Citation Format: Lior H. Katz, Andrea Cortes, Vivek Shukla, Keigo Machida, Hidekazu Tsukamoto, Kirty Shetty, Aiwu R. He, Lynt B. Johnson, Jian Chen, Randa El-Zein, Ju-Seog Lee, Lopa Mshra. Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway. [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 4111. doi:10.1158/1538-7445.AM2014-4111

Abstract 5198: Targeting E3 ligase PRAJA1 in hepatocellular cancer .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Hepatocellular Cancer (HCC) is a rising and lethal disease, that is difficult to treat due to late diagnosis and few viable targeted therapeutics. Recent studies support a key role for TGF-β signaling in suppressing these tumors. Expression of the E3 ubiquitin ligase PRAJA, a negative regulator of TGF-β signaling via Smad3 and its adaptor β2-spectrin (β2SP), is dramatically raised in up to 70% of human HCCs. This indicates that PRAJA1 overexpression suppresses Smad3 and β2SP mediated TGF-β tumor suppressor signaling hence promoting HCC progression. Hypothesis: Disruption of TGF-β/β2SP tumor suppressor pathway by PRAJA1 leads to uncontrolled activation of chromatin and tumor formation. Therefore, small molecule inhibitors that specifically target PRAJA1 could be very useful in HCC therapy. Materials & Methods: Cancer patient datasets were retrieved from Gene Expression Omnibus and analyzed with Oncomine analysis tools. We analyzed 3 group-arrays of liver tumor tissues (total 298 cancer tissues vs. 249 normal tissues, p-value<1E-4). We also analyzed 55 arrays for other cancers (total 2850 cancer tissues vs. 779 normal tissues, p-value<1E-4). To validate protein expression, specimens from 13 HCC and normal liver tissues were immunostained with anti-PRAJA1. Whole mount in situ hybridization histochemistry (ISHH) assay was used to determine knock down PRAJA1in zebrafish embryos. Soft agar assay and colony formation assay were performed to elucidate PRAJA1 oncogenic activity in HCC cells. Additionally, we screened a compound library for E3 ligase inhibitors targeting PRAJA1. Results: (1) PRAJA1 expression is dramatically raised in human HCCs with loss of TGF-β signaling. (2) PRAJA1 interacts with β2SP/Smad3 and inhibits TGF-β tumor suppressor target gene activation. (3) Inhibition of PRAJA1 in the developing Zebrafish embryo and HCCs leads to high levels of apoptosis. (4) Overexpression of PRAJA1 leads to increased HCC growth; conversely knock down of PRAJA1 suppresses its oncogenic activities. (5) TGF-β promotes complex of CTCF and β2SP in nucleus. (6) RTA402/RTA405 inhibits PRAJA1 and restores TGF-β tumor suppressor function in HCC cells. (7) RTA402/RTA405 induces apoptosis and inhibits HCC cell growth and tumorigenesis. Conclusions: PRAJA1 disrupts the TGF-β tumor suppressor pathway, and its overexpression promotes cancer cell survival and proliferation. We have identified 2 novel triterpenoids that exhibit PRAJA1 inhibitory activity, which have demonstrated that suppression of PRAJA1 by specific small molecule inhibitors restores TGF-β tumor suppressor function and suppresses growth of HCC cells. Our results provide a better understanding of the mechanisms that regulate HCC development. Importantly, this study may lead to new therapeutics targeting this lethal cancer and potentially to a Phase I clinical trial in HCC. Citation Format: Jian Chen, Jiun-Sheng Chen, Vivek Shukla, Zhixing Yao, Hai-Long Piao, Wilma Jogunoori, Bibhuti Mishra, Lopa Mishtra. Targeting E3 ligase PRAJA1 in hepatocellular cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2013-5198

Abstract 1775: High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: A crucial factor in protection from liver injury, fibrosis and cancer by agents such as alcohol, aflatoxins and viral hepatitis is the enforcement of genomic stability. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Because inactivation of the TGF-β pathway results in DNA damage, alcohol toxicity and HCC, TGF-β has been implicated as a critical promoter of genomic stability and tumor suppression; however, the framework by which this occurs is not known. We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples and identified inactivation of TGF-beta pathway members as a prominent characteristic of alcohol induced HCC, potentially through inactivation of a TGF-b adaptor, β2SP. We therefore hypothesized that the TGF-β/β2SP/Smad3 pathway is crucial for protection against aldehyde genotoxicity through enforcing genomic stability. Materials & Methods: We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples. β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. Primary MEFs from β2SP+/+, β2SP+/− and β2SP−/− mice were treated with colcemid for cytogenetic analysis, and they were also treated with multiple DNA damaging agents and γ-irradiation to evaluate Mdc1, NBS1 and Rad51 foci formation. ChIP assays were performed to determine the recruitment of β2SP/Smad3/Smad4 at FancD2 promoter. Results: (1) Through our WGS and WES analyses, we discovered a novel inactivating mutation of TGF-β/β2SP pathway in alcohol-associated HCC. This β2SP mutation results in functional disruption of TGF-β signaling. (2) Alcohol treatment in the absence of β2SP mice results in teratogenicity and spontaneous fetal alcohol-like phenotype (FAS). (3) Loss of β2SP results in premature replicative senescence and marked hypersensitivity to DNA interstrand crosslinking agents, such as mitomycin C (MMC). (4) DNA damage response induces nuclear localization of β2SP in a TGF-β-dependent manner. (5) Suppression of β2SP impairs the recruitment of DNA repair proteins in the nucleus and consequently the repair of DNA double-strand breaks. (6) Loss of β2SP correlates with loss of FANCD2 expression, but not of any of the other 12 Fanconi anemia complementation factors. Moreover, our results indicate that β2SP/Smad3/Smad4 regulate expression of FANCD2 at the transcriptional level. (7) The expression of β2SP and FANCD2 is correlated in alcoholic hepatitis and cirrhotic livers. Conclusions: Through whole genome and Exome sequencing we found a novel inactivating mutation in β2SP which results in loss of TGF-β signaling pathway. Importantly, these results could potentially lead to new therapeutics targeting toxin-induced DNA damage and tumorigenesis. Citation Format: Vivek Shukla, Jian Chen, Jiun-Sheng Chen, Ying Li, Lior Katz, Avijit Majumdar, Lei Li, Walter Hittleman, Xiaoping Su, Junjie Chen, Xifeng Wu, Patrizia Farci, Lopa Mishra. High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2013-1775

Abstract 3692: Vitamin D as a chemoprophylactic agent for liver cancer.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. One third of patients with cirrhosis will develop HCC, thus comprising a well-characterized population at high-risk for the development of this deadly, yet potentially preventable cancer. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-beta signaling. Recently, inactivation of TGF-beta signaling has been associated with HCC. The identification of subgroups of patients responsive to treatment is vital for preventing this disease effectively. In an effort to understand the role of Vit D in HCC and to identify clear responders, we analyzed the role of Vit D in the context of TGF-beta inactivation. Methods: The effect of calcitriol on cell proliferation was measured in HCC cell lines as well as in Hep-G2 cells that were knocked down to the adaptor protein β2-spectrin (Spnb2). To evaluate in vivo effects of VD on animals predisposed to developing liver cancer, Spnb2 +/ − mice were fed with special diets containing 1000 IU VD/kg/D or 10,000 IU VD/kg/D. After 6 weeks, the animals were euthanized and hepatocyte proliferation and the content of fat in the liver were analyzed through Ki67 staining and oil red staining, respectively. The expression of cyclin D1 was evaluated by Western blotting (WB). Lastly, liver samples from patients with HCC who had received VD supplementation in the diet were also evaluated by immunohistochemistry. Results: We observed a dramatic response to treatment with calcitriol as indicated by a significant increase in the expression of CYP24A1. Additionally, treatment with VD and calcitriol suppressed the growth of HCC cell lines. Absence of Spnb2 caused more prominent suppression of the growth of Hep-G2. From our animal studies we found that steatosis and hepatocyte proliferation were higher in Spnb2 +/ − mice fed with a low dose of VD, and that the high dose VD significantly reduced both parameters. The increased proliferation in Spnb2 +/ − mice fed with a low dose of VD was associated with higher levels of cyclin D1 expression, which was restored the level almost to that seen in the wild type mice with the higher dose of VD. Lastly, VD supplementation to patients with HCC was associated with higher expression of β2-spectrin (p Conclusions: VD suppresses the proliferation of several HCC cell lines, and in agreement with this, high doses of VD in the diet suppress hepatocyte proliferation and steatosis in the HCC prone Spnb2 +/ − mice. Our data also indicate that VD administration appears to restore the expression of key cell cycle proteins even in the setting of TGF-β signaling disruption. Taken together, these preliminary results suggest that VD treatment strategies could potentially be pivotal in preventing obesity-induced HCC. Citation Format: Lior H. Katz, Vivek Shukla, Sara Peleg, Kirti shetty, Jian Chen, Steven Curley, Powel H. Brown, Lopa Mishra. Vitamin D as a chemoprophylactic agent for liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2013-3692

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

Abstract 4695: TGF-β enforces genomic instability in liver cancers

Background: Over 90% of liver cancers, from viral hepatitis to alcohol injury, occur against a background of chronic liver disease and cirrhosis. Genomic stability, G1/S cell cycle arrest through c-Myc and CDK suppression, have been some of the mechanisms proposed in tumor suppression by TGF-β. The Smad3/4 adaptor protein, β2SP, is emerging as potent regulator of tumor genesis by its ability to affect TGF-β tumor suppressor function. Deletion of β2SP results in a dramatic and spontaneous formation of liver cancer and gastrointestinal cancers. Moreover, β2SP+/− and β2SP+/− /Smad3+/− mutant mice phenocopy human Beckwith-Wiedemann syndrome (BWS), a hereditary human cancer stem cell syndrome imprinting disorder with loss of p57, increase in IGF2, and an association with 800 fold increase of cancers that include those of the liver. Delayed liver regeneration and extensive DNA damage was observed in β2SP heterozygote mice. In addition, spectrins have been observed to associate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionally regulate FANC genes. Hypothesis: We therefore hypothesised that TGF-β is a crucial enforcer of genomic stability, and that β2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the DNA repair pathway. Materials & Methods: To determine the role of β2SP and Smad3 in the DNA damage response, and whether β2SP and β2SP/Smad3 loss plays a causal role in the downregulation of the Fanc/Brca pathway, we used our mouse models as well as the mouse embryonic fibroblasts. Oxidative DNA damage was induced by H2O2 treatment and assayed tail length analysis. Irradiation was induced by 8Gy IR and DNA was studied by immunofluorescence of DNA damage sensors H2AX, 532BP1, and pChk2. DNA repair was assayed by Mdc1, NBS1, and Rad51 foci formation at different time points after irradiation. Results: β2SP+/− and β2SP+/−/Smad3+/− mice exhibited an increased prevalence of HCC and GI cancer and many phenotypic characteristics observed in BWS patients. Loss of β2SP results in premature replicative senescence with spontaneous DNA damage. β2SP deficiency results in increased sensitivity to exogenous DNA damage and insufficient DNA repair. Depletion of TGF-β adaptor protein β2SP impairs the loading of repair proteins and repair of DNA double-strand breaks. Loss of FancD2 expression in HCC and GI cancer cell lines correlates with loss of β2SP. Moreover, β2SP−/− and β2SP+/−/Smad3+/− cell lines accumulate DNA damage that is further exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Conclusions: TGF-β confers genomic stability through regulation of DNA impairment of the Fanc/Brca DNA damage response and interstrand link repair and inactivation of TGF- ≤ pathway results in alcohol toxicity, cirrhosis and liver cancers. 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 4695. doi:1538-7445.AM2012-4695