Wilma Jogunoori

Pathogenesis of Hepatocellular Carcinoma Development in Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is being recognized as an increasingly important contributor to the burden of hepatocellular carcinoma (HCC) worldwide. It is often accompanied by obesity and diabetes mellitus and is believed to be the hepatic representation of the metabolic syndrome. HCC development in NAFLD is multifactorial and complex. It is dependent on not only the well-described mechanisms noted in chronic liver injury but also on the molecular derangements associated with obesity and dysmetabolism. These include adipocyte remodeling, adipokine secretion, lipotoxicity, and insulin resistance. Recent advances focus on the importance of the gut-liver axis in accelerating the process of oncogenesis in NAFLD. The Farnesoid X receptor (FXR) has been demonstrated to have important metabolic effects, and its pharmacological activation by obeticholic acid has been recently reported to produce histological improvement in non-alcoholic steatohepatitis (NASH). It is hoped that delineating the mechanisms of hepatic fibrosis and oncogenesis in NASH will lead to enhanced strategies for cancer prevention, surveillance, and therapy in this population.

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.

Loss of the transforming growth factor-β effector β2-Spectrin promotes genomic instability

Exposure to genotoxins such as ethanol‐derived acetaldehyde leads to DNA damage and liver injury and promotes the development of cancer. We report here a major role for the transforming growth factor β/mothers against decapentaplegic homolog 3 adaptor β2‐Spectrin (β2SP, gene Sptbn1) in maintaining genomic stability following alcohol‐induced DNA damage. β2SP supports DNA repair through β2SP‐dependent activation of Fanconi anemia complementation group D2 (Fancd2), a core component of the Fanconi anemia complex. Loss of β2SP leads to decreased Fancd2 levels and sensitizes β2SP mutants to DNA damage by ethanol treatment, leading to phenotypes that closely resemble those observed in animals lacking both aldehyde dehydrogenase 2 and Fancd2 and resemble human fetal alcohol syndrome. Sptbn1‐deficient cells are hypersensitive to DNA crosslinking agents and have defective DNA double‐strand break repair that is rescued by ectopic Fancd2 expression. Moreover, Fancd2 transcription in response to DNA damage/transforming growth factor β stimulation is regulated by the β2SP/mothers against decapentaplegic homolog 3 complex. Conclusion: Dysfunctional transforming growth factor β/β2SP signaling impacts the processing of genotoxic metabolites by altering the Fanconi anemia DNA repair pathway. (Hepatology 2017;65:678‐693)Exposure to genotoxins such as ethanol-derived acetaldehyde leads to DNA damage and liver injury, and promotes the development of cancer. We report here a major role for the TGF-β/Smad3 adaptor β2-Spectrin (β2SP, gene Sptbn1) in maintaining genomic stability following alcohol-induced DNA damage. β2SP supports DNA repair through β2SP-dependent activation of Fancd2, a core component of the Fanconi anemia complex. Loss of β2SP leads to decreased Fancd2 levels and sensitizes β2SP mutants to DNA damage by ethanol treatment, leading to phenotypes that closely resemble those observed in animals lacking both Aldh2 and Fancd2, and resemble human fetal alcohol syndrome. Sptbn1-deficient cells are hypersensitive to DNA cross-linking agents, and have defective DNA double-strand break repair that is rescued by ectopic Fancd2 expression. Moreover, Fancd2 transcription in response to DNA damage/TGF-β stimulation is regulated by the β2SP/Smad3 complex. Thus, dysfunctional TGF-β/β2SP signaling impacts the processing of genotoxic metabolites by altering the Fanconi anemia DNA repair pathway. This article is protected by copyright. All rights reserved.

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.

Transforming growth factor-β in liver cancer stem cells and regeneration

Cancer stem cells have established mechanisms that contribute to tumor heterogeneity as well as resistance to therapy. Over 40% of hepatocellular carcinomas (HCCs) are considered to be clonal and arise from a stem‐like/cancer stem cell. Moreover, HCC is the second leading cause of cancer death worldwide, and an improved understanding of cancer stem cells and targeting these in this cancer are urgently needed. Multiple studies have revealed etiological patterns and multiple genes/pathways signifying initiation and progression of HCC; however, unlike the transforming growth factor β (TGF‐β) pathway, loss of p53 and/or activation of β‐catenin do not spontaneously drive HCC in animal models. Despite many advances in cancer genetics that include identifying the dominant role of TGF‐β signaling in gastrointestinal cancers, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support effective targeted therapies for these common and lethal cancers. Moreover, pathways involved in stem cell transformation into gastrointestinal cancers remain largely undefined. Identifying the key mechanisms and developing models that reflect the human disease can lead to effective new treatment strategies. In this review, we dissect the evidence obtained from mouse and human liver regeneration, and mouse genetics, to provide insight into the role of TGF‐β in regulating the cancer stem cell niche. (Hepatology Communications 2017;1:477–493)

Role of TGF-β in Alcohol-Induced Liver Disease

Over 90% of hepatocellular carcinoma (HCC) occurs against a background of chronic liver disease or cirrhosis induced from viral hepatitis to alcohol injury. One third of patients with cirrhosis will develop HCC during their lifetime, with a 3-5% annual incidence. However, little is known about the key mechanisms by which toxins mediate DNA damage in the liver. Recent studies support a central role for TGF-β signaling in conferring genomic stability yet the precise mechanism of action and the specific stages of tumor suppression remain unclear (Bornstein S, White R, Malkoski S, Oka M, Han G, Cleaver T, Reh D, Andersen P, Gross N, Olson S, Deng C, Lu SL, Wang XJ. J Clin Invest 119:3408-3419 (2009); Korc M. J Clin Invest 119:3208-3211 (2009); Glick A, Popescu N, Alexander V, Ueno H, Bottinger E, Yuspa SH. Proc Natl Acad Sci U S A 96:14949-14954 (1999)). Furthermore, it has recently been shown that β2SP+/- and β2SP+/-/Smad3+/- mice phenocopy a hereditary human cancer syndrome, the Beckwith-Wiedemann syndrome (BWS), which has an 800 fold risk of cancers including HCC, hepatoblastoma, and a range of liver disorders. Identifying key biological pathways and mechanisms for suppressing alcohol-induced stem cell injury and HCC will be critical for enhancing patient care and the employment of new therapeutic approaches.

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art, Challenges and Research Directions.

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA’s research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC.

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

Abstract 2540: Disruption of a TGF-β-CTCF regulated pathway leads to cancer stem cells

Background: Sporadic cancer formation from stem cells, and molecular switches leading to this remain poorly understood. Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder with an 800-fold increased risk of developing tumors. Imprinting of the IGF2/H19 and the CDKN1C/KCNQ1 loci on chromosome 11p15.5 is mediated by the chromatin insulator CCCTC-binding factor (CTCF). This regulation is lost in BWS, leading to aberrant overexpression of the growth promoting genes. Epigenetic silencing of β2-spectrin (β2SP, gene SPTBN1), a SMAD adaptor for Transforming Growth Factor-beta (TGF-β) signaling, is causally associated with BWS. With this new model for BWS, the mechanisms though which disruption of TGF-β signaling leads to tumorigenesis could be elucidated further. We further sought to determine the roles of the TGF-β-mediated β2SP/SMAD3/CTCF complex in stem cell biological function. Experimental procedures: 1. Tumorigenesis analysis in Sptbn1+/− /Smad3+/− mice. 2. Whole-transcriptome sequencing were performed in the BWS cells and Sptbn1+/−; Sptbn1−/−; Smad3+/−; and Sptbn1+/−/Smad3+/− MEFs. 3. Tumor-initiating-cell (TIC) tumorigenesis, sphere formation assays were performed for stem cell like characteristics. Results: 1. We observed that double heterozygous Sptbn1+/−/Smad3+/− mice with defective TGF-β signaling develop multiple tumors phenotypically similar to those of BWS. 2. CTCF is TGF-β-inducible and facilitates TGF-β-mediated repression of hTERT transcription via β2SP/SMAD3/CTCF interactions. This regulation is abrogated in the TGF-β defective mice and BWS, causing hTERT overexpression. 3. Whole-transcriptome RNA sequencing of MEFs isolated from these three TGF-β-deficient strains displayed increased levels of stem cell-associated genes, including PDPN, EPAS1, CXCL1 and ALDH2. 4. CTCF, β2SP, and SMAD3, and modulate stem cell like characteristics such as CD133+/CD49+ TIC tumorigenesis, sphere formation and nanog expression. 5. Further, knock-down of β2SP, Smad3, or CTCF in HepG2 cells resulted in an increase of sphere formation, further supporting a role of these elements in regulating stemness. Conclusions: Loss of CTCF-dependent imprinting of tumor promoter genes such as IGF2 and TERT is caused by a defective TGF-β pathway, giving new insight into BWS-associated tumorigenesis and sporadic human cancers that are associated with mutations in SPTBN1 and SMAD3. Dysfunction of the β2SP/SMAD3/CTCF complex increases stem-like properties and enhances TICs. Our analysis provides important insight into the switches involved in sporadic cancer formation, particularly those associated with this human stem cell disorder. Citation Format: Jian Chen, Zhixing Yao, Jiun-Sheng Chen, Young Jin Gi, Yun Seong Jeong, Wilma Jogunoori, Mitchell Belkin, Bibhuti Mishra, Jon White, Abhisek Mitra, Shulin Li, Milton Finegold, Marta Davila, Jerry Shay, Keigo Machida, Hidekazu Tsukamoto, Lopa Mishra. Disruption of a TGF-β-CTCF regulated pathway leads to cancer stem cells. [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 2540.

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.