Mice with dysfunctional TGF-ß signaling develop altered intestinal microbiome and colorectal cancer resistant to 5FU.

Emerging data show a rise in colorectal cancer (CRC) incidence in young men and women that is often chemoresistant. One potential risk factor is an alteration in the microbiome. Here, we investigated the role of TGF-ß signaling on the intestinal microbiome and the efficacy of chemotherapy for CRC induced by azoxymethane and dextran sodium sulfate in mice. We used two genotypes of TGF-ß-signaling-deficient mice (Smad4+/- and Smad4+/-Sptbn1+/-), which developed CRC with similar phenotypes and had similar alterations in the intestinal microbiome. Using these mice, we evaluated the intestinal microbiome and determined the effect of dysfunctional TGF-ß signaling on the response to the chemotherapeutic agent 5-Fluoro-uracil (5FU) after induction of CRC. Using shotgun metagenomic sequencing, we determined gut microbiota composition in mice with CRC and found reduced amounts of beneficial species of Bacteroides and Parabacteroides in the mutants compared to the wild-type (WT) mice. Furthermore, the mutant mice with CRC were resistant to 5FU. Whereas the abundances of E. boltae, B.dorei, Lachnoclostridium sp., and Mordavella sp. were significantly reduced in mice with CRC, these species only recovered to basal amounts after 5FU treatment in WT mice, suggesting that the alterations in the intestinal microbiome resulting from compromised TGF-ß signaling impaired the response to 5FU. These findings could have implications for inhibiting the TGF-ß pathway in the treatment of CRC or other cancers.

Role of TGF-ß 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-ß 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 ß2SP+/- and ß2SP+/-/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.

A Pan-Cancer Analysis Reveals High-Frequency Genetic Alterations in Mediators of Signaling by the TGF-ß Superfamily.

We present an integromic analysis of gene alterations that modulate transforming growth factor ß (TGF-ß)-Smad-mediated signaling in 9,125 tumor samples across 33 cancer types in The Cancer Genome Atlas (TCGA). Focusing on genes that encode mediators and regulators of TGF-ß signaling, we found at least one genomic alteration (mutation, homozygous deletion, or amplification) in 39% of samples, with highest frequencies in gastrointestinal cancers. We identified mutation hotspots in genes that encode TGF-ß ligands (BMP5), receptors (TGFBR2, AVCR2A, and BMPR2), and Smads (SMAD2 and SMAD4). Alterations in the TGF-ß superfamily correlated positively with expression of metastasis-associated genes and with decreased survival. Correlation analyses showed the contributions of mutation, amplification, deletion, DNA methylation, and miRNA expression to transcriptional activity of TGF-ß signaling in each cancer type. This study provides a broad molecular perspective relevant for future functional and therapeutic studies of the diverse cancer pathways mediated by the TGF-ß superfamily.

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.

Loss of the transforming growth factor-ß effector ß2-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 ß/mothers against decapentaplegic homolog 3 adaptor ß2-Spectrin (ß2SP, gene Sptbn1) in maintaining genomic stability following alcohol-induced DNA damage. ß2SP supports DNA repair through ß2SP-dependent activation of Fanconi anemia complementation group D2 (Fancd2), a core component of the Fanconi anemia complex. Loss of ß2SP leads to decreased Fancd2 levels and sensitizes ß2SP 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 ß stimulation is regulated by the ß2SP/mothers against decapentaplegic homolog 3 complex. CONCLUSION: Dysfunctional transforming growth factor ß/ß2SP signaling impacts the processing of genotoxic metabolites by altering the Fanconi anemia DNA repair pathway. (Hepatology 2017;65:678-693).

Transforming growth factor-ß 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 ß (TGF-ß) pathway, loss of p53 and/or activation of ß-catenin do not spontaneously drive HCC in animal models. Despite many advances in cancer genetics that include identifying the dominant role of TGF-ß 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-ß in regulating the cancer stem cell niche. (Hepatology Communications 2017;1:477-493).

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

Disruption of the TGF-ß 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-ß 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-ß pathway and VDD were validated clinically, where an absence of VD supplementation was associated with low TGF-ß pathway member expression levels and ß-catenin activation in fibrotic/cirrhotic human liver tissues. Subsequent supplementing VD led to restoration of TGF-ß member expression with lower ß-catenin levels. Bioinformatics analysis provides positive supportive correlation between somatic mutations for VD-related genes and the TGF-ß pathway. We conclude that VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and ß-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-ß-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-ß 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-ß signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-ß 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-ß in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

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 nuclear 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 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.

Epigenetic silencing of beta-spectrin, a TGF-beta signaling/scaffolding protein in a human cancer stem cell disorder: Beckwith-Wiedemann syndrome.

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy. However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. The non-pleckstrin homology domain ß-spectrin (ß2SP) (the official name for human is Spectrin, beta, nonerythrocytic 1 (SPTBN1), isoform 2; the official name for mouse is Spectrin beta 2 (Spnb2), isoform 2), a scaffolding protein, functions as a potent TGF-ß signaling member adaptor in tumor suppression and development. Yet, the role of the ß2SP in human tumor syndromes remains unclear. Here, we report that ß2SP(+/-) mice are born with many phenotypic characteristics observed in BWS patients, suggesting that ß2SP mutant mice phenocopy BWS, and ß2SP loss could be one of the mechanisms associated with BWS. Our results also suggest that epigenetic silencing of ß2SP is a new potential causal factor in human BWS patients. Furthermore, ß2SP(+/-) mice provide an important animal model for BWS, as well as sporadic cancers associated with it, including lethal gastrointestinal and pancreatic cancer. Thus, these studies could lead to further insight into defects generated by dysfunctional stem cells and identification of new treatment strategies and functional markers for the early detection of these lethal cancers that otherwise cannot be detected at an early stage.

Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis.

UNLABELLED: We have previously demonstrated that 40%-70% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) within 15 months, revealing the importance of the transforming growth factor-beta (TGF-beta) signaling pathway in suppressing tumorigenesis in the liver. The current study was carried out to investigate mechanisms by which embryonic liver fodrin (ELF), a crucial Smad3/4 adaptor, suppresses liver tumor formation. Histological analysis of hyperplastic liver tissues from elf(+/-) mice revealed abundant newly formed vascular structures, suggesting aberrant angiogenesis with loss of ELF function. In addition, elf(+/-) mice displayed an expansion of endothelial progenitor cells. Ectopic ELF expression in fetal bovine heart endothelial (FBHE) cells resulted in cell cycle arrest and apoptosis. Further analysis of developing yolk sacs of elf(-/-) mice revealed a failure of normal vasculature and significantly decreased endothelial cell differentiation with embryonic lethality. Immunohistochemical analysis of hepatocellular cancer (HCC) from the elf(+/-) mice revealed an abnormal angiogenic profile, suggesting the role of ELF as an angiogenic regulator in suppressing HCC. Lastly, acute small interfering RNA (siRNA) inhibition of ELF raised retinoblastoma protein (pRb) levels nearly fourfold in HepG2 cells (a hepatocellular carcinoma cell line) as well as in cow pulmonary artery endothelial (CPAE) cells, respectively. CONCLUSION: Taken together these results, ELF, a TGF-beta adaptor and signaling molecule, functions as a critical adaptor protein in TGF-beta modulation of angiogenesis as well as cell cycle progression. Loss of ELF in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers. Our studies propose that ELF is potentially a powerful target for mimetics enhancing the TGF-beta pathway tumor suppression of HCC.

Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.

Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ "cancer stem cells," such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000-50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-beta-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF. elf(+/-) mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-beta signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC in elf(+/-) mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-beta signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-beta pathway.

Cell cycle deregulation and loss of stem cell phenotype in the subventricular zone of TGF-beta adaptor elf-/- mouse brain.

The mammalian forebrain subependyma contains neural stem cells and other proliferating progenitor cells. Recent studies have shown the importance of TGF-beta family members and their adaptor proteins in the inhibition of proliferation in the nervous system. Previously, we have demonstrated that TGF-beta induces phosphorylation and association of ELF (embryonic liver fodrin) with Smad3 and Smad4 resulting in nuclear translocation. Elf(-/-) mice manifest abnormal neuronal differentiation, with loss of neuroepithelial progenitor cell phenotype in the subventricular zone (SVZ) with dramatic marginal cell hyperplasia and loss of nestin expression. Here, we have analyzed the expression of cell cycle-associated proteins cdk4, mdm2, p21, and pRb family members in the brain of elf(-/-) mice to verify the role of elf in the regulation of neural precursor cells in the mammalian brain. Increased proliferation in SVZ cells of the mutant mice coincided with higher levels of cdk4 and mdm2 expression. A lesser degree of apoptosis was observed in the mutant mice compared to the wild-type control. Elf(-/-) embryos showed elevated levels of hyperphosphorylated forms of pRb, p130 and p107 and decreased level of p21 compared to the wild-type control. These results establish a critical role for elf in the development of a SVZ neuroepithelial stem cell phenotype and regulation of neuroepithelial cell proliferation, suggesting that a mutation in the elf locus renders the cells susceptible to a faster entry into S phase of cell cycle and resistance to senescence and apoptotic stimuli.

Inactivation of ELF/TGF-beta signaling in human gastrointestinal cancer.

TGF-beta/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. ELF, a beta-spectrin, plays a key role in the transmission of TGF-beta-mediated transcriptional response through Smads. ELF was originally identified as a key protein involved in endodermal stem/progenitor cells committed to foregut lineage. Also, as a major dynamic adaptor and scaffolding protein, ELF is important for the generation of functionally distinct membranes, protein sorting and the development of polarized differentiated epithelial cells. Disruption of elf results in the loss of Smad3/Smad4 activation and, therefore, a disruption of the TGF-beta pathway. These observations led us to pursue the function of ELF in gastrointestinal (GI) epithelial cell-cell adhesion and tumor suppression. Here, we show a significant loss of ELF and reduced Smad4 expression in human gastric cancer tissue samples. Also, of the six human gastric cancer cell lines examined, three show deficient ELF expression. Furthermore, we demonstrate the rescue of E-cadherin-dependent homophilic cell-cell adhesion by ectopic expression of full-length elf. Our results suggest that ELF has an essential role in tumor suppression in GI cancers.

Loss of cooperative function of transforming growth factor-beta signaling proteins, smad3 with embryonic liver fodrin, a beta-spectrin, in primary biliary cirrhosis.

UNLABELLED: Modulation of fibrogenesis, epithelial, and mesenchymal cell fates are prominent effects of transforming growth factor-beta (TGF-beta) signaling by Smad proteins. We have previously shown that Smad2 and Smad3 insufficiency leads to a loss of bile ducts. In addition, Smad3/4 activity is mediated by embryonic liver fodrin (ELF), a beta-Spectrin. In mouse elf(-/-) mutants and in liver explant cultures, loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. A similar phenotype is seen in a number of cholestatic diseases with progressive loss of intrahepatic bile ducts and fibrosis. However, the expression patterns of Smads or role of ELF in cholestatic and fibrotic liver diseases are not yet known. METHODS/RESULTS: We investigated the role of ELF in primary biliary cirrhosis (PBC), autoimmune hepatitis C, chronic viral hepatitis and in livers from mice deficient in Smad2/Smad3. We generated elf(+/-) mutant mice and analyzed for chronic liver disease and hepatocellular cancer (HCC) from 6 to 12 months. Perturbations in ELF expression were consistently seen only in PBC tissues. ELF expression was similarly aberrant in tissues from Smad2(+/-)/Smad3(+/-) mutant mice. Further studies indicated that ELF mislocalization is correlated with aberrant localization of Smad3 in some PBC tissues. Thirteen of 17 elf(+/-) mutant mice developed steatosis, fibrosis, hepatic dysplasia, with HCC in two mice. CONCLUSIONS: These results suggest that a compromised cytoarchitecture and polarized trafficking of TGF-beta signaling molecules, ELF and Smad3 are involved in the pathogenesis of PBC as well as HCC.