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.

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.

Analysis of Genomes and Transcriptomes of Hepatocellular Carcinomas Identifies Mutations and Gene Expression Changes in the Transforming Growth Factor-ß Pathway.

BACKGROUND & AIMS: Development of hepatocellular carcinoma (HCC) is associated with alterations in the transforming growth factor-beta (TGF-ß) signaling pathway, which regulates liver inflammation and can have tumor suppressor or promoter activities. Little is known about the roles of specific members of this pathway at specific of HCC development. We took an integrated approach to identify and validate the effects of changes in this pathway in HCC and identify therapeutic targets. METHODS: We performed transcriptome analyses for a total of 488 HCCs that include data from The Cancer Genome Atlas. We also screened 301 HCCs reported in the Catalogue of Somatic Mutations in Cancer and 202 from Cancer Genome Atlas for mutations in genome sequences. We expressed mutant forms of spectrin beta, non-erythrocytic 1 (SPTBN1) in HepG2, SNU398, and SNU475 cells and measured phosphorylation, nuclear translocation, and transcriptional activity of SMAD family member 3 (SMAD3). RESULTS: We found somatic mutations in at least 1 gene whose product is a member of TGF-ß signaling pathway in 38% of HCC samples. SPTBN1 was mutated in the largest proportion of samples (12 of 202, 6%). Unsupervised clustering of transcriptome data identified a group of HCCs with activation of the TGF-ß signaling pathway (increased transcription of genes in the pathway) and a group of HCCs with inactivation of TGF-ß signaling (reduced expression of genes in this pathway). Patients with tumors with inactivation of TGF-ß signaling had shorter survival times than patients with tumors with activation of TGF-ß signaling (P = .0129). Patterns of TGF-ß signaling correlated with activation of the DNA damage response and sirtuin signaling pathways. HepG2, SNU398, and SNU475 cells that expressed the D1089Y mutant or with knockdown of SPTBN1 had increased sensitivity to DNA crosslinking agents and reduced survival compared with cells that expressed normal SPTBN1 (controls). CONCLUSIONS: In genome and transcriptome analyses of HCC samples, we found mutations in genes in the TGF-ß signaling pathway in almost 40% of samples. These correlated with changes in expression of genes in the pathways; up-regulation of genes in this pathway would contribute to inflammation and fibrosis, whereas down-regulation would indicate loss of TGF-ß tumor suppressor activity. Our findings indicate that therapeutic agents for HCCs can be effective, based on genetic features of the TGF-ß pathway; agents that block TGF-ß should be used only in patients with specific types of HCCs.

Identifying low density lipoprotein cholesterol associated variants in the Annexin A2 (ANXA2) gene.

BACKGROUND AND AIMS: Annexin-A2 (AnxA2) is an endogenous inhibitor of proprotein convertase subtilisin/kexin type-9 (PCSK9). The repeat-one (R1) domain of AnxA2 binds to PCSK9, blocking its ability to promote degradation of low-density lipoprotein cholesterol-receptors (LDL-R) and thereby regulate low-density lipoprotein cholesterol (LDL-C) levels. Here we identify variants in ANXA2 influencing LDL-C levels and we determine the molecular mechanisms of their effects. RESULTS: The ANXA2 single nucleotide polymorphism (SNP) genotype-phenotype association was examined using the Second-Northwick-Park Heart Study (NPHSII) (n∼2700) and the UCL-LSHTM-Edinburgh-Bristol (UCLEB) consortium (n∼14,600). The ANXA2-R1 domain coding-SNP rs17845226 (V98L) associated with LDL-C, homozygotes for the minor allele having ≈18.8% higher levels of LDL-C (p = 0.004), and higher risk of coronary heart disease (CHD) (p = 0.04). The SNP is in modest linkage disequilibrium (r2 > 0.5) with two intergenic SNPs, rs17191344 and rs11633032. Both SNPs showed allele-specific protein binding, and the minor alleles caused significant reduction in reporter gene expression (≈18%, p < 0.001). In the expression quantitative trait loci (eQTL) study, minor allele homozygotes have significantly lower levels of ANXA2-mRNA expression (p = 1.36 × 10-05). CONCLUSIONS: Both rs11633032 and rs17191344 SNPs are functional variants, where the minor alleles create repressor-binding protein sites for transcription factors that contribute to reduced ANXA2 gene expression. Lower AnxA2 levels could increase plasma levels of PCSK9 and thus increase LDL-C levels and risk of CHD. This supports, for the first time in humans, previous observations in mouse models that changes in the levels of AnxA2 directly influence plasma LDL-C levels, and thus implicate this protein as a potential therapeutic target for LDL-C lowering.

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.

TGF-ß/ß2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of ß2-spectrin (ß2SP, encoded by SPTBN1), a SMAD adaptor for TGF-ß signaling, is causally associated with BWS; however, a role of TGF-ß deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-ß signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-ß-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-ß inducible and facilitates TGF-ß-mediated repression of TERT transcription via interactions with ß2SP and SMAD3. This regulation was abrogated in TGF-ß-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-ß pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Anterior thoracolumbar spine exposure: critical review and analysis.

BACKGROUND: Indications for anterior thoracolumbar spine interbody fusion have expanded because of safe and expeditious surgical exposure that can be provided by the approach surgeon. In our practice, previous anterior interbody instrumentation, multiple disc level exposure, patient age, and body habitus are not surgical deterrents despite the potential for increased complications. The arterial and venous complications of anterior spine exposure have been well documented; however, the purpose of this study is to document the incidence of other complications, such as deep vein thrombosis (DVT), lymphedema, seroma/hematoma, wound infection, and hospital readmission and to determine whether outcome is influenced by the factors mentioned above. METHODS: Six hundred seventeen consecutive patients had anterior thoracolumbar spine exposure performed by a single vascular surgeon between January 2007 and June 2012. Office and hospital records were reviewed with institutional review board approval. RESULTS: The mean patient age was 56 years, and 16% were >69 years of age. The mean body mass index (BMI) was 29.27 kg/m(2) (range: 16-53 kg/m(2)); 39% were considered obese, with BMI measurements of >30 kg/m(2). The overwhelming majority of cases were performed for varying grades of spondylolisthesis and/or degenerative disc disease; in 8 cases (1.3%), the indication for disc exposure was diskitis/osteomyelitis. One disc level was exposed in 36% of cases, 2 in 43%, and ≥3 in 21%. Six percent of patients had previous anterior spine exposure, 42% had previous posterior laminectomy and/or diskectomy, and 3% required anterior disc reexposure to remove hardware or an artificial disc. There was 1 major arterial dissection and 3 major venous injuries. Other complications included extensive DVT (2%), debilitating lymphedema (0.5%), wound seroma/hematoma requiring treatment (2%), wound infection (3%), and readmission within 60 days (8%). Multilevel (>2 levels) disc exposure was associated with an increased rate of lymphedema, posterior lumbar wound infection, and hospital readmission (all P values ≤ 0.01; chi-squared analysis). A BMI >30 kg/m(2) was associated with an increased rate of DVT, posterior lumbar wound infection, and hospital readmission (all P values ≤ 0.018; chi-squared analysis). Age >69 years was associated with an increased rate of wound hematoma (P = 0.002; chi-squared analysis). Logistic regression analysis revealed that BMI >30, multilevel disc exposure, and removal of an artificial disc or hardware were all associated with an increased rate of any nonvessel complication (P values < 0.001); however, no specific variable was associated with an increased rate of a major vessel complication, including those cases where the surgical indication was diskitis/osteomyelitis. CONCLUSIONS: The overall incidence of nonvessel injury complications after anterior thoracolumbar spine exposure is low. Redo anterior spine exposure and redo disc exposure cases, including those that require hardware or artificial disc removal, can be performed safely. Multidisc level exposure is, however, associated with an increased incidence of lymphedema, wound infection, and hospital readmission. Patients with BMI >30 kg/m(2) should be approached with caution because there is a significantly increased rate of DVT, wound infection, and hospital readmission.

Hyperglycemia is associated with increased risk of morbidity and mortality after colectomy for cancer.

BACKGROUND: The relationship of hyperglycemia to general surgery outcomes is not well-understood. We studied the association of operative day and postoperative day 1 (POD1) blood glucose (BG) with outcomes after open colectomy for cancer. STUDY DESIGN: We retrospectively analyzed the 2000-2005 Veterans Affairs Surgical Quality Improvement Program database, linked with Veterans Affairs Decision Support System BG values. Median BG was categorized as hypoglycemic (<80 mg/dL); normoglycemic (BG 80-120 mg/dL); or mildly (BG 121-160 mg/dL), moderately (BG 161-200 mg/dL), or severely (BG >200 mg/dL) hyperglycemic. The relationship of BG to postoperative outcomes was assessed with multivariable logistic regression. RESULTS: We identified 9,638 colectomies. We excluded 511 procedures for emergency status or preoperative coma, mechanical ventilation, or sepsis. After excluding patients without recorded BG, we analyzed operative day and POD1 BG in 7,576 and 5,773 procedures, respectively. On multivariable analysis, operative day moderate hyperglycemia was associated with surgical site infection (odds ratio = 1.44; 95% CI, 1.10-1.87). POD1 severe hyperglycemia was associated with cardiac arrest (odds ratio = 2.31; 95% CI, 1.08-4.98) and death (odds ratio = 1.97; 95% CI, 1.23-3.15). POD1 mild (odds ratio = 2.20; 95% CI, 1.05-4.60), moderate (odds ratio = 3.44; 95% CI, 1.51-7.84), and severe (odds ratio = 3.94; 95% CI, 1.64-9.58) hyperglycemia and hypoglycemia (odds ratio = 6.74; 95% CI, 1.75-25.97) were associated with myocardial infarction. Associations were similar in diabetic and nondiabetic patients. CONCLUSIONS: Even mild hyperglycemia was associated with adverse outcomes after colectomy, suggesting that a perioperative BG target of 80 to 120 mg/dL, although avoiding hypoglycemia, might be appropriate. Randomized clinical trials are needed to confirm these findings.

Urachal remnant small bowel obstruction: report of two adult cases.

This report describes two separate cases of adult patients with intestinal obstruction caused by an urachal remnant. While reports of infected urachal cysts causing intra-abdominal pathology are not uncommon, intestinal obstruction caused by urachal remnants is exceedingly rare in the pediatric population and has never been described in adults. Both of these patients required surgical intervention with excision of the urachal remnant and subsequent resolution of the small bowel obstruction.