Tumor necrosis factor alpha (TNF-α) is a critical mediator of inflammation, and its production is tightly regulated, with control points operating at nearly every step of its biosynthesis. We sought to identify uncharacterized TNF-α 3′ untranslated region (3′UTR)-interacting proteins utilizing a novel screen, termed the RNA capture assay. We identified CARHSP1, a cold-shock domain-containing protein. Knockdown of CARHSP1 inhibits TNF-α protein production in lipopolysaccharide (LPS)-stimulated cells and reduces the level of TNF-α mRNA in both resting and LPS-stimulated cells. mRNA stability assays demonstrate that CARHSP1 knockdown decreases TNF-α mRNA stability from a half-life ( t 1/2 ) of 49 min to a t 1/2 of 22 min in LPS-stimulated cells and from a t 1/2 of 29 min to a t 1/2 of 24 min in resting cells. Transfecting CARHSP1 into RAW264.7 cells results in an increase in TNF-α 3′UTR luciferase expression in resting cells and CARHSP1 knockdown LPS-stimulated cells. We examined the functional effect of inhibiting Akt, calcineurin, and protein phosphatase 2A and established that inhibition of Akt or calcineurin but not PP2A inhibits CARHSP1 function. Subcellular analysis establishes CARHSP1 as a cytoplasmic protein localizing to processing bodies and exosomes but not on translating mRNAs. We conclude CARHSP1 is a TNF-α mRNA stability enhancer required for effective TNF-α production, demonstrating the importance of both stabilization and destabilization pathways in regulating the TNF-α mRNA half-life.

The purpose of this study is to determine the genetic frequency of GNAS activating mutations in colorectal cancer and the corresponding pathology of GNAS mutant tumors. Oncogenic mutations in GNAS have been described in a number of neoplasms including those of the pituitary, kidney, pancreas, and, more recently, in colon cancer. To ascertain the frequency in colon cancer we employed a sensitive pyrosequencing platform for mutation detection of the R201C and R201H GNAS hotspots in tumor samples representing all clinical stages. We additionally assayed for KRAS and BRAF mutations as previous reports have shown that these often co-occur with activating GNAS mutations. Of the 428 colon tumors assayed, mutations in GNAS were present in 10 of the samples (2.3%), indicating this is a significant, albeit infrequent, mutation in colorectal tumors. Nine GNAS mutant tumors (90%) harbored concomitant activating mutations in either the KRAS or BRAF oncogene, which was significantly greater than the mutation frequency of these genes in the tumor population (56%, p<0.0305). All ten of the GNAS mutant tumors arose in the right (proximal) colon (p<0.007), and 7 of 8 reviewed cases exhibited a marked villous morphology. Taken together, these data indicate that GNAS mutant colon tumors commonly have synchronous mutations in KRAS or BRAF, are right-sided in location, and are associated with a villous morphology.

The molecular basis of aberrant protein glycosylation, a pathological alteration widespread in colorectal cancers (CRC), and the mechanisms by which it contributes to tumor progression remain largely unknown. We performed targeted re-sequencing of 430 glycosylation-associated genes in a series of patient-derived CRC cell lines (N = 31) and matched primary tumor tissues, identifying 12 new significantly mutated glycosylation-associated genes in colon cancer. In particular, we observed an enrichment of mutations in genes (B3GNT2, B4GALT2, ST6GALNAC2) involved in the biosynthesis of N- and Cores 1–3 O-linked glycans in the colon, accounting for ~16% of the CRCs tested. Analysis of independent large-scale tumor tissue datasets confirmed recurrent mutations within these genes in colon and other gastrointestinal cancers. Systematic biochemical and phenotypic characterization of the candidate wild-type and mutant glycosyltransferases demonstrated these mutations as either markedly altering protein localization, post-translational modification, encoded enzymatic activities and/or the migratory potential of colon carcinoma cells. These findings suggest that functionally deleterious mutations in glycosyltransferase genes in part underlie aberrant glycosylation, and contribute to the pathogenesis of molecular subsets of colon and other gastrointestinal malignancies.

Tumor necrosis factor-␣ (TNF-␣) production is regulated by transcriptional and posttranscriptional mechanisms. Lipopolysaccharide activates the NFB pathway increasing TNF-␣ transcription. Lipopolysaccharide also activates the mitogenactivated protein kinase pathways, resulting in stabilization and enhanced translation of the TNF-␣ message. In addition, nuclear export of the TNF-␣ mRNA is a posttranscriptionally regulated process involving the Tpl2-ERK pathway and requiring the presence of the TNF-␣ AU-rich element (ARE). We demonstrate that nuclear export of the TNF-␣ message requires not only the TNF-␣ ARE but also the interaction of the proteins TAP and NxT1, both of which are involved in nucleocytoplasmic transport of mRNA. Through the use of dominant negative ERK1 and ERK2, we establish that control of TNF-␣ mRNA nuclear export operates specifically through ERK1. Finally, we examined the role of two established TNF-␣ ARE-binding proteins, HuR and tristetraprolin, that shuttle between the nucleus and cytoplasm. These data demonstrate that neither tristetraprolin nor HuR is required for TNF-␣ mRNA export. It is unclear at this time if ARE-binding protein(s) directly interact with the TAP-NxT1 complex, if each complex is independently targeted by ERK1, or if only one complex is targeted.

IMPORTANCE Esophageal adenocarcinoma and its precursor lesion Barrett esophagus have seen a dramatic increase in incidence over the past 4 decades yet marked genetic heterogeneity of this disease has precluded advances in understanding its pathogenesis and improving treatment. OBJECTIVE To identify novel disease susceptibility variants in a familial syndrome of esophageal adenocarcinoma and Barrett esophagus, termed familial Barrett esophagus, by using high-throughput sequencing in affected individuals from a large, multigenerational family. DESIGN, SETTING, AND PARTICIPANTS We performed whole exome sequencing (WES) from peripheral lymphocyte DNA on 4 distant relatives from our multiplex, multigenerational familial Barrett esophagus family to identify candidate disease susceptibility variants. Gene variants were filtered, verified, and segregation analysis performed to identify a single candidate variant. Gene expression analysis was done with both quantitative real-time polymerase chain reaction and in situ RNA hybridization. A 3-dimensional organotypic cell culture model of esophageal maturation was utilized to determine the phenotypic effects of our gene variant. We used electron microscopy on esophageal mucosa from an affected family member carrying the gene variant to assess ultrastructural changes. MAIN OUTCOMES AND MEASURES Identification of a novel, germline disease susceptibility variant in a previously uncharacterized gene. RESULTS A multiplex, multigenerational family with 14 members affected (3 members with esophageal adenocarcinoma and 11 with Barrett esophagus) was identified, and whole-exome sequencing identified a germline mutation (S631G) at a highly conserved serine residue in the uncharacterized gene VSIG10L that segregated in affected members. Transfection of S631G variant into a 3-dimensional organotypic culture model of normal esophageal squamous cells dramatically inhibited epithelial maturation compared with the wild-type. VSIG10L exhibited high expression in normal squamous esophagus with marked loss of expression in Barrett-associated lesions. Electron microscopy of squamous esophageal mucosa harboring the S631G variant revealed dilated intercellular spaces and reduced desmosomes. CONCLUSIONS AND RELEVANCE This study presents VSIG10L as a candidate familial Barrett esophagus susceptibility gene, with a putative role in maintaining normal esophageal homeostasis. Further research assessing VSIG10L function may reveal pathways important for esophageal maturation and the pathogenesis of Barrett esophagus and esophageal adenocarcinoma.