TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn’s disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin–mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf^−/−) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM–derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2^−/− BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca²⁺ homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca²⁺ remodeling includes critical changes in store-operated Ca²⁺ entry (SOCE) and Ca²⁺ store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca²⁺ transport, a comprehensive view of Ca²⁺ remodeling in CRC is lacking. We have used Next Generation Sequencing (NGS) to investigate differences in expression of 77 selected gene transcripts involved in intracellular Ca²⁺ transport in CRC. To this end, mRNA from normal human colonic NCM460 cells and human colon cancer HT29 cells was isolated and used as a template for transcriptomic sequencing and expression analysis using Ion Torrent technology. After data transformation and filtering, exploratory analysis revealed that both cell types were well segregated. In addition, differential gene expression using R and bioconductor packages show significant differences in expression of selected voltage-operated Ca²⁺ channels and store-operated Ca²⁺ entry players, transient receptor potential (TRP) channels, Ca²⁺ release channels, Ca²⁺ pumps, Na⁺/Ca²⁺ exchanger isoforms and genes involved in mitochondrial Ca²⁺ transport. These data provide the first comprehensive transcriptomic analysis of Ca²⁺ remodeling in CRC.

In the western world, colorectal cancer (CRC) is the third most common cause of cancer-related deaths. Survival is closely related to the stage of cancer at diagnosis striking the clinical need for biomarkers capable of early detection. To search for possible biological parameters for early diagnosis of CRC we evaluated protein expression for three CREC (acronym: Cab45, reticulocalbin, ERC-55, calumenin) proteins: reticulocalbin, calumenin, and ERC-55 in a cellular model consisting of a normal derived colon mucosa cell line, NCM460, and a primary adenocarcinoma cell line of the colon, SW480. Furthermore, this cellular model was analyzed by a top-down proteomic approach, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for novel putative diagnostic markers by identification of differentially expressed proteins between the two cell lines. A different colorectal carcinoma cell line, HCT 116, was used in a bottom-up proteomic approach with label-free quantification (LFQ) LC–MS/MS. The two cellular models gave sets of putative diagnostic CRC biomarkers. Various of these novel putative markers were verified with increased expression in CRC patient neoplastic tissue compared to the expression in a non-involved part of the colon, including reticulocalbin, calumenin, S100A6 and protein SET. Characterization of these novel identified biological features for CRC patients may have diagnostic potential and therapeutic relevance in this malignancy characterized by a still unmet clinical need.

We compared morphological, biological and molecular biological patterns of a newly established, spontaneously immortalized pancreatic ductal cell line, TAKA-1, with a hamster pancreatic ductal adenocarcinoma cell line, PC-1. PC-1 cells grew in a monolayer on plastic tissue culture flasks, whereas TAKA-1 cells required type I collagen gel matrix to propagate. The growth rate and argyrophilic nuclear organizer region (Ag-NOR) counts were greater in PC-1 cells than in TAKA-1 cells. More TAKA-1 cells were in G0/G1 and less were in the S cell cycle phase than PC-1 cells. Karyotypically, the consistent change in TAKA-1 cells was an abnormal no. 3 chromosome, whereas additional chromosomal abnormalities were found in PC-1 cells. Ultrastructurally, TAKA-1 cells formed ductal structures and were composed of two types of cells, as in the normal hamster pancreatic ducts, whereas PC-1 cells were pleomorphic, showed evidence for loss of differentiation and contained intracytoplasmic lumens. Unlike the PC-1, TAKA-1 cells did not show a point mutation at codon 12 in the c-Ki-ras oncogene and did not grow in soft agar. Receptor binding assay showed specific epidermal growth factor binding to both cell lines, but secretin binding only to TAKA-1 cells. Both cells produced and released transforming growth factor-α in serum-free medium. Both cell lines expressed blood group A antigen, carbonic anhydrase, coexpressed cytokeratin and vimentin, and reacted with tomato and Phaseolus vulgaris leucoagglutinin (L-PHA) lectins. The results demonstrate that chromosomal abnormalities, cell cycle patterns, expression of cytokeratin 18, lectin bindings and the c-Ki-ras mutation are the features that distinguish the benign from the malignant pancreatic ductal cells in Syrian hamster.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer.Prevention Relevance: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/β-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Background: Wnt signaling in the colon cancer tumor microenvironment (TME) may affect cancer biologic properties including invasion and metastatic dissemination. Prior reports have suggested that the expression of select frizzled (Fz) receptors may be altered in cancers and in the TME. Methods: Colon cancer, colonic adenoma and normal colonic mucosal specimens were obtained under institutional review board approval and analyzed for the expression of Fz1 and Fz2 by confocal fluorescent immunohistochemistry and Wnt-specific membrane array. In vitro, the effect of Wnt3a on Fz1 expression was examined in normal-derived NCM460 cells by qRT-PCR and immunohistochemistry. Results: Fz1 was expressed in colon cancer and villous adenomas but not in more benign tubular adenomas. Fz1 expression was seen in normal colonic mucosa in close proximity to colon cancer, but not villous or tubular adenomas. Normal colonic mucosa distant from colon cancer did not express Fz1. Fz2 was expressed ubiquitously in cancer, adenomas and normal colonic mucosa. Fz1 expression was induced by Wnt3a in a normal colon mucosa-derived cell line in vitro. Conclusions: Fz1 is a Wnt responsive gene in colon-derived tissues. Fz1 expression exhibited increased expression in normal mucosa only in close proximity to colon cancer. This field effect was not seen with pre-malignant adenomas and may be due to Wnt/β-catenin signaling within the TME. Fz1 may represent a new TME-directed therapeutic target for patients with colon cancer.