BVES regulates c-Myc stability via PP2A and suppresses colitis-induced tumourigenesis.

OBJECTIVE: Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. DESIGN: We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. RESULTS: BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. CONCLUSION: Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.

Activin A balance regulates epithelial invasiveness and tumorigenesis.

Activin A (Act A) is a member of the TGFß superfamily. Act A and TGFß have multiple common downstream targets and have been described to merge in their intracellular signaling cascades and function. We have previously demonstrated that coordinated loss of E-cadherin and TGFß receptor II (TßRII) results in epithelial cell invasion. When grown in three-dimensional organotypic reconstruct cultures, esophageal keratinocytes expressing dominant-negative mutants of E-cadherin and TßRII showed activated Smad2 in the absence of functional TßRII. However, we could show that increased levels of Act A secretion was able to induce Smad2 phosphorylation. Growth factor secretion can activate autocrine and paracrine signaling, which affects crosstalk between the epithelial compartment and the surrounding microenvironment. We show that treatment with the Act A antagonist Follistatin or with a neutralizing Act A antibody can increase cell invasion in organotypic cultures in a fibroblast- and MMP-dependent manner. Similarly, suppression of Act A with shRNA increases cell invasion and tumorigenesis in vivo. Therefore, we conclude that maintaining a delicate balance of Act A expression is critical for homeostasis in the esophageal microenvironment.

Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction.

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.

Co-overexpression of AXL and c-ABL predicts a poor prognosis in esophageal adenocarcinoma and promotes cancer cell survival.

Background: Esophageal adenocarcinoma (EAC) is highly aggressive and characterized by poor prognosis. AXL expression has been linked to Barrett's tumorigenesis and resistance to chemotherapy, which is associated with c-ABL intracellular localization. However, the molecular and functional relationship between AXL and c-ABL and the clinical significance of the co-expression of these proteins in EAC remain unclear. Methods: We used immunohistochemical analysis (IHC) on tissue microarrays containing human EAC samples (n=53) and normal esophageal tissues (n=11) in combination with corresponding deidentified clinicopathological information to evaluate the expression and the prognostic significance of AXL and c-ABL in EAC. The data were statistically analyzed using Kruskal-Wallis, the chi-square, the Fisher's exact, and Pearson tests. The Kaplan-Meier method and Cox proportional hazards regression model were used to evaluate cancer patient survival. We used a serum deprivation EAC cell model to investigate the pro-survival function of AXL and c-ABL using cell viability, apoptosis, and lactate dehydrogenase activity assays. We performed in vitro assays, including Western blotting, quantitative real-time PCR, and translational chromatin immunoprecipitation (TrIP-Chip) to study the molecular relationship between AXL and c-ABL in EAC cells. Results: IHC analysis revealed that AXL and c-ABL were overexpressed in 55% and 66% of EAC samples, respectively, as compared to normal tissues. Co-overexpression of the two proteins was observed in 49% of EAC samples. The chi-square test indicated a significant association between AXL and c-ABL expression in the EAC samples (χ2 = 6.873, p = 0.032), and the expression of these proteins was significantly associated with EAC patient age (p < 0.001), tumor stage (p < 0.01), and lymph node status (p < 0.001). AXL and c-ABL protein expression data analysis exhibited an identical clinicopathological association profile. Additionally, we found a significant association between expression of AXL (χ2 = 16.7, p = 0.002) or c-ABL (χ2 = 13.4, p = 0.001) and survival of EAC patients. The Cox proportional hazards model and log rank test predicted a significant increase in mortality of patients with high expression of AXL [hazard ratio (HR): 2.86, 95% confidence interval (CI): 1.53 - 5.34, p = 0.003] or c-ABL [HR: 3.29, 95% CI: 1.35 - 8.03, p = 0.001] as compared to those patients with low expression of AXL or c-ABL proteins. Molecular investigations indicated that AXL positively regulates c-ABL protein expression through increased cap-dependent protein translation involving phosphorylation of EIF4E in EAC cells. Next, we investigated the functional relationship between AXL and c-ABL in EAC cells. We demonstrated that the pro-survival activity of AXL requires c-ABL expression in response to serum deprivation. Conclusion: This study highlights the importance of the co-overexpression of AXL and c-ABL proteins as a valuable prognostic biomarker and targeting these proteins could be an effective therapeutic approach in EAC or other solid tumors expressing high levels of AXL and c-ABL proteins.

Serine Threonine Kinase 17A Maintains the Epithelial State in Colorectal Cancer Cells.

Serine threonine kinase 17A (STK17A) is a ubiquitously expressed kinase originally identified as a regulator of apoptosis; however, whether it functionally contributes to colorectal cancer has not been established. Here, we have analyzed STK17A in colorectal cancer and demonstrated decreased expression of STK17A in primary tumors, which is further reduced in metastatic lesions, indicating a potential role in regulating the metastatic cascade. Interestingly, changes in STK17A expression did not modify proliferation, apoptosis, or sensitivity of colorectal cancer cell lines to treatment with the chemotherapeutic 5-fluorouracil. Instead, STK17A knockdown induced a robust mesenchymal phenotype consistent with the epithelial-mesenchymal transition, including spindle-like cell morphology, decreased expression of adherens junction proteins, and increased migration and invasion. Additionally, overexpression of STK17A decreased cell size and induced widespread membrane blebbing, a phenotype often associated with activation of cell contractility. Indeed, STK17A-overexpressing cells displayed heightened phosphorylation of myosin light chain in a manner dependent on STK17A catalytic activity. Finally, patient-derived tumor organoid cultures were used to more accurately determine STK17A's effect in primary human tumor cells. Loss of STK17A induced morphologic changes, decreased E-cadherin, increased invasion, and augmented organoid attachment on 2D substrates, all together suggesting a more metastatic phenotype. Collectively, these data indicate a novel role for STK17A in the regulation of epithelial phenotypes and indicate its functional contribution to colorectal cancer invasion and metastasis. IMPLICATIONS: Loss of serine threonine kinase 17A occurs in colorectal cancer metastasis, induces mesenchymal morphologies, and contributes to tumor cell invasion and migration in colorectal cancer.

Kaiso is required for MTG16-dependent effects on colitis-associated carcinoma.

The myeloid translocation gene family member MTG16 is a transcriptional corepressor that relies on the DNA-binding ability of other proteins to determine specificity. One such protein is the ZBTB family member Kaiso, and the MTG16:Kaiso interaction is necessary for repression of Kaiso target genes, such as matrix metalloproteinase-7. Using the azoxymethane and dextran sodium sulfate (AOM/DSS) murine model of colitis-associated carcinoma, we previously determined that MTG16 loss accelerates tumorigenesis and inflammation. However, it was unknown whether this effect was modified by Kaiso-dependent transcriptional repression. To test for a genetic interaction between MTG16 and Kaiso in inflammatory carcinogenesis, we subjected single and double knockout (DKO) mice to the AOM/DSS protocol. Mtg16-/- mice demonstrated increased colitis and tumor burden; in contrast, disease severity in Kaiso-/- mice was equivalent to wild-type controls. Surprisingly, Kaiso deficiency in the context of MTG16 loss reversed injury and pro-tumorigenic responses in the intestinal epithelium following AOM/DSS treatment, and tumor numbers were returned to near to wild-type levels. Transcriptomic analysis of non-tumor colon tissue demonstrated that changes induced by MTG16 loss were widely mitigated by concurrent Kaiso loss, and DKO mice demonstrated downregulation of metabolism and cytokine-associated gene sets with concurrent activation of DNA damage checkpoint pathways as compared with Mtg16-/-. Further, Kaiso knockdown in intestinal enteroids reduced stem- and WNT-associated phenotypes, thus abrogating the induction of these pathways observed in Mtg16-/- samples. Together, these data suggest that Kaiso modifies MTG16-driven inflammation and tumorigenesis and suggests that Kaiso deregulation contributes to MTG16-dependent colitis and CAC phenotypes.

High dietary salt-induced dendritic cell activation underlies microbial dysbiosis-associated hypertension.

Excess dietary salt contributes to inflammation and hypertension via poorly understood mechanisms. Antigen presenting cells including dendritic cells (DCs) play a key role in regulating intestinal immune homeostasis in part by surveying the gut epithelial surface for pathogens. Previously, we found that highly reactive γ-ketoaldehydes or isolevuglandins (IsoLGs) accumulate in DCs and act as neoantigens, promoting an autoimmune-like state and hypertension. We hypothesized that excess dietary salt alters the gut microbiome leading to hypertension and this is associated with increased immunogenic IsoLG-adduct formation in myeloid antigen presenting cells. To test this hypothesis, we performed fecal microbiome analysis and measured blood pressure of healthy human volunteers with salt intake above or below the American Heart Association recommendations. We also performed 16S rRNA analysis on cecal samples of mice fed normal or high salt diets. In humans and mice, high salt intake was associated with changes in the gut microbiome reflecting an increase in Firmicutes, Proteobacteria and genus Prevotella bacteria. These alterations were associated with higher blood pressure in humans and predisposed mice to vascular inflammation and hypertension in response to a sub-pressor dose of angiotensin II. Mice fed a high salt diet exhibited increased intestinal inflammation including the mesenteric arterial arcade and aorta, with a marked increase in the B7 ligand CD86 and formation of IsoLG-protein adducts in CD11c+ myeloid cells. Adoptive transfer of fecal material from conventionally housed high salt-fed mice to germ-free mice predisposed them to increased intestinal inflammation and hypertension. These findings provide novel insight into the mechanisms underlying inflammation and hypertension associated with excess dietary salt and may lead to interventions targeting the microbiome to prevent and treat this important disease.

Correction: Kaiso is required for MTG16-dependent effects on colitis-associated carcinoma.

In the original version of this article the authors noted that the GEO accession number for the relevant dataset was listed incorrectly as GSE12454.

MTG16 is a tumor suppressor in colitis-associated carcinoma.

MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16-/- mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16-/- mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16-/- mice failed to rescue the Mtg16-/- protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.

The Applicability of a Human Immunohistochemical Panel to Mouse Models of Hepatocellular Neoplasia.

Various immunohistochemical panels are used as aids to distinguish between primary hepatocellular malignancies and metastatic tumors and between benign lesions and carcinomas. We compared the immunohistochemical spectrum of hepatocellular lesions in mice with that of human hepatocellular carcinoma (HCC). Specifically, we compared the staining parameters of 128 murine foci of cellular alteration (FCA) and tumors (adenoma and HCC) from archival tissue blocks of 3 transgenic mouse models (LFABP-cyclin D1, Alb1-TGFß1, and LFABP-cyclin D1 × Alb1-TGFß1) with those of archival human HCC (n = 5). Antibodies were chosen according to their published performance and characterization in human hepatocellular tumor diagnosis and included: arginase 1 (Arg1), ß-catenin, glutamine synthetase (GS), glypican 3, hepatocyte paraffin 1 (HepPar1), and cytokeratin 19 (CK19). GS was the single best immunostain for identifying hepatocellular tumors in mice, with 100% positive staining. Data showed a trend toward loss of normal function (staining) with Arg1, with a higher percentage of positive staining in FCA than in adenomas and HCC. All FCA lacked murine ß-catenin nuclear translocation, which was present in 2 of the 7 adenomas and 22 of the 96 HCC tested. HepPar1 staining was lower than anticipated, except in trabecular HCC (16 of 22 samples were positive). Glyp3 stained very lightly, and only scattered CK19-positive cells were noted (4 of 44 cases of mouse trabecular HCC). Thus, GS appears to be the most useful marker for identifying neoplasia in the transgenic mouse models we tested and should be included in immunohistochemistry assessing hepatocellular neoplasia development.

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes.

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate ß cell/islet dysfunction and ß cell loss.

Diagnostic and therapeutic implications of a novel immunohistochemical panel detecting duodenal mucosal invasion by pancreatic ductal adenocarcinoma.

BACKGROUND: We investigated a series of pancreaticoduodenectomy and duodenal biopsies with a panel of immunohistochemical markers to identify duodenal mucosal invasion by pancreatic ductal adenocarcinoma (PDAC), including markers of poor prognosis and targets of promising novel immunotherapies. MATERIALS AND METHODS: Eighteen consecutive pancreaticoduodenectomy specimens with duodenal mucosal invasion by PDAC were examined for expression of MUC1, MUC4, MUC5AC, MUC6, mesothelin, MUC2, CDX2, and DPC4 on formalin-fixed, paraffin-embedded sections of duodenal-ampullary-pancreatic junctions. Expression of all but MUC6 was also assessed in duodenal biopsies from 12 patients with duodenal mucosal invasion by PDAC. RESULTS: The duodenal mucosa expressed MUC1 (crypts), MUC2 (goblet cells), MUC6 (Brunner glands), CDX2, and DPC4. PDACs in the duodenal mucosa from the resection (n=16-18) and biopsy (n=12) specimens were marked as follows: MUC1 100% (30/30), MUC4 83% (24/29), MUC5AC 83% (25/30), mesothelin 82% (23/28), MUC2 7% (2/30), and CDX2 36% (10/28). Loss of DPC4 expression was seen in 16 of 29 (55%) cases. Reactive mucosa adjacent to PDAC expressed MUC4, MUC5AC and mesothelin in 65% (17/26), 19% (5/27), and 19% (5/26) of cases, respectively. While MUC5AC and mesothelin had high diagnostic accuracy for detection of PDAC, MUC2, CDX2 and DPC4 expression demonstrated negative correlation with PDAC, with absent expression being highly specific for PDAC. CONCLUSION: Immunohistochemical labeling for PDAC biomarkers may aid the diagnosis of PDAC in duodenal biopsy, especially in situations where diagnosis of a pancreatic mass is challenging.

STAT6 activation in ulcerative colitis: a new target for prevention of IL-13-induced colon epithelial cell dysfunction.

BACKGROUND: Interleukin 13 (IL-13) is upregulated in ulcerative colitis (UC) and increases colon epithelial permeability by inducing apoptosis and expression of the pore-forming tight junction protein claudin-2. IL-13 induces activation of signal transducer and activator of transcription 6 (STAT6). However, the STAT6 phosphorylation status in patients with UC is unknown, as is the effect of STAT6 inhibition on colonic epithelium exposed to IL-13. The study aims were to determine if mucosal STAT6 phosphorylation is increased in patients with UC, and if STAT6 inhibition attenuates IL-13-induced colon epithelial cell dysfunction. METHODS: Immunohistochemical staining for phosphorylated (p) STAT6 was performed on colonic tissue from newly diagnosed pediatric subjects with UC (early UC) or Crohn's disease (CD), colectomy tissue from adults with UC (advanced UC), and controls. Colon HT-29 and T84 cells were transfected with STAT6 small interfering RNA (siRNA), or treated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor that inhibits STAT6, prior to IL-13 treatment. RESULTS: The median score for epithelial pSTAT6 was 0 in control subjects, 2 in early UC (versus control P = 0.019), 4 in advanced UC (P = 0.003), and 0 in CD (P = 0.4). Cell transfection with STAT6 siRNA prevented IL-13-induced apoptosis and claudin-2 expression. SAHA inhibited IL-13-induced STAT6 phosphorylation, apoptosis, and claudin-2 expression, and mitigated IL-13-induced reductions in transepithelial resistance. CONCLUSIONS: UC is associated with increased colonic epithelial STAT6 phosphorylation, and STAT6 inhibition prevents IL-13-induced apoptosis and barrier disruption. These data identify STAT6 as a novel target for UC treatment and support further study of SAHA as a therapeutic agent.

Dopamine and cAMP regulated phosphoprotein MW 32 kDa is overexpressed in early stages of gastric tumorigenesis.

BACKGROUND: Gastric adenocarcinoma is a leading cause of cancer mortality. The role of dopamine and cAMP regulated phosphoprotein MW 32 kDa (DARPP-32) overexpression in the gastric tumorigenesis cascade remains unclear. METHODS: The expression of DARPP-32 in the multistep carcinogenesis cascade was examined using immunohistochemistry analysis on 533 samples. The contribution of DARPP-32 in cellular transformation and molecular signaling was investigated using NIH3T3, AGS, and SNU16 cells. RESULTS: The composite expression score (CES), calculated from immunostaining patterns, increased significantly from normal or gastritis to metaplasia, dysplasia, and adenocarcinoma (P < .001). In patients with normal stomach or gastritis and tumor samples, a 76% and 77% chance, respectively, was found (P < .001) that CES was higher in the tumor. High median CES correlated with well- or moderately differentiated (P = .03) gastric adenocarcinomas. NIH3T3 cells transfected with DARPP-32 demonstrated increased levels of phospho-AKT and a 5-fold increase in the number of foci as compared with the control (P = .02). DARPP-32 expression in AGS cells led to increased protein levels of phospho-AKT and BCL-2. For validation, the knockdown of endogenous DARPP-32 expression in SNU16 cells using shRNA resulted in decreased levels of phospho-AKT phosphorylation and BCL-2. CONCLUSION: Our results suggest that DARPP-32 overexpression may participate in the transition to intestinal metaplasia and in the progression to neoplasia. The ability of DARPP-32 to transform NIH3T3 cells and to regulate AKT and BCL-2 underscores its possible oncogenic potential.