DUOX2 ACTIVATION ALTERS INTESTINAL PERMEABILITY AND DRIVES DISTINCT METAGENOMIC AND METABOLOMIC SIGNATURES THAT PARALLEL ALTERATIONS IN IBD DYSPLASIA.

We have shown that Activin A (activin), a TGF-β superfamily member, has pro-metastatic effects on colon cancer cells. Recent evidence indicates activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. We recently provided evidence that activin signaling is required for TGF-β stimulation of metastasis indicating significant cross-talk in the tumor microenvironment (TME). Understanding the link between stimulation of metastasis and inflammation via activin and/or TGF-β may provide a novel opportunity for targeted therapy in colorectal cancer (CRC) via anti-activin treatment. Here, we hypothesized that activin exerts cell-specific effects in the TME to both promote anti-tumoral activity of immune cells and pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. Western blot and transwell migration assays with and without activin were performed in human microsatellite instability (MSI) epithelial colon cancer cells with varying expression levels of ACVR2. The influence of the canonical Smad4 pathway was elucidated in a Ts4-Cre;cApc^flox;Smad4^flox mouse model for CRC. We stained and analyzed IHC images from the TMA of 1055 stage II and III CRC patients from the QUASAR2 cohort for CD4, TGF-β, and activin. TMA samples were also analyzed via Digital Spatial Profiling (DSP, NanoString) to determine the immune cell heterogeneity and cell signaling patterns within the TME relative to activin. We found that activin leads to activation of the non-canonical pAkt pathway in ACVR2 restored HCT116+chr2 colon cancer cells, but not in ACVR2 mutated HCT116 cells and leads to tumor cell migration in a PI3K-dependent manner. IHC analysis of the TMA samples indicated that increased levels of CD4, activin, or TGF-β were associated with increased survival time. Interestingly, low levels of both activin and TGF-β expression was associated with the worst survival time suggesting critical cross-talk and highly specific cellular signaling outcomes. In vivo, ablation of canonical SMAD signaling is associated with elevated activin, α-SMA and pAkt and increases dysplasia, mortality and stromal disorganization in intestinal tissue. DSP analysis identified activin co-localization in the stroma was coupled to increases in T-cell exhaustion markers while activin-negative regions of the tissue were associated with increases in markers for antigen presenting cells (APCs). Activin co-localization in the tumoral compartment of the tissue was associated with increases in markers of the MAPK and PI3K/Akt pathways. Taken together, these results suggest that the effects of activin in CRC are highly context-dependent and cellular-specific to promote a tumor-tolerant TME while enhancing tumor cell survival via PI3K/Akt and MAPK.

Colorectal cancer (CRC) development is modulated by the crosstalk between tumor cells and extrinsic factors, such as immune cells. CD4+ T cells, in particular, are important players in CRC, due to their dual role in activating the cytotoxic CD8+ T cells, while participating in tumor killing as well. To protect against extensive DNA damage due to constant exposure to tumor antigens, T cells can activate p21, which induces cell cycle arrest. However, the functional role of p21 in CD4+ T cells during the antitumor response agaings CRC has not yet been evaluated.
p21 expression in CD4+ T cells was evaluated in murine CRC tumor models, as well as in tumor biopsy samples from patients with CRC. To evaluate effector function of p21-expressing CD4+ T cells, in vivo orthotopic MC38 tumor models and in vitro co-culture models were employed.
Evaluation of tumor microenvironment in murine and human CRC samples revealed that p21 is upregulated in both nucleus and cytoplasm of CD4+ T cells. Interestingly, p21 expression in CD4+ T cells was negatively regulated with stage in tumor samples from patients with CRC. In in vivo murine CRC models, p21-deficiency in CD4+ T cells resulted in increased tumor growth compared to wildtype mice. Further evaluation of the tumor microenvironment revealed that p21-/- CD4+ T cells have a reduced cytotoxic potential, as shown by the low relative numbers of Tbet+ T cells and decreased IFNγ production. Co-culture experiments using MC38 tumor cells and in vitro polarized Th1 cells confirmed that the p21-deficient CD4+ T cells have a reduced effector function compared to the B6/J ones, therefore leading to decreased tumor cell death. Furthermore, persistent proliferation of p21-/- Th1 T cells led to accumulation of effector / effector memory T cells and loss of CD27/CD28 co-stimulatory molecules. These changes together with increased expression of DNA repair gene BRCA1 and checkpoint molecule CTLA4 suggest that p21-deficiency in CD4+ T cells promotes their exhaustion.
All in all, our data describe the previously unknown role of p21 in regulating CD4+ T cell exhaustion during the antitumor response against colorectal cancer. A better understanding of these cells could help improve the therapies used for treatment of CRC patients.

Background: The sorting nexin (SNX) family plays a crucial role in protein trafficking and endocytosis. SNX27 is a unique member containing an additional PDZ domain and recycles transmembrane proteins in a PDZ-dependent manner. SNX27 has been explored in neurological disorders, however its impact on intestinal homeostasis, inflammation, and tumorigenesis is unknown. Here, we aim to determine the tissue-specific role of SNX27 in intestinal barriers and inflammation.
Methods: We generated a novel model of intestinal epithelial cells (IECs) SNX27 conditional knockout and analyzed the basal level changes in the SNX27^ΔIEC mice. We then investigated the role of SNX27 in intestinal chronic inflammation and tumorigenesis using a DSS-colitis model and an AOM/DSS colon cancer model. Furthermore, we analyzed the changes of SNX27 in patients with inflammatory bowel disease and colon cancer using human datasets.
Results: The SNX27^ΔIEC mice had shorter colon and higher intestinal permeability without any treatment, compared to the SNX27^loxp mice. We analyzed junction proteins in the colon and observed downregulation of Claudin10, upregulation of Claudin15, and rearranged distribution of ZO-1 and E-cadherin in SNX27^ΔIEC mice. We observed that SNX27^ΔIEC mice were more susceptible to DSS-induced colitis as they lost more body weight, had shorter colon and small intestine, and significantly increased intestinal permeability. Dysregulated ZO-1 and E-cadherin were observed by immunostaining and western blots. Cytokines, such as TNF-alpha, significantly increased in the SNX27^ΔIEC mice compared to SNX27^Loxp mice after challenging DSS. Moreover, SNX27^ΔIEC mice were more susceptible to AOM/DSS with a higher mortality rate. AOM/DSS-SNX27^ΔIEC mice had earlier-onset of tumorigenesis, larger small intestine and spleen, smaller cecum, and higher intestinal permeability. The overall tumor burden was higher in SNX27^ΔIEC mice with larger tumors, compared to the SNX27^loxp mice. For the physiological relevance, we compared human datasets GSE102134, -9452, -10616, and -1710 to analyze SNX27 expression in Crohn’s disease (CD) and ulcerative colitis (UC) patients. We showed a trend of downregulated SNX27 mRNA expression in human CD, and significant reduced SNX27 in UC patients. We identified reduction in SNX27 expression in early and late onset CRC patients (GSE25071).
Conclusion: We showed a disrupted epithelial barrier integrity and increased inflammation upon loss of SNX27. Intestinal SNX27 deletion led to earlier on-set of colonic tumors and increased tumor burden, resulting in poor survival outcomes in mice. There was downregulated SNX27 mRNA in human IBD and colon cancer. Functional studies will be done using human tissue samples and organoids. Overall, our study will provide novel insights into intestinal homeostasis and pathophysiology of colitis and cancer.

NFAT5 is a transcription factor that belongs to the NFAT family, a group of proteins sharing a Rel-like homology region. NFAT5-regulated genes play a central role in inflammatory responses. Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Previously, we have found that NFAT5 is upregulated in the intestinal epithelium of IBD patients. The purpose of this study was to investigate: the role for NFAT5 in the regulation of tight junction proteins (TJs) claudin-1 and ZO-1 expression. Methods. i) Human small intestine and colon crypt cells were isolated from adjacent normal human small bowel and colon obtained from patients following intestinal resection; intestinal organoids were cultured and treated with IFN-gamma or TNFa. ii) To determine the role of NFAT5 in intestinal epithelium in vivo, we generated NFAT5^loxp/loxp; Villin-CreERT2 mice. Mice were injected with tamoxifen to induce NFAT5 knockout (KO). In addition, small intestine and colon were also isolated from NFAT5^loxp/loxp; Villin-CreERT2 mice, and intestinal organoids were cultured and treated with the active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT), to induce NFAT5 KO. iii) The human colon cancer (CRC) cell lines Caco-2 and HCT116 were treated with IFN-gamma or TNFa, two critical pro-inflammatory cytokines. iv) To knock down NFAT5 in HCT116 cells, cells were infected with lentiviral vectors containing shRNA directed to NFAT5 or non-targeting control (NTC) shRNA; stably expressing cells were selected with puromycin. Expression of NFAT5, claudin-1, and ZO-1 was determined by either immunohistochemistry, western blot, or real-time RT-PCR. Results. i) Treatment of human colon and small intestinal organoids with IFN-gamma or TNFa increased the expression of NFAT5, claudin-1, and ZO-1. ii) NFAT5 KO decreased mRNA and protein expression of intestinal NFAT5, claudin-1, and ZO-1. iii) Treatment of Caco-2 and HCT116 cells with IFN-gamma or TNFa increased mRNA and protein expression of NFAT5, claudin-1, and ZO-1; these increases were attenuated by NFAT5 knockdown, suggesting a role for NFAT5 in the regulation of TJ proteins. Conclusions. We demonstrate that NFAT5 modulates the expression of critical tight junction proteins ZO-1 and claudin-1 in intestinal cells. Importantly, our findings suggest that NFAT5 may play an important role in the maintenance of intestinal epithelial barrier function.

Background: Inflammatory bowel diseases (IBD) are characterized by dysbiosis, defects in the epithelial barrier, and increased redox stress and may result in the development of dysplasia or colitis-associated cancer (CAC). The epithelial NADPH oxidase dual oxidase 2 (DUOX2) is consistently altered in IBD patients before the onset of disease and in association with dysbiosis and dysplasia. We have shown that chronic activation of DUOX2 leads to microbiome-dependent colonic tumors, which are abrogated by inactivating DUOX2. However, the mechanisms by DUOX2 activity leads to dysplasia are not well addressed. Alterations in the gut microbiome/metabolome play a role in IBD and CAC. Though microbiome changes between IBD patients and healthy subjects are well described, few studies have interrogated the alterations between IBD patients with and without dysplasia. In the current study, we used patient samples to test the hypothesis that microbiome and metabolome alterations were associated with dysplasia in IBD patients. Mechanistically, we address the effect of constitutive DUOX2 activation on the host and microbiome that may drive IBD and CAC.
Methods: Shotgun sequencing and metabolomic analysis were performed on stool from 6 IBD patients with dysplasia and 12 without dysplasia. villin-TLR4 (vTLR4) mice, which have increased activation of DUOX2, vTLR4-DUOX2-KO mice, and their wild-type littermates were euthanized at baseline in both specific-pathogen free (SPF) and germ free (GF) conditions. Blood and livers were collected for permeability assessment. Colonic tissue and colonic epithelial cells were collected for RNA-sequencing, qPCR and microbiome analysis. Stool was collected for metabolome analysis.
Results: In IBD dysplasia, our data show a decrease in alpha diversity and an increase in deoxycholic acid and hypoxanthine/xanthine, which have been linked to redox stress and CAC. In mice, activation of DUOX2 induced pro-inflammatory pathway activation without overt inflammation, which was confirmed by qPCR. vTLR4 mice had altered permeability and increased bacterial translocation, which was rescued by deletion of DUOX2. GF vTLR4 mice had increased DUOX2 activity and permeability but reduced inflammatory response as compared to their SPF counterparts. Microbiome and metabolome analysis revealed increased proportions of cholic and taurocholic acids and reduced amounts of inosine with DUOX2 activation. These molecules are implicated in the dampening of anti-tumor response and formation of genotoxic substances.
Conclusion: Our results establish an altered microbiome and metabolome profile in IBD dysplasia. We show that dysregulated DUOX2 activity alters barrier function and drives alterations in both the microbiome and metabolome that can be further explored for their diagnostic or therapeutic potential in IBD and IBD-associated dysplasia.