Probes for INS

Abstract

Background and Aims

Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal sodium/hydrogen exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in ulcerative colitis (UC)-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors.

Methods

We assessed NHE8 expression in human CRCs by IHC and studied tumor burden in NHE8KO mice using an AOM/DSS colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NSG mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by PCR and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of β-catenin and c-Myc were also analyzed to evaluate Wnt/β-catenin activation.

Results

NHE8 was undetectable in human CRC tissues. Whereas only 9% of NHE8WT mice exhibited tumorigenesis in the AOM/DSS colon cancer model, almost ten times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared to HT29NHE8WT cells. Furthermore, NHE8 deficiency resulted in elevated Lgr5 expression in the colon, in HT29 derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/β-catenin activation.

Conclusions

NHE8 might be an intrinsic factor that regulates Wnt/β-catenin in the intestine.

Although cancer tissue generally shows limited immune responses, some cancers abound with lymphocytes, which generally show favorable prognosis. These cancers, despite their rarity, are important in analyzing immune responses in cancer tissue. Transforming growth factor β1 (TFGβ1) is a multifunctional cytokine, generally having an immunosuppressive function. The present study analyzes the in situ TGFβ1 expression in 23 cases of lymphocyte-rich gastric carcinomas (Ly-rich GCs) using immunohistochemistry and in situ hybridization. Immunohistochemistry revealed that latency-associated peptide (LAP) of TGFβ1 was localized in mainly immune cells in all cases, which was more abundant than in control GCs. Expression of LAP by cancer cells was only focal. In situ hybridization also confirmed abundant TGFβ1 mRNA expression in the lymphoid stroma. Double immunofluorescent microscopy identified LAP+ cells as macrophages, dendritic cells, and part of T cells. Close cell-to-cell contact was observed between LAP+ dendritic-shaped cells and FoxP3+ regulatory T cells (Treg cells). Mature dendritic cells in Ly-rich GCs expressed LAP more frequently than those in the secondary lymphoid organs. Our data revealed abundant expression of TGFβ1 in immune cells with contact to Treg cells in lymphoid stroma, which is consistent with the notion that TGFβ1 is one of the immunosuppressive factors in cancer stroma.

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.

Endothelial cells (ECs) are a key component of the tumor microenvironment. They have abnormal characteristics compared to the ECs in normal tissues. Here, we found a marked increase in lipocalin-type prostaglandin D synthase (L-PGDS) mRNA (Ptgds) expression in ECs isolated from mouse melanoma. Immunostaining of mouse melanoma revealed expression of L-PGDS protein in the ECs. In situhybridization also showed L-PGDS (PTGDS) mRNA expression in the ECs of human melanoma and oral squamous cell carcinoma. In vitro experiments showed that stimulation with tumor cell-derived IL-1 and TNF-α increased L-PGDS mRNA expression and its product prostaglandin D2 (PGD2 ) in human normal ECs. We also investigated the contribution of L-PGDS-PGD2 to tumor growth and vascularization. Systemic or EC-specific deficiency of L-PGDS accelerated the growth of melanoma in mice, whereas treatment with an agonist of the PGD2receptor, DP1 (BW245C, 0.1 mg/kg, injected intraperitoneally twice daily), attenuated it. Morphological and in vivo studies showed that endothelial L-PGDS deficiency resulted in functional changes of tumor ECs such as accelerated vascular hyperpermeability, angiogenesis, and endothelial-to-mesenchymal transition (EndMT) in tumors, which in turn reduced tumor cell apoptosis. These observations suggest that tumor cell-derived inflammatory cytokines increase L-PGDS expression and subsequent PGD2 production in the tumor ECs. This PGD2 acts as a negative regulator of the tumorigenic changes in tumor ECs.

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.