Nfe2l2/NRF2 deletion attenuates tumorigenesis and increases bacterial diversity in a mouse model of Lynch syndrome.

Lynch syndrome (LS) is the most prevalent heritable form of colorectal cancer (CRC). Its early onset and high lifetime risk for CRC emphasize the necessity for effective chemoprevention. NFE2L2 (NRF2) is often considered a potential druggable target, and many chemopreventive compounds do induce NRF2. However, while NRF2 counteracts oxidative stress, it is also overexpressed in CRC and may promote tumorigenesis. Herein, we evaluated the role of NRF2 in prevention of LS-associated neoplasia. We found an increased levels of NRF2 in intestinal epithelia of mice with intestinal epithelial-specific Msh2 deletion (MSH2ΔIEC) as compared to C57BL/6 (wild type) mice, as well as an increase in downstream NRF2 targets Nqo1 and Gclc. Likewise, NRF2 levels were increased in human MSH2-deficient LS tumors compared to healthy controls. In silico analysis of a publicly accessible RNA-sequencing LS dataset also found an increase in downstream NRF2 targets. Upon crossing MSH2ΔIEC with Nrf2null mice (MSH2ΔIECNrf2null), we unexpectedly found reduced tumorigenesis in MSH2ΔIECNrf2null compared to MSH2ΔIEC after 40 weeks. This occurred despite an increase in oxidative damage in MSH2ΔIECNrf2null mice. Loss of NRF2 impaired proliferation as seen by Ki67 intestinal staining and in organoid cultures. This was accompanied by diminished WNT/ß-catenin signaling. Apoptosis was unaffected. Microbial alpha-diversity increased over time with loss of NRF2 based upon 16S rRNA gene amplicon sequencing of murine fecal samples. Altogether, we show that NRF2 protein levels are increased in MSH2-deficiency and associated neoplasia, but loss of NRF2 attenuates tumorigenesis. Activation of NRF2 may not be a feasible strategy for chemoprevention in LS.

Intravenous iron is non-inferior to oral iron regarding cell growth and iron metabolism in colorectal cancer associated with iron-deficiency anaemia.

Oral iron promotes intestinal tumourigenesis in animal models. In humans, expression of iron transport proteins are altered in colorectal cancer. This study examined whether the route of iron therapy alters iron transport and tumour growth. Colorectal adenocarcinoma patients with pre-operative iron deficiency anaemia received oral ferrous sulphate (n = 15), or intravenous ferric carboxymaltose (n = 15). Paired (normal and tumour tissues) samples were compared for expression of iron loading, iron transporters, proliferation, apoptosis and Wnt signalling using immunohistochemistry and RT-PCR. Iron loading was increased in tumour and distributed to the stroma in intravenous treatment and to the epithelium in oral treatment. Protein and mRNA expression of proliferation and iron transporters were increased in tumours compared to normal tissues but there were no significant differences between the treatment groups. However, intravenous iron treatment reduced ferritin mRNA levels in tumours and replenished body iron stores. Iron distribution to non-epithelial cells in intravenous iron suggests that iron is less bioavailable to tumour cells. Therefore, intravenous iron may be a better option in the treatment of colorectal cancer patients with iron deficiency anaemia due to its efficiency in replenishing iron levels while its effect on proliferation and iron metabolism is similar to that of oral iron treatment.

IL10R2 Overexpression Promotes IL22/STAT3 Signaling in Colorectal Carcinogenesis.

The mucosal immune response in the setting of intestinal inflammation contributes to colorectal cancer. IL10 signaling has a central role in gut homeostasis and is impaired in inflammatory bowel disease (IBD). Out of two IL10 receptor subunits, IL10R1 and IL10R2, the latter is shared among the IL10 family of cytokines and activates STAT signaling. STAT3 is oncogenic in colorectal cancer; however, knowledge about IL10 signaling upstream of STAT3 in colorectal cancer is lacking. Here, expression of IL10 signaling genes was examined in matched pairs from normal and tumor tissue from colorectal cancer patients showing overexpression (mRNA, protein) of IL10R2 and STAT3 but not IL10R1. IL10R2 overexpression was related to microsatellite stability. Transient overexpression of IL10R2 in HT29 cells increased proliferation upon ligand activation (IL10 and IL22). IL22, and not IL10, phosphorylated STAT3 along with increased phosphorylation of AKT and ERK. A significantly higher expression of IL22R1 and IL10R2 was also confirmed in a separate cohort of colorectal cancer samples. IL22 expression was elevated in gut mucosa from patients with IBD and colitis-associated cancer, which also exhibited increased expression of IL22R1 but not its coreceptor IL10R2. Overall, these data indicate that overexpression of IL10R2 and STAT3 contributes to colorectal carcinogenesis in microsatellite-stable tumors through IL22/STAT3 signaling.

Overexpression of PAK1 promotes cell survival in inflammatory bowel diseases and colitis-associated cancer.

BACKGROUND: Chronic gut inflammation predisposes to the development of colorectal cancer and increased mortality. Use of mesalamine (5-ASA) in the treatment of ulcerative colitis modulates the risk of neoplastic progression. p21 activated kinase 1 (PAK1) mediates 5-ASA activity by orchestrating MAPK signaling, Wnt-ß catenin pathway, and cell adhesion; all implicated in the colon carcinogenesis. We evaluated the role of PAK1 in IBD and in colitis-associated cancer (CAC). METHODS AND RESULTS: PAK1 expression was scored by immunohistochemistry in human samples from IBD, CAC, and in normal mucosa. Compared with controls, a higher PAK1 expression was detected in IBD which further increased in CAC. The consequence of PAK1 overexpression was investigated using normal diploid colon epithelial cells (HCEC-1CT), which showed higher proliferation and decreased apoptosis on overexpression of PAK1. Analysis of IBD and CAC samples showed activation of AKT (p-AKT). However, mTOR pathway was activated in IBD but not in CAC. Treatment of cells with specific inhibitors (PD98059/LY294002/rapamycin) of growth signaling pathways (MEK/PI3K/mTOR) demonstrated that in HCEC-1CT, PAK1 expression is regulated by MEK, PI3K, and mTOR. In colorectal cancer cell lines, PAK1, and beta-catenin expression correlated and inhibition of PAK1 and addition of 5-ASA elicited similar molecular affects by reducing ERK and AKT activation. Moreover, 5-ASA disrupted PAK1 interaction and colocalization with ß-catenin. CONCLUSIONS: Our data indicate that (1) PAK1 is upregulated in IBD and CAC (2) PAK1 overexpression is associated with activation of PI3K-AKT/mTOR prosurvival pathways in IBD.

Thymoquinone attenuates tumor growth in ApcMin mice by interference with Wnt-signaling.

BACKGROUND: Patients with familial adenomatous polyposis (FAP) are at increased risk for the development of colorectal cancer. Surgery and chemoprevention are the most effective means to prevent cancer development. Thymoquinone (TQ) is considered the main compound of the volatile Nigella sativa seed oil and has been reported to possess anticarcinogenic properties. In this study we evaluated the chemopreventive properties of TQ in a mouse model of FAP. METHODS: APCMin mice were fed with chow containing 37.5 mg/kg or 375 mg/kg TQ for 12 weeks. H&E stained intestine tissue sections were assessed for tumor number, localization, size, and grade. Immunohistochemistry for ß-catenin, c-myc, Ki-67 and TUNEL-staining was performed to investigate TQ's effect on major colorectal cancer pathways. TQ's impact on GSK-3ß and ß-catenin were studied in RKO cells. RESULTS: 375 mg/kg but not 37.5 mg/kg TQ decreased the number of large polyps in the small intestine of APCMin mice. TQ induced apoptosis in the neoplastic tissue but not in the normal mucosa. Furthermore, upon TQ treatment, ß-catenin was retained at the membrane and c-myc decreased in the nucleus, which was associated with a reduced cell proliferation in the villi. In vitro, TQ activated GSK-3ß, which induced membranous localization of ß-catenin and reduced nuclear c-myc expression. CONCLUSIONS: In summary, TQ interferes with polyp progression in ApcMin mice through induction of tumor-cell specific apoptosis and by modulating Wnt signaling through activation of GSK-3ß. Nigella sativa oil (or TQ) might be useful as nutritional supplement to complement surgery and chemoprevention in FAP.