| Colorectal cancer (CRC) is the third most common cancer diagnosed among men and women. Many factors, such as diet, life style and genetic predisposition, have been identified to contribute to the development of colorectal cancer. Inflammatory responses play important roles at different stages of tumor development, including initiation, progression and conversion to malignant state. Both inflammatory bowel disease (IBD) and ulcerative colitis(UC), which display chronic inflammation of the gastrointestinal mucosa, are associated with increased risk for the development of colon cancer.Interferon-γ(IFN-γ), a proinflammatory cytokine which is mainly produced by T cells and natural killer (NK) cell, plays many critical roles in promoting host resistance to microbial infection, innate immunity or adaptive immune system, inflammatory responses and hematopoiesis. It has been shown that IFN-γ also participates in regulation of cell proliferation, apoptosis and senescence. Moreover, a role for IFN-y in protecting against tumor development has also been identified recently. IFN-γ signaling pathway has been found to associate with colon and rectal cancer risk and subsequent survival. IFN-γ can exert direct anti-proliferative effects on colon cancer cells. IFN-y can also induce apoptosis of human colon cancer cells through inhibiting Bcl-x. However, opposing role of IFN-γ signaling during tumorigenesis has also been documented, as illustrated by the observation that transgenic expression of IFN-y in mouse stomach leads to increased inflammation and tumor formation. Development of colorectal carcinomas in Socs1-deficient mice is dependent on IFN-y signaling. Therefore, the exact role of IFN-y signaling during tumorigenesis remains to be clarified. In order to explore the role of IFN-y signaling in intestinal tumor progression, we generated the Ifngr1-/-ApcMin/+ mice and studied the effect of Ifngri deficiency on the intestinal tumorigenesis.Part one. IFN-γ receptor deficiency enhances intestinal tumorigenesis in ApcMin/+mice.The ApcMin/+ mouse is an animal model of human familial adenomatous polyposis. ApcMin/+ mice develop numerous intestinal adenomas, which arise as a result of loss or mutation of the wildtype Ape allele, and consequently the upreguation of (3-catenin. ApcMin/+ mice have been frequently used to study the development of CRC. Many factors have been identified that either promote or suppress intestinal tumorigenesis. Tumor development in ApcMin/+ mice were independent of inflammation, but Inflammatory responses contribute to carcinogenesis. There have been numerous reports that described the role of inflammatory factors, such as Cox2, MyD88, Gstp, IL6, Stat3 and Gas6, in tumorigenesis in ApcMin mice.To determine the role of IFN-y signaling in the development of intestinal polyposis in ApcMin/+mice, we introduced Ifngr1 deficiency into ApcMin/+mice. The Ifngr1-/- ApcMin/+mice showed significantly shorter survival compared with the ApcMin/+mice. We also determined the hemoglobin levels in these mice, and found that anemia was more severe in lfngr1-/-ApcMin/+mice. Since most of the Ifngr1-/- pcMin/+mice die by 20 weeks, we sacrificed the animals and scored the polyps when they were around 18 weeks. The numbers of polyps in the small intestine and colon of the Ifngr1-/- ApcMin/+ mice were significantly greater than those in the ApcMin/+mice (p<0.01). In addition, the polyps in Ifngr1-/- ApcMin/+mice were larger in size than those in age-matched ApcMin/+ mice (p<0.01).We next characterized proliferation and apoptosis of the intestinal tumors. Ki67 immunostaining indicated that there were more proliferating cells in tumors of lfngr1-/-ApcMin/+mice than in those of Ifngr1+/+ApcMin/+mice. Expression level of Pcna, another marker of proliferation, showed the same trend. In contrast, the level of apoptosis, as assessed by TUNEL-staining, was not significantly different between Ifngr1-/-ApcMin/+ and Ifngr1-/-ApcMin/+ mice.In a C57BL/6J background, most tumors in the ApcMin/+mice are benign adenomas and do not demonstrate aggressive invasion or metastasis. We performed histological examination of the polyps in lfngr1’AApcMin/+mice and observed that> 30%of them were of invasive nature. These results indicate that the IFN-y receptor deficiency renders increased invasiveness to the intestinal tumors.In order to gain insight into the molecular basis underlying the enhanced tumorigenesis in Ifngr1-/-ApcMin/+mice, we interrogated the global gene expression in tumors from Ifngr1-/-ApcMin/+and Ifngr1-/-ApcMin/+mice by RNA sequencing.4021 genes were differentially expressed(adjusted p value <0.05) between the two types of tumors. KEGG analysis revealed a significant enrichment of multiple pathways that are known to enhance tumor progression. Inflammatory response, cell proliferation and growth pathway appeared to be upregulated in tumors of Ifngr1-/-ApcMin/+mice when compared to Ifngr1-/-ApcMin/+mice. Citric acid cycle and oxidative phosphorylation, processes that generate ATP, were downregulated in tumors of Ifngr1-/-ApcMin/+mice. Thus, the gene expression patterns in tumors of Ifngr1-/-ApcMin/+mice generally indicate an increased degree of malignancy when compared to those in Ifngr1-/-ApcMin/+mice. We next performed qRT-PCR to validate the gene expression results obtained with RNA-seq. Representative genes, such as Mmp3, Mmp7, Mmp9 and Hifia, were all confirmed to be more pronouncedly upregulated in tumors of Ifngr1-/-ApcMin/+mice than in those of Ifngr1-/-ApcMin/+mice. The expression levels of Cdhr2 and Cdx2, which act as tumor suppressors, were more pronouncedly downregulated in tumors from Ifngr1-/-ApcMin/+mice. We next performed immunohistochemical analysis of CD31, a marker of endothelial cells and angiogenesis, in intestinal tumors. CD31-positive cells were indeed more abundant in tumors of Ifngr1-/-ApcMin/+mice than in those of Ifngr1-/-ApcMin/+mice, suggesting that the tumors of Ifngr1-/-ApcMin/+mice were more vascularized.We also surveyed the genes that are differentially expressed between tumors and mucosal tissues in Ifngr1-/-ApcMin/+mice and Ifngr1-/-ApcMin/+mice by RNA-seq. The upregulated genes were compared to those previously reported to be upregulated in human colorectal cancer specimens when compared to mucosa(GSE20842). Interestingly, the upregulated genes in tumors of Ifngr1-/-ApcMin/+mice were found to be more likely to overlap with those upregulated in human colorectal cancers(Figure3F, p-value= 0.0018).This result further supports that tumors of Ifngr1-/-ApcMin/+mice more resemble human colorectal cancers than those in Ifngr1-/-ApcMin/+mice.Part two. Increased inflammation in the intestine of Ifngr1-/-ApcMin/+ mice.There have been numerous reports that described the role of inflammatory factors in tumorigenesis in ApcMin/+mice. Tumor development in ApcMin/+ mice was independent of inflammation, but inflammatory responses accelerates tumorigenesis. Because IFN-y is a pro-inflammatory cytokine, lack of Ifngri would be expected to bring about changes in the repertoire of cytokines. We determined the expression levels of genes encoding various cytokines in the intestine of Ifngr1-/-ApcMin/+mice. Cxcl2, Cxcl5, Reg3b, IL-1fi, Saa3, Tnfa, and Cox-2(Ptgs2) were significantly upregulated in tumors from Ifngr1-/-ApcMin/+mice when compared to those from Ifngr1-/-ApcMin/+mice. The expression level of Cd74, which inhibits cytokine signaling, was significantly reduced in tumors from Ifngr1-/-ApcMin/+mice.Macrophages are a major component of the leukocyte infiltrates in various tumor stroma. Immunohistochemistry showed that polyp stromas of Ifngr1-/-ApcMin/+mice were infiltrated with more F4/80-positive macrophages. IFN-y was reported to promote the phenotypic conversion of tumor-associated macrophages (TAM) from M2 to M1 in ApcMin/+mice polyps. We next determined the expression levels of representative M1 and M2-associated genes by qRT-PCR. Expression levels of Cxcl9, which are key effector molecules produced by M1 macrophages, were significantly lower in tumors of Ifngr1-/-ApcMin/+mice. In contrast, expression levels of Arginasei, Trem2, and Ym1, which are typical M2 markers, were higher in tumors of Ifngr1-/-ApcMin/+mice. Thus, the impaired IFN-y signaling due to lack of Ifngr1 generated a pro-tumor cytokine milieu. We next examined CD4+cells in the tumors of ApcMin/+mice by immunofluorescence staining, but did not detect a significant difference in abundance between lfngr-/- ApcMin/+mice and Ifngr1+/+ApcMin/+mice. The function of Th1 cells is presumably impaired due to disruption of IFN-y signaling in lfngr-/- mice. The expression level of IL-4, which induces Th2 differentiation and also functions as an effector of Th2 cells, was not significantly increased Ifngr/-ApcMin/+mice. However, the expression levels of IL-23a and IL-17a, which induces Th17 differentiation and functions as Th17 effector, respectively, were significantly increased in tumors of lfngrAApcMin/+ mice. The upregulation of IL-23a/IL-17a axis further substantiates the notion of increased inflammation in in tumors of Ifngr-/- ApcMin/+mice. Mutation or loss of the Ape gene is thought to represent the initiating event in the process of intestinal tumorigenesis. It is the functional loss of Ape that leads to the nuclear accumulation of β-catenin. In C57BL/6 ApcMm/+mice, all the tumors form as a result of loss of the wide type allele of Ape gene, mainly via mitotic recombination. To determine whether wildtype Ape allele is also lost in tumors from Ifngr1-/-ApcMin/+mice, we performed Ape loss of heterozygosity (LOH) analysis. We observed that only the ApcMin/+allele, but not the wildtype allele, could be detected in tumors arising from Ifngr1-/-ApcMtn/+mice, as in those from ApcMm/+mice, suggesting that lack of Ifngri led to increased tumor multiplicity similarly via loss of the wildtype allele.Part three. IFN-y inhibited β-catenin/TCF function by upregulating Dkk1. Aberrant activation of the β-catenin in ApcM/7/+mice drives the initiation and progression of colorectal neoplasia. We next evaluated the cellular localization of β-catenin in tumors using immunohistochemistry. The mean nuclear β-catenin signal intensity was stronger in Ifngr1-/-ApcMm/+mice than in ApcMin/+mice. Measurement of expression levels of target genes of β-catenin/TCF confirmed the upregulation of P-catenin function. The levels of cyclin D and c-myc were significantly increased in Ifngr1-/-ApcMm/Jr mice. We determined the level of Dkk1 by qRT-PCR and Western blot analysis and found that the level of Dkk1 was substantially reduced in Ifngr1-/- ApcMin/+mice.We also studied the effect of IFN-y on Wnt signaling in colorectal cancer cells HCT15. We found that the mRNA level of DKK1 was dramatically increased by IFN-y treatment. A β-catenin/TCF reporter assay (TOPflash) revealed that TCF activity in HCT15 cells could be substantially inhibited by IFN-y. Importantly, addition of anti-DKK1 antibodies significantly attenuated the inhibitory effect of IFN-y on TOPflash activity, suggesting that inhibitory effect of IFN-y signaling on β-catenin/TCF function is at least partially mediated by an upregulation of DKK1.We next tested whether the induction of DKK1 by IFN-y is via STAT1, a classical IFN-y signaling molecule. However, knockdown of Stat1 by siRNA or STAT1 inhibitor did not affect the stimulatory effect of IFN-y on DKK1 expression in HCT15 cells, suggesting that the upregulation of DKK1 is not mediated by STAT1.Part four. IFNGR1 was down regulated in human colorectal cancers Since tumorigenesis in Apcmin/+ mice is enhanced in the absence of IFN-γ receptor, we speculated that IFN-y receptor may function to impede intestinal tumorigenesis and that the intestinal tumors may exhibit a reduced expression of IFN-y receptor. We first determined the transcript levels of Ifngr1 in adenomas and adjacent normal tissues in ApcMin/+ mice by qRT-PCR. Indeed, Ifngr1 expression was significantly downregulated in tumors when compared to that in mucosa tissues. We next compared the expression levels of IFNGR1 in 65 pairs of human colorectal cancers and normal mucosa tissues (GSE20842). Consistently, IFNGR1 was significantly downregulated in cancer specimens when compared to mucosa tissues. It is noteworthy that the single nucleotide polymorphismrs3799488 in IFNGR1 was reported to be associated with increased risk of rectal cancers Since IFNGR1 expression was downregulated in human colorectal cancers, we next interrogated whether the expression levels of IFNGR1 were correlated with prognosis of cancer patients using the public available databases GSE14333 and GSE17536. Although no statistically significant correlations between IFNGR1 expression levels and survival were detected, the results suggest a trend that the group with the intermediate levels of IFNGR1 had the longest median survival time.In summary, we showed that lack of IFN-y receptor could promote cancer development in ApcMin/+ Mice. Lack of IFN-y receptor resulted in a tumor permissive cytokine milieu and an upregulation of Wnt signaling. Our results provided further insight into pathogenesis of colorectal cancer and should bear implications in the clinical application of IFN-y in the management of colon cancer. |
