miR-7/TGF-β2 axis sustains acidic tumor microenvironment-induced lung cancer metastasis

Acidosis, regardless of hypoxia involvement, is recognized as a chronic and harsh tumor microenvironment (TME) that educates malignant cells to thrive and metastasize. Although overwhelming evidence supports an acidic environment as a driver or ubiquitous hallmark of cancer progression, the unrevealed core mechanisms underlying the direct effect of acidification on tumorigenesis have hindered the discovery of novel therapeutic targets and clinical therapy. Here, chemical-induced and transgenic mouse models for colon, liver and lung cancer were established, respectively. miR-7 and TGF-β2 expressions were examined in clinical tissues (n = 184). RNA-seq, miRNA-seq, proteomics, biosynthesis analyses and functional studies were performed to validate the mechanisms involved in the acidic TME-induced lung cancer metastasis. Our data show that lung cancer is sensitive to the increased acidification of TME, and acidic TME-induced lung cancer metastasis via inhibition of miR-7-5p. TGF-β2 is a direct target of miR-7-5p. The reduced expression of miR-7-5p subsequently increases the expression of TGF-β2 which enhances the metastatic potential of the lung cancer. Indeed, overexpression of miR-7-5p reduces the acidic pH-enhanced lung cancer metastasis. Furthermore, the human lung tumor samples also show a reduced miR-7-5p expression but an elevated level of activated TGF-β2; the expressions of both miR-7-5p and TGF-β2 are correlated with patients' survival. We are the first to identify the role of the miR-7/TGF-β2 axis in acidic pH-enhanced lung cancer metastasis. Our study not only delineates how acidification directly affects tumorigenesis, but also suggests miR-7 is a novel reliable biomarker for acidic TME and a novel therapeutic target for non-small cell lung cancer (NSCLC) treatment. Our study opens an avenue to explore the pH-sensitive subcellular components as novel therapeutic targets for cancer treatment.

Suppression of AOM/DSS-induced colorectal cancer by scutellarin through downregulation of Wnt signaling pathway activity / 中国药理学与毒理学杂志

OBJECTIVE To investigate the therapeutic effect of scutellarin on colitis-associated cancer (CAC) and its underlying mechanism based on Wnt/β-catenin signaling pathway. METHODS The mouse model of CAC was estab?lished by azomethane oxide (AOM) and sodium dextran sulfate (DSS), followed by scutellarin treatment, with recording the body weight, diarrhea and hematochezia. After sacrificing the mice, the colorectal length and colorectal tumor were assessed. The levels of pro-inflammatory factors TNF-α and IL-6 in mice's sera were measured by the enzyme-linked immunosorbent assay (ELISA). The colorectal lesions were appraised by hematoxylin and eosin (H&E) staining. Theβ-catenin level in CAC tissues was probed by immunofluorescent analysis. The apoptosis-related genes Bax and Bcl-2, and Wnt signaling pathway-related genes β-catenin, GSK-3β, TCF4, c-Myc and cyclin D1 were detected by real-time quantitative RT-PCR (RT-qPCR). Finally, Western blotting analysis (WB) was employed to examine the expressions of the apoptosis and Wnt signaling pathway-related proteins. RESULTS Scutellarin significantly improved AOM/DSS-caused weight loss, colorectal length shortening, and tumor growth in mice (P<0.01). Meanwhile, colorectal lesions could be substantially alleviated by scutellarin. ELISA results showed that the levels of pro-inflammatory factors TNF-αand IL-6 were drastically lessened (P<0.01). Scutellarin also sharply inhibited the nuclear translocation of β-catenin, as evidenced by the reduction in the nuclear level ofβ-catenin protein. In addition, scutellarin attenuated the mRNA expres?sion of Wnt signaling pathway-relatedβ-catenin, TCF4, c-Myc and cyclin D1, whereas it heightened GSK-3βmRNA level. These results were consolidated by WB analysis, which indicated that scutellarin could mitigate the protein levels of phospho-GSK-3β,β-catenin, TCF4, c-Myc and cyclin D1, with the increase in GSK-3β protein in CAC tissue. Moreover, scutellarin could induce the apoptosis of CAC, demonstrated by enhanced expression of Bax and diminished expression of Bcl-2 in both mRNA and protein levels. CONCLUSION Scutellarin may ameliorate colitis-associated colorectal cancer by weakening Wnt/β-catenin signaling cascade.

Protective effect of Pai-Nong-San against AOM/DSS-induced CAC in mice through inhibiting the Wnt signaling pathway / 中国天然药物

Pai-Nong-San (PNS), a prescription of traditional Chinese medicine, has been used for years to treat abscessation-induced diseases including colitis and colorectal cancer. This study was aimed to investigate the preventive effects and possible protective mechanism of PNS on a colitis-associated colorectal cancer (CAC) mouse model induced by azoxymethane (AOM)/dextran sodium sulfate (DSS). The macroscopic and histopathologic examinations of colon injury and DAI score were observed. The inflammatory indicators of intestinal immunity were determined by immunohistochemistry and immunofluorescence. The high throughput 16S rRNA sequence of gut microbiota in the feces of mice was performed. Western blot was used to investigate the protein expression of the Wnt signaling pathway in colon tissues. PNS improved colon injury, as manifested by the alleviation of hematochezia, decreased DAI score, increased colon length, and reversal of pathological changes. PNS treatment protected against AOM/DSS-induced colon inflammation by regulating the expression of CD4

Effect of Estradiol in an Azoxymethane/Dextran Sulfate Sodium-Treated Mouse Model of Colorectal Cancer: Implication for Sex Difference in Colorectal Cancer Development / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. MATERIALS AND METHODS: AOM/DSS-treated male and female mice were sacrificed at weeks 2, 10, and 16, to assess estrogen effects on colitis and carcinogenesis. Macroscopic and histologic severity of colitis and Western blot and quantitative real-time polymerase chain reaction were evaluated, to measure inflammatory mediators and cytokines. RESULTS: Compared with AOM/DSS-treated male mice (M-AOM/DSS group), AOM/DSS-treated male mice with estradiol administration (M-AOM/DSS+estr group) displayed at week 2 significantly decreased severity of colitis. At weeks 10 and 16, AOM/DSS-treated female mice (F-AOM/DSS group) and the M-AOM/DSS+estr group showed significantly lower tumor multiplicity compared with the M-AOM/DSS group. At week 2, F-AOM/DSS group had a lower level of nuclear factor-κB (NF-κB) expression and higher level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression, compared to the M-AOM/DSS group. At week 2, expression levels of NF-κB and its related mediators decreased in the M-AOM/DSS+estr group, while levels of Nrf2 and Nrf2-related anti-oxidant enzymes increased. In addition, estradiol significantly increased Nod-like receptor protein 3 (NLRP3) inflammasome expressions in AOM/DSS-treated male mice. In contrast, at weeks 10 and 16, Nrf2 and its-related anti-oxidant enzymes and NLRP3 inflammasome were highly expressed in M-AOM/DSS group and in F-AOM/DSS group, who developed cancer. CONCLUSION: The data suggest that estradiol inhibits the initiation of CRC by regulating Nrf2-related pathways. Moreover, these imply the dual role of Nrf2 and NLRP3 inflammasome, including promotion of tumor progression upon tumor initiation.

Effects of 17β-Estradiol on Colonic Permeability and Inflammation in an Azoxymethane/Dextran Sulfate Sodium-Induced Colitis Mouse Model

BACKGROUND/AIMS: Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBDs) such as ulcerative colitis. This dysfunction is caused by increased permeability and the loss of tight junctions in intestinal epithelial cells. The aim of this study was to investigate whether estradiol treatment reduces colonic permeability, tight junction disruption, and inflammation in an azoxymethane (AOM)/dextran sodium sulfate (DSS) colon cancer mouse model. METHODS: The effects of 17β-estradiol (E2) were evaluated in ICR male mice 4 weeks after AOM/DSS treatment. Histological damage was scored by hematoxylin and eosin staining and the levels of the colonic mucosal cytokine myeloperoxidase (MPO) were assessed by enzyme-linked immunosorbent assay (ELISA). To evaluate the effects of E2 on intestinal permeability, tight junctions, and inflammation, we performed quantitative real-time polymerase chain reaction and Western blot analysis. Furthermore, the expression levels of mucin 2 (MUC2) and mucin 4 (MUC4) were measured as target genes for intestinal permeability, whereas zonula occludens 1 (ZO-1), occludin (OCLN), and claudin 4 (CLDN4) served as target genes for the tight junctions. RESULTS: The colitis-mediated induced damage score and MPO activity were reduced by E2 treatment (p < 0.05). In addition, the mRNA expression levels of intestinal barrier-related molecules (i.e., MUC2, ZO-1, OCLN, and CLDN4) were decreased by AOM/DSS-treatment; furthermore, this inhibition was rescued by E2 supplementation. The mRNA and protein expression of inflammation-related genes (i.e., KLF4, NF-κB, iNOS, and COX-2) was increased by AOM/DSS-treatment and ameliorated by E2. CONCLUSIONS: E2 acts through the estrogen receptor β signaling pathway to elicit anti-inflammatory effects on intestinal barrier by inducing the expression of MUC2 and tight junction molecules and inhibiting pro-inflammatory cytokines.

Expression of tyrosine and threonine protein kinase in carcinogenic process of colorectal cancer and its relationship with prognosis / 中华肿瘤杂志

Objective@#To investigate the expression of TTK (tyrosine and threonine protein kinase) in the process of colorectal cancer (CRC) development and its relationship to prognosis in CRC patients.@*Methods@#Colitis-associated colon cancer model was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. Mice at four different stages of colon cancer development were obtained, named AD1 (inflammation of the recovery), AD2 (mild dysplasia), AD3 (adenoma) and AD4 (adenocarcinoma), as well as negative control (no treatment). The expression of TTK was measured by real time fluorescent quantitative PCR (qPCR) and immunohistochemical staining in mouse colon tissues and 24 pairs of CRC specimens. The relationship between TTK and prognosis was analyzed in a set of CRC genome-wide gene expression microarray data that was obtained from Gene Expression Omnibus (GEO) of National Center for Biotechnology Information (NCBI).@*Results@#The genome-wide microarray data from mouse AOM-DSS model indicated that the expression of TTK mRNA was gradually elevated during the development of colon cancer. The subsequent qPCR results showed that TTK mRNA levels in negative control, AD1, AD2, AD3 and AD4 groups were 1.05±0.42, 1.10±0.03, 1.38±0.15, 1.33±0.17 and 2.12±0.22, respectively. And TTK expression in AD2, AD3 and AD4 groups were significantly higher than that in negative control (P<0.05). The protein expression of TTK showed by immunohistochemical staining had similar tendency as the results of TTK mRNA. Besides that, the TTK mRNA levels in tumor tissues (0.71±0.10) from 24 CRC patients were significantly higher than those in paired adjacent normal tissues (0.18±0.04; P<0.001). The positive expression rate of TTK protein in 5 pairs of CRC clinical samples was 80.0%, and it was significantly higher than that in adjacent normal tissues (30.8%, P=0.014). Furthermore, according to a public transcriptomic data (GSE17536), the high levels of TTK were associated with poor prognosis in CRC patients.@*Conclusions@#Elevated expression of TTK is related to colonic carcinogenesis in both of mouse model and human CRC samples. TTK is a poor prognostic factor in CRC.