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Supplementary Figures and Legends

Primers used for validation the gene expression level

The interplay between gut microbiota and the host immune system is emerging as a factor in the pathogenesis of colorectal cancer. Here, we set out to identify the effect of Akkermansia muciniphila (A. muciniphila) on colorectal cancer pathogenesis. A. muciniphila abundance was significantly reduced in patients with colorectal cancer from two independent clinical cohorts and the GMrepo dataset. Supplementation with A. muciniphila suppressed colonic tumorigenesis in ApcMin/+ mice and the growth of implanted HCT116 or CT26 tumors in nude mice. Mechanistically, A. muciniphila facilitated enrichment of M1-like macrophages in an NLRP3-dependent manner in vivo and in vitro. As a consequence, NLRP3 deficiency in macrophages attenuated the tumor-suppressive effect of A. muciniphila. In addition, we revealed that TLR2 was essential for the activation of the NF-κB/NLRP3 pathway and A. muciniphila induced M1-like macrophage response. We observed positive correlations between M1-like macrophages, NLRP3/TLR2 and A. muciniphila in patients with colorectal cancer, which corroborated these findings. In summary, A. muciniphila–induced M1-like macrophages provide a therapeutic target in the colorectal cancer tumor microenvironment.

Figure S1-S8

Gene amplification is a hallmark of cancer and is frequently observed in colorectal cancer. Previous whole-genome sequencing of colorectal cancer clinical specimens identified amplification of Ring finger protein 6 (RNF6), a RING-domain E3 ubiquitin ligase. In this study, we showed that RNF6 is upregulated in 73.5% (147/200) of patients with colorectal cancer and was positively associated with RNF6 gene amplification. Furthermore, RNF6 expression and its gene amplification were independent prognostic factors for poor outcome of patients with colorectal cancer. RNF6 promoted cell growth, cell-cycle progression, and epithelial-to-mesenchymal transition in colorectal cancer cells; RNF6 also promoted colorectal tumor growth and lung metastasis in mouse models. Mechanistic investigations revealed that RNF6 bound and ubiquitylated transducin-like enhancer of split 3 (TLE3), a transcriptional repressor of the β-catenin/TCF4 complex. RNF6-mediated degradation of TLE3 significantly suppressed the association of TLE3 with TCF4/LEF, which in turn led to recruitment of β-catenin to TCF4/LEF, triggering Wnt/β-catenin activation. Restoration of TLE3 expression abolished the oncogenic effects of RNF6. Taken together, these results demonstrate that RNF6 plays a pivotal oncogenic role in colorectal tumorigenesis.Significance: RNF6-mediated ubiquitination and degradation of TLE3 activates the Wnt/β-catenin pathway in colorectal carcinogenesis. Cancer Res; 78(8); 1958–71. ©2018 AACR.

Supplementary Video S4

RNF6 up-regulates in CRC and indicates a poor outcome of CRC patients.

Supplementary Materials and Methods, Figures, Tables

Purpose:Chromosomal instability (CIN) is a common phenomenon in colorectal cancer, but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) is a critical regulator of CIN in colorectal cancer. We thus studied the effect of MAP9 loss on colorectal cancer in Map9-knockout mice and in cell lines.Experimental Design:We generated colon epithelial–specific Map9-knockout mice and evaluated colorectal cancer development. Effect of Map9 knockout on colorectal cancer progression was determined in chemical or ApcMin/+-induced colorectal cancer. Molecular mechanism of MAP9 was determined using spectral karyotyping, microtubule assays, and whole-genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 patients with CRC.Results:Spontaneous colonic tumors (9.1%) were developed in colon epithelium–specific Map9-knockout mice at 17 months, but none was observed in wild-type littermates. Map9 deletion accelerated colorectal cancer formation both in ApcMin/+ mice and azoxymethane-treated mice, and reduced survival in ApcMin/+ mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fiber from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors, and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from Map9 knockout ApcMin/+ mice. In patients with colorectal cancer, MAP9 was frequently silenced and its downregulation was associated with poor survival.Conclusions:MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in colorectal cancer.

Supplementary Table S6

There remains a paucity of functional biomarkers in gastric cancer. Here, we report the identification of the sodium channel subunit SCNN1B as a candidate biomarker in gastric cancer. SCNN1B mRNA expression was silenced commonly by promoter hypermethylation in gastric cancer cell lines and primary tumor tissues. Tissue microarray analysis revealed that high expression of SCNN1B was an independent prognostic factor for longer survival in gastric cancer patients, especially those with late-stage disease. Functional studies demonstrated that SCNN1B overexpression was sufficient to suppress multiple features of cancer cell pathophysiology in vitro and in vivo. Mechanistic investigations revealed that SCNN1B interacted with the endoplasmic reticulum chaperone, GRP78, and induced its degradation via polyubiquitination, triggering the unfolded protein response (UPR) via activation of PERK, ATF4, XBP1s, and C/EBP homologous protein and leading in turn to caspase-dependent apoptosis. Accordingly, SCNN1B sensitized gastric cancer cells to the UPR-inducing drug tunicamycin. GRP78 overexpression abolished the inhibitory effect of SCNN1B on cell growth and migration, whereas GRP78 silencing aggravated growth inhibition by SCNN1B. In summary, our results identify SCNN1B as a tumor-suppressive function that triggers UPR in gastric cancer cells, with implications for its potential clinical applications as a survival biomarker in gastric cancer patients. Cancer Res; 77(8); 1968–82. ©2017 AACR.

Clinicopathological features of CRC patients

Cox regression analysis of potential prognostic predictors for patients in the colorectal cancer cohort (HongKong cohort 1 and BeiJing cohort 1, n=160)

Knockdown of RNF6 inhibits tumor growth.

Table S1-S6

RNF6 depletion arrests cell cycle progression, increases cell apoptosis and inhibits tumor migration.

Supplementary Video S3

Supplementary Video S5

Supplementary Video S6

Supplementary Table S5

The interplay between gut microbiota and the host immune system is emerging as a factor in the pathogenesis of colorectal cancer. Here, we set out to identify the effect of Akkermansia muciniphila (A. muciniphila) on colorectal cancer pathogenesis. A. muciniphila abundance was significantly reduced in patients with colorectal cancer from two independent clinical cohorts and the GMrepo dataset. Supplementation with A. muciniphila suppressed colonic tumorigenesis in ApcMin/+ mice and the growth of implanted HCT116 or CT26 tumors in nude mice. Mechanistically, A. muciniphila facilitated enrichment of M1-like macrophages in an NLRP3-dependent manner in vivo and in vitro. As a consequence, NLRP3 deficiency in macrophages attenuated the tumor-suppressive effect of A. muciniphila. In addition, we revealed that TLR2 was essential for the activation of the NF-κB/NLRP3 pathway and A. muciniphila induced M1-like macrophage response. We observed positive correlations between M1-like macrophages, NLRP3/TLR2 and A. muciniphila in patients with colorectal cancer, which corroborated these findings. In summary, A. muciniphila–induced M1-like macrophages provide a therapeutic target in the colorectal cancer tumor microenvironment.

Purpose: Chromosomal instability (CIN) is a common phenomenon in colorectal cancer, but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) is a critical regulator of CIN in colorectal cancer. We thus studied the effect of MAP9 loss on colorectal cancer in Map9-knockout mice and in cell lines. Experimental Design: We generated colon epithelial–specific Map9-knockout mice and evaluated colorectal cancer development. Effect of Map9 knockout on colorectal cancer progression was determined in chemical or ApcMin/+-induced colorectal cancer. Molecular mechanism of MAP9 was determined using spectral karyotyping, microtubule assays, and whole-genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 patients with CRC. Results: Spontaneous colonic tumors (9.1%) were developed in colon epithelium–specific Map9-knockout mice at 17 months, but none was observed in wild-type littermates. Map9 deletion accelerated colorectal cancer formation both in ApcMin/+ mice and azoxymethane-treated mice, and reduced survival in ApcMin/+ mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fiber from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors, and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from Map9 knockout ApcMin/+ mice. In patients with colorectal cancer, MAP9 was frequently silenced and its downregulation was associated with poor survival. Conclusions: MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in colorectal cancer.

Tripartite motif (TRIM) family proteins participate in a variety of important cellular processes, including apoptosis, cell-cycle arrest, DNA repair, and senescence. In this study, we demonstrated that a novel TRIM family member, TRIM67, was commonly silenced in colorectal cancer and its downregulation was associated with poor survival. Trim67 knockout in ApcMin/+ mice increased the incidence, multiplicity, and burden of colorectal tumors. Similarly, colon-specific knockout of Trim67 significantly accelerated azoxymethane-induced colorectal cancer in mice. RNA sequencing revealed that the antitumor effect of TRIM67 was mediated by activation of the p53 signaling pathway. TRIM67 interacted directly with the C-terminus of p53, inhibiting p53 degradation by its ubiquitin ligase MDM2. TRIM67 was also a transcriptional target of p53; upon cellular stress, p53 bound to the TRIM67 promoter and induced significant upregulation of TRIM67, thereby forming a TRIM67/p53 self-amplifying loop that boosts p53-induced cell growth inhibition and apoptosis. Consequently, loss of this p53-positive regulatory program profoundly compromised p53-mediated responses to chemotherapy-induced DNA damage. Dampened p53 response was also observed in tumors of Trim67 knockout mice and Trim67 knockout embryonic fibroblasts. TRIM67 reactivation restored p53 activation and sensitized colorectal cancer cells to chemotherapy in vitro and in vivo. TRIM67 thus functions as a pivotal tumor suppressor in colorectal cancer and is a potential target for improving chemotherapy responsiveness. Significance: The TRIM67/p53 axis represents a novel therapeutic target that could be harnessed to improve chemotherapy efficacy in colorectal cancer expressing wild-type p53 but with repressed p53 signaling.

There remains a paucity of functional biomarkers in gastric cancer. Here, we report the identification of the sodium channel subunit SCNN1B as a candidate biomarker in gastric cancer. SCNN1B mRNA expression was silenced commonly by promoter hypermethylation in gastric cancer cell lines and primary tumor tissues. Tissue microarray analysis revealed that high expression of SCNN1B was an independent prognostic factor for longer survival in gastric cancer patients, especially those with late-stage disease. Functional studies demonstrated that SCNN1B overexpression was sufficient to suppress multiple features of cancer cell pathophysiology in vitro and in vivo. Mechanistic investigations revealed that SCNN1B interacted with the endoplasmic reticulum chaperone, GRP78, and induced its degradation via polyubiquitination, triggering the unfolded protein response (UPR) via activation of PERK, ATF4, XBP1s, and C/EBP homologous protein and leading in turn to caspase-dependent apoptosis. Accordingly, SCNN1B sensitized gastric cancer cells to the UPR-inducing drug tunicamycin. GRP78 overexpression abolished the inhibitory effect of SCNN1B on cell growth and migration, whereas GRP78 silencing aggravated growth inhibition by SCNN1B. In summary, our results identify SCNN1B as a tumor-suppressive function that triggers UPR in gastric cancer cells, with implications for its potential clinical applications as a survival biomarker in gastric cancer patients. Cancer Res; 77(8); 1968–82. ©2017 AACR.

Phospho-sulindac is a sulindac derivative with promising anticancer activity in lung cancer, but its limited metabolic stability presents a major challenge for systemic therapy. We reasoned that inhalation delivery of phospho-sulindac might overcome first-pass metabolism and produce high levels of intact drug in lung tumors. Here, we developed a system for aerosolization of phospho-sulindac and evaluated the antitumor efficacy of inhaled phospho-sulindac in an orthotopic model of human non–small cell lung cancer (A549 cells). We found that administration by inhalation delivered high levels of phospho-sulindac to the lungs and minimized its hydrolysis to less active metabolites. Consequently, inhaled phospho-sulindac (6.5 mg/kg) was highly effective in inhibiting lung tumorigenesis (75%; P < 0.01) and significantly improved the survival of mice bearing orthotopic A549 xenografts. Mechanistically, phospho-sulindac suppressed lung tumorigenesis by (i) inhibiting EGF receptor (EGFR) activation, leading to profound inhibition of Raf/MEK/ERK and PI3K/AKT/mTOR survival cascades; (ii) inducing oxidative stress, which provokes the collapse of mitochondrial membrane potential and mitochondria-dependent cell death; and (iii) inducing autophagic cell death. Our data establish that inhalation delivery of phospho-sulindac is an efficacious approach to the control of lung cancer, which merits further evaluation. Mol Cancer Ther; 12(8); 1417–28. ©2013 AACR.