ELK4 Promotes Colorectal Cancer Progression by Activating the Neoangiogenic Factor LRG1 in a Noncanonical SP1/3-Dependent Manner.

Although the MAPK/MEK/ERK pathway is prevalently activated in colorectal cancer (CRC), MEK/ERK inhibitors show limited efficiency in clinic. As a downstream target of MAPK, ELK4 is thought to work primarily by forming a complex with SRF. Whether ELK4 can serve as a potential therapeutic target is unclear and the transcriptional regulatory mechanism has not been systemically analyzed. Here, it is shown that ELK4 promotes CRC tumorigenesis. Integrated genomics- and proteomics-based approaches identified SP1 and SP3, instead of SRF, as cooperative functional partners of ELK4 at genome-wide level in CRC. Serum-induced phosphorylation of ELK4 by MAPKs facilitated its interaction with SP1/SP3. The pathological neoangiogenic factor LRG1 is identified as a direct target of the ELK4-SP1/SP3 complex. Furthermore, targeting the ELK4-SP1/SP3 complex by combination treatment with MEK/ERK inhibitor and the relatively specific SP1 inhibitor mithramycin A (MMA) elicited a synergistic antitumor effect on CRC. Clinically, ELK4 is a marker of poor prognosis in CRC. A 9-gene prognostic model based on the ELK4-SP1/3 complex-regulated gene set showed robust prognostic accuracy. The results demonstrate that ELK4 cooperates with SP1 and SP3 to transcriptionally regulate LRG1 to promote CRC tumorigenesis in an SRF-independent manner, identifying the ELK4-SP1/SP3 complex as a potential target for rational combination therapy.

Innovative molecular subtypes of multiple signaling pathways in colon cancer and validation of FMOD as a prognostic-related marker.

PURPOSE: Colon cancer is highly heterogeneous in terms of the immune and stromal microenvironment, genomic integrity, and oncogenic properties; therefore, molecular subtypes of the four characteristic dimensions are expected to provide novel clues for immunotherapy of colon cancer. METHODS: According to the enrichment of four dimensions, we performed consensus cluster analysis and identified three robust molecular subtypes for colon cancer, namely immune enriched, immune deficiency, and stroma enriched. We characterized and validated the immune infiltration, gene mutations, copy number variants, methylation, protein expression, and clinical features in different datasets. Finally, we developed an 8-gene risk prognostic model and proposed the innovative RiskScore. In addition, a nomogram model was constructed combining clinical characteristics and RiskScore to validate its excellent clinical predictive power. RESULTS: Combining clinical patient tissue samples and histochemical microarray data, we found that high FMOD expression in tumor epithelial cells was associated with poorer patient prognosis, but FMOD expression in the mesenchyme was not associated with prognosis. In pan-cancer, RiskScore, a prognostic model constructed based on characteristic pathway scores, was a poor prognostic factor for malignancy and was negatively associated with immunotherapy response. CONCLUSION: The identification of molecular subtypes could provide innovative ideas for immunotherapy of colon cancer.

Metabolic pathway-based molecular subtyping of colon cancer reveals clinical immunotherapy potential and prognosis.

PURPOSE: Colon cancer presents challenges to clinical diagnosis and management due to its high heterogeneity. For more efficient and convenient diagnosis and treatment of colon cancer, we are committed to characterizing the molecular features of colon cancer by pioneering a classification system based on metabolic pathways. METHODS: Based on the 113 metabolic pathways and genes collected in the previous stage, we scored and filtered the metabolic pathways of each sample in the training set by ssGSEA, and obtained 16 metabolic pathways related to colon cancer recurrence. In consistent clustering of training set samples with recurrence-related metabolic pathway scores, we identified two robust molecular subtypes of colon cancer (MC1 and MC2). Furthermore, we performed multi-angle analysis on the survival differences of subtypes, metabolic characteristics, clinical characteristics, functional enrichment, immune infiltration, differences with other subtypes, stemness indices, TIDE prediction, and drug sensitivity, and finally constructed colon cancer prognostic model. RESULTS: The results showed that the MC1 subtype had a poor prognosis based on higher immune activity and immune checkpoint gene expression. The MC2 subtype is associated with high metabolic activity and low expression of immune checkpoint genes and a better prognosis. The MC2 subtype was more responsive to PD-L1 immunotherapy than the MC1 subclass. However, we did not observe significant differences in tumor mutational burden between the two. CONCLUSION: Two molecular subtypes of colon cancer based on metabolic pathways have distinct immune signatures. Constructing prognostic models based on subtype differential genes provides valuable reference for personalized therapy targeting unique tumor metabolic signatures.

CK2-induced cooperation of HHEX with the YAP-TEAD4 complex promotes colorectal tumorigenesis.

Dysregulation of Hippo pathway leads to hyperactivation of YAP-TEAD transcriptional complex in various cancers, including colorectal cancer (CRC). In this study, we observed that HHEX (Hematopoietically expressed homeobox) may enhance transcription activity of the YAP-TEAD complex. HHEX associates with and stabilizes the YAP-TEAD complex on the regulatory genomic loci to coregulate the expression of a group of YAP/TEAD target genes. Also, HHEX may indirectly regulate these target genes by controlling YAP/TAZ expression. Importantly, HHEX is required for the pro-tumorigenic effects of YAP during CRC progression. In response to serum stimulation, CK2 (Casein Kinase 2) phosphorylates HHEX and enhances its interaction with TEAD4. A CK2 inhibitor CX-4945 diminishes the interaction between HHEX and TEAD4, leading to decreased expression of YAP/TEAD target genes. CX-4945 synergizes the antitumor activity of YAP-TEAD inhibitors verteporfin and Super-TDU. Elevated expression of HHEX is correlated with hyperactivation of YAP/TEAD and associated with poor prognosis of CRC patients. Overall, our study identifies HHEX as a positive modulator of YAP/TEAD to promote colorectal tumorigenesis, providing a new therapeutic strategy for targeting YAP/TEAD in CRC.

Rational design of a sensitivity-enhanced tracer for discovering efficient APC-Asef inhibitors.

The adenomatous polyposis coli (APC)-Rho guanine nucleotide exchange factor 4 (Asef) protein-protein interaction (PPI) is essential for colorectal cancer metastasis, making it a promising drug target. Herein, we obtain a sensitivity-enhanced tracer (tracer 7) with a high binding affinity (Kd = 0.078 µM) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescence-polarization assays for APC-Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC50 of 0.041 ± 0.004 µM and a conjugated transcriptional transactivating sequence for generating cell-permeable MAIT-516. MAIT-516 inhibits CRC cell migration by specifically hindering the APC-Asef PPI. Furthermore, MAIT-516 exhibits no cytotoxic effects on normal intestinal epithelial cell and colorectal cancer cell growth. Overall, we develop a sensitivity-enhanced tracer for fluorescence polarization assays, which is used for the precise quantification of high-activity APC-Asef inhibitors, thereby providing insight into PPI drug development.

Increased expression of yes-associated protein/YAP and transcriptional coactivator with PDZ-binding motif/TAZ activates intestinal fibroblasts to promote intestinal obstruction in Crohn's disease.

BACKGROUND: Intestinal obstruction caused by intestinal fibrosis is a common and serious complication of Crohn's disease (CD). Intestinal fibroblasts, the main effector cells mediating gastrointestinal fibrosis, are activated during chronic inflammation. However, the mechanism of fibroblast activation in CD has not been well elucidated. METHODS: Fibroblasts isolated from stenotic and nonstenotic intestines of CD patients were used for RNA sequencing. Immunohistochemical and immunofluorescent staining was performed to evaluate the correlation between intestinal fibrosis and YAP/TAZ expression in our CD cohort and a DSS-induced chronic colitis murine model. A Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) inhibitor was used to explore the ROCK1-YAP/TAZ axis in intestinal fibroblasts in vitro and DSS-induced chronic colitis murine model in vivo. FINDINGS: The expression of YAP/TAZ was significantly upregulated in stenotic fibroblasts, which was associated with the YAP/TAZ target gene signature. YAP/TAZ knockdown suppressed the activation of intestinal fibroblasts. In intestinal fibroblasts, YAP/TAZ were activated by the Rho-ROCK1 signalling pathway. High YAP/TAZ expression was positively correlated with ROCK1 expression, which is a prognostic marker for intestinal obstruction in CD patients. INTERPRETATION: YAP/TAZ activation can lead to fibroblast activation and intestinal obstruction in CD. The effect of ROCK1 inhibitor on alleviating intestinal fibrosis is associated with YAP/TAZ inhibition. Targeted inhibition of YAP/TAZ in fibroblasts may be a potential therapeutic strategy to suppress intestinal fibrosis in CD. FUNDING: This work was supported by the National Key R&D Program of China (2019YFC1316002), the NSFC (81873547, 82073201, 81874177, 82000481) and the Shanghai Sailing Program (20YF1429400).

LAMB3 promotes tumour progression through the AKT-FOXO3/4 axis and is transcriptionally regulated by the BRD2/acetylated ELK4 complex in colorectal cancer.

Aberrant expression of laminin-332 promotes tumour growth and metastasis in multiple cancers. However, the dysregulated expression and mechanism of action of LAMB3, which encodes the ß3 subunit of laminin-332, and the mechanism underlying dysregulated LAMB3 expression in CRC remain obscure. Here, we show that LAMB3 is overexpressed in CRC and that this overexpression is correlated with tumour metastasis and poor prognosis. Overexpression of LAMB3 promoted cell proliferation and cell migration in vitro and tumour growth and metastasis in vivo, while knockdown of LAMB3 elicited opposing effects. LAMB3 inhibited the tumour suppressive function of FOXO3/4 by activating AKT in CRC. Both the BET inhibitor JQ1 and the MEK inhibitor U0126 decreased the mRNA level of LAMB3 in multiple CRC cells. Mechanistically, ELK4 cooperated with BRD2 to regulate the transcription of LAMB3 in CRC by directly binding to the ETS binding motifs in the LAMB3 promoter. ELK4 was as acetylated at K125, which enhanced the interaction between ELK4 and BRD2. JQ1 disrupted the interaction between ELK4 and BRD2, resulting in decreased binding of BRD2 to the LAMB3 promoter and downregulation of LAMB3 transcription. Both ELK4 and BRD2 expression was associated with LAMB3 expression in CRC. LAMB3 expression was also negatively correlated with FOXO3/4 in CRC. Our study reveals the pro-tumorigenic role of LAMB3 through the AKT-FOXO3/4 axis and the transcriptional mechanism of LAMB3 in CRC, demonstrating that LAMB3 is a potential therapeutic target that can be targeted by BET inhibitors and MEK inhibitors.

Small heat shock protein CRYAB inhibits intestinal mucosal inflammatory responses and protects barrier integrity through suppressing IKKß activity.

Alpha B-crystallin (CRYAB) is an important member of the small heat shock protein family, and plays a protective and therapeutic role in neurological inflammation. CRYAB expression was assessed in cultured HT29 and Caco-2 cells and inflamed mucosa of patients with inflammatory bowel disease (IBD) and colitis models in mice. Lentivirus-overexpressing and CRSIPR/Cas9 systems were used in different cells to upregulate and silence CRYAB expression, respectively. Cell permeable recombined fusion protein TAT-CRYAB was injected intraperitoneally into dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice to assess its anti-inflammatory effects. CRYAB was found to be significantly decreased in the inflamed mucosa from IBD patients and DSS-induced colitis in mice, and negatively correlated with the levels of TNF-α and IL-6, respectively. Enforced expression of CRYAB suppressed expression of proinflammatory cytokines (e.g., TNF-α, IL-6, IL-1ß, and IL-8) via inhibiting the IKK complex formation, whereas lack of CRYAB expression markedly enhanced proinflammatory responses. Consistently, administration of TAT-CRYAB fusion protein significantly alleviated DSS- or TNBS-induced colitis in mice and protected intestinal barrier integrity. CRYAB regulates inflammatory response in intestinal mucosa by inhibiting IKKß-mediated signaling and may serve as a novel therapeutic approach in the treatment of IBD.

Clinical outcomes and risk factors of secondary extraintestinal manifestation in ulcerative colitis: results of a multicenter and long-term follow-up retrospective study.

BACKGROUND: Extraintestinal manifestations (EIM) are common in ulcerative colitis (UC). In Shanghai, China, data on the incidence rate and risk factors of EIM in UC patients remain scarce. METHODS: The study population consisted of UC patients who were identified from a prospectively maintained, institutional review board-approved database at our institutes from June 1986 to December 2018. The demographic and clinical characteristics of the study participants were analyzed. The study included secondary EIM in UC patients and follow-up, while primary EIM was excluded. The diagnosis of EIM was based on clinical, radiological, endoscopic, and immunologic examination and histological findings. RESULTS: In total, 271 eligible patients were included in the current study, with a median follow-up time of 13.0 years (interquartile range, 9.0-17.0), and including 31 cases (11.4%) that developed EIM. EIM was associated with clinical outcomes in UC patients and the following factors were identified as contributing factors for the development of EIM: a disease duration of >5 years (odds ratio (OR), 3.721; 95% confidence interval (CI) [1.209-11.456]), age at diagnosis >40 years (OR, 2.924, 95% CI [1.165-7.340]), refractory clinical symptoms (OR, 4.119; 95% CI [1.758-9.650]), and moderate or severe anemia (OR, 2.592; 95% CI [1.047-6.413]). CONCLUSION: In this study, approximately 11.4% UC patients go on to develop at least one EIM. Clinicians should prioritize early control of the disease and treatment of anemia in UC in order to prevent the development of EIM and improve disease prognosis.

Increased IGF2BP3 expression promotes the aggressive phenotypes of colorectal cancer cells in vitro and vivo.

Previous literatures reported insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) is a poor prognostic marker for colorectal cancer (CRC) patients. However, basic research on the effect and biological role of IGF2BP3 in CRC was still scare. Real-time quantitative polymerase chain reaction and western blot analysis were used to examine IGF2BP3 expression level in tumors and paired normal tissues from CRC patients. Tissue microarrays with 192 CRC patients were subjected to immunohistochemical staining to analyze the prognostic value of IGF2BP3. Proliferation assays, migration assays, and xenograft tumor formation in nude mice were performed to assess the biological role of IGF2BP3 in CRC cells. IGF2BP3 expression was significantly upregulated in tumor tissues compared with the matched normal tissues both in messenger RNA and protein level and was associated with worse prognosis. IGF2BP3 knockdown made cell cycle arrest to impair the proliferation ability of CRC cells and further inhibited the xenograft tumor growth in nude mice, also inhibited the migration ability of CRC cells via inducing epithelial-mesenchymal transition. Therefore, the research demonstrated that increased IGF2BP3 expression promoted the aggressive phenotypes of CRC cells. Targeted IGF2BP3 could be a novel and effective gene therapy for CRC patients to make a better prognosis.

Combination of CDX2 expression and T stage improves prognostic prediction of colorectal cancer.

OBJECTIVE: Prognostic prediction of colorectal cancer (CRC) remains challenging because of its heterogeneity. Aberrant expression of caudal-type homeobox transcription factor 2 (CDX2) is strongly correlated with the prognosis of CRC. METHODS: Tissue samples of patients with CRC who underwent surgery in Xinhua Hospital (Shanghai, China) from January 2010 to January 2013 were collected. CDX2 expression was semiquantitatively evaluated via immunohistochemistry. RESULTS: In total, 138 patients were enrolled in this study from a prospectively maintained institutional cancer database. The median follow-up duration was 57.5 months (interquartile range, 17.0-71.0 months). In the Cox proportional hazards model, low CDX2 expression combined with stage T4 CRC was significantly the worst prognostic factor for disease-free survival (hazard ratio = 7.020, 95% confidence interval = 3.922-12.564) and overall survival (hazard ratio = 5.176, 95% CI = 3.237-10.091). In the Kaplan-Meier survival analysis, patients with low CDX2 expression and stage T4 CRC showed significantly worse disease-free survival and overall survival than those with low CDX2 expression alone. CONCLUSION: CDX2 expression combined with the T stage was more accurate for predicting the prognosis of CRC. Determining the prognosis of CRC using more than one variable is valuable in developing appropriate treatment and follow-up strategies.

Reduced Risk of Inflammatory Bowel Disease-associated Colorectal Neoplasia with Use of Thiopurines: a Systematic Review and Meta-analysis.

BACKGROUND AND AIMS: The association between thiopurines and colorectal neoplasia risk remains controversial in inflammatory bowel disease [IBD] patients. We performed a systematic review and meta-analysis examining this association. METHODS: A comprehensive search of the PubMed, EMBASE and Cochrane Library databases was performed to identify relevant literature. Random-effects models were applied to calculate the pooled odds ratio [OR] and relative risk [RR] with corresponding 95% confidence intervals [CIs] among case-control and cohort studies. RESULTS: Eleven cohort and 16 case-control studies involving 95397 patients were included in this study. Overall, the use of thiopurines was associated with a reduced risk of colorectal neoplasia both in case-control [OR = 0.49, 95% CI: 0.34-0.70] and cohort studies [RR = 0.96, 95% CI: 0.94-0.98]. Moreover, a protective effect of thiopurines against advanced neoplasia [high-grade dysplasia and cancer] [OR = 0.51, 95% CI: 0.31-0.84 for case-control studies; RR = 0.96, 95% CI: 0.94-0.98 for cohort studies] and colorectal cancer [CRC] [OR = 0.56, 95% CI: 0.34-0.93 for case-control studies; RR = 0.96, 95% CI: 0.94-0.98 for cohort studies] was also observed. Furthermore, when the analysis was conducted on patients at a high risk for colorectal neoplasia, the chemopreventive effect was confirmed in patients with long disease duration [> 8 years] but not in those with extensive colitis or primary sclerosing cholangitis. CONCLUSIONS: This study demonstrated that thiopurine use was associated with a reduced risk of colorectal neoplasia, advanced neoplasia and CRC in IBD patients, especially those with long disease duration [> 8 years].

The hepatoprotective effect of the probiotic Clostridium butyricum against carbon tetrachloride-induced acute liver damage in mice.

Previous studies have revealed that the probiotic Clostridium butyricum (C. butyricum) can attenuate cirrhosis in chronic non-alcoholic liver disease. However, the effects of C. butyricum on acute liver injury (ALI) remain unclear. Therefore, the present study aims to examine the hepatoprotective effects and the underlying mechanisms employed by C. butyricum in a carbon tetrachloride (CCl4)-induced ALI murine model. Here, we evaluated the survival rate and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), anti-oxidants, cytokines and the gut microbiota to elucidate the potential mechanisms by which C. butyricum is hepatoprotective. Our results show that five days of prophylactic C. butyricum treatment significantly reduced mortality by 40% and decreased the CCl4-induced levels of ALT and AST in the serum of these mice. Additionally, prophylactic treatment with C. butyricum increased the activity of both superoxide dismutase (SOD) and catalase (CAT), and substantially reduced malondialdehyde (MDA) levels, which were deteriorated in the untreated ALI mice compared to normal control mice. Furthermore, C. butyricum up-regulated the nuclear factor (erythroid-derived 2)-like 2 (NRF2) content. CCl4-induced mice also exhibited considerable increases of phosphorylation of nuclear factor-kappa B (NF-κB) p65 and pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). However, the inflammatory responses of the liver induced by CCl4 were significantly alleviated by C. butyricum pretreatment. Additionally, we found that interleukin-10 (IL-10), an anti-inflammatory mediator, was increased in the C. butyricum-pretreated group. Microbiota analysis in these mice revealed crosstalk between the gut microbial metabolites and ALI. The intestinal flora was changed by CCl4 administration and was shifted by the probiotic C. butyricum toward more beneficial bacteria, particularly the Clostridia orders, which are the known producers of the anti-inflammatory and anti-oxidative metabolite butyrate. In conclusion, we found that the intestinal flora changes after the intraperitoneal injection of CCl4. We also offer novel insights into the mechanism by which probiotic C. butyricum pretreatment alleviates the CCl4-induced inflammation and oxidative stress of the liver via the modulation of NRF2, NF-κB p65, IL-10 and the intestinal microbiota in mice.