Wnt/β-catenin signaling plays a critical role in colorectal cancer (CRC) tumorigenesis and the homeostasis of colorectal cancer stem cells (CSCs), but its molecular mechanism remains unclear. B-cell lymphoma 3 (Bcl-3), a member of the IκB family, is overexpressed in CRC and promotes tumorigenicity. Here, we report a novel function of Bcl-3 in maintaining colorectal CSC homeostasis by activating Wnt/β-catenin signaling. Silencing Bcl-3 suppresses the self-renewal capacity of colorectal CSCs and sensitizes CRC cells to chemotherapeutic drugs through a decrease in Wnt/β-catenin signaling. Moreover, our data show that Bcl-3 is a crucial component of Wnt/β-catenin signaling and is essential for β-catenin transcriptional activity in CRC cells. Interestingly, Wnt3a increases the level and nuclear translocation of Bcl-3, which binds directly to β-catenin and enhances the acetylation of β-catenin at lysine 49 (Ac-K49-β-catenin) and transcriptional activity. Bcl-3 depletion decreases the Ac-K49-β-catenin level by increasing the level of histone deacetylase 1 to remove acetyl groups from β-catenin, thus interrupting Wnt/β-catenin activity. In CRC clinical specimens, Bcl-3 expression negatively correlates with the overall survival of CRC patients. A significantly positive correlation was found between the expression of Bcl-3 and Ac-K49-β-catenin. Collectively, our data reveal that Bcl-3 plays a crucial role in CRC chemoresistance and colorectal CSC maintenance via its modulation of the Ac-K49-β-catenin, which serves as a promising therapeutic target for CRC.

The prognosis of advanced colon adenocarcinoma (COAD) remains poor. However, existing methods are still difficult to assess patient prognosis. Pyroptosis, a lytic and inflammatory process of programmed cell death caused by the gasdermin protein, is involved in the development and progression of various tumors. Moreover, there are no related studies using pyroptosis-related genes to construct a model to predict the prognosis of COAD patients. Thus, in this study, bioinformatics methods were used to analyze the data of COAD patients downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to construct a risk model for the patient prognosis. TCGA database was used as the training set, and GSE39582 downloaded from GEO was used as the validation set. A total of 24 pyroptosis-related genes shown significantly different expression between normal and tumor tissues in COAD and seven genes (CASP4, CASP5, CASP9, IL6, NOD1, PJVK, and PRKACA) screened by univariate and LASSO cox regression analysis were used to construct the risk model. The receiver operating characteristic (ROC) and Kaplan–Meier (K–M curves) curves showed that the model based on pyroptosis-related genes can be used to predict the prognosis of COAD and can be validated by the external cohort well. Then, the clinicopathological factors were combined with the risk score to establish a nomogram with a C-index of 0.774. In addition, tissue validation results also showed that CASP4, CASP5, PRKACA, and NOD1 were differentially expressed between tumor and normal tissues from COAD patients. In conclusion, the risk model based on the pyroptosis-related gene can be used to assess the prognosis of COAD patients well, and the related genes may become the potential targets for treatment.

Background: Pyroptosis belongs to a unique type of programmed cell death among which GSDME is reported to exert anti-tumor immunity. However, the underlying mechanisms of how to boost tumor-infiltrating lymphocytes and whether it could benefit the efficacy of ICIs are still unknown. Methods: CRC samples were used to analyze its relationship with CD8⁺T cells. GSDME in mouse CRC cell lines CT26/MC38 was overexpressed. The infiltration of CD8⁺T cells in grafted tumors was determined by multiplex flow cytometric analysis and immunohistochemistry. Transcriptomic analysis was performed in cell lines to define key signatures related to its overexpression. The mechanism of how mtDNA was released by GSDME-induced mitochondrial damage and activated cGAS-STING pathway was observed. Whether GSDME benefited ICIs and the relationships with the genotypes of CRC patients were investigated. Results: It had favorable prognostic value in CRC and was positively associated with increased number and functionality of CD8⁺T cells both in human samples and animal models. This was due to mitochondrial damage and activation of cGAS-STING-IFNβ pathway for the recruitment of CD8⁺T cells. Mechanically, GSDME overexpression enhanced N-GSDME level, leading to the mitochondrial damage and mtDNA was released into cytosol. Finally, GSDME benefited with ICIs and exhibited positive relationships with MSI in CRC patients. Conclusion: We presented the mechanism of GSDME in anti-tumor immunity through activating cGAS-STING-IFNβ axis mediated by mitochondrial damage, leading to more infiltration of CD8⁺T cells with synergistic efficacy with ICIs.

Colorectal cancer (CRC) is one of the prevalent malignant tumors. This study is aimed at evaluating the mechanism of anlotinib (anlo) on tumor microenvironment (TME) in CRC, and its effects in combination with immune checkpoint inhibitors (ICIs) therapy. Firstly, MC38 and CT26 cells were both exposed to different gradient concentrations of anlo for 72 h, to investigate the cell viability and synergetic therapy efficacy with ICIs by CCK8. The results showed that anlo could obviously inhibit cell growth and showed no synergistic efficacy therapy in combination with αPDL1 in vitro. Then, we found the upregulation of programmed cell death ligand 1(PDL1) expression both in vitro and in vivo after anlo treatment. In vivo, anlo could enhance the percentage of natural killer (NK) cells and M1 macrophage cells and decrease the percentage of M2 macrophage cells in TME. Moreover, we explored the mechanism and we proved that anlo could activate reactive oxygen species (ROS)/c‐Jun N‐terminal kinase (JNK)/activator protein‐1 (AP‐1) signaling pathway to increase the expression levels of PDL1, IFN‐α/β/γ, and CXCL2 in two cell lines in vitro. We also proved that anlo had synergistic effects with ICIs in vivo. Finally, it could also increase the mRNA and protein PDL1 expression levels in human cell lines, which was consistent with mouse CRC cell lines. However, there are still a few limitations. On one hand, the ROS/JNK/AP‐1 pathway needs to be proved whether it can be activated in human cell lines. On the other hand, the mechanism behind ROS promoting phosphorylation of JNK needs to be explored.

The prognosis of colon adenocarcinoma (COAD) needs to be improved. Cuproptosis is a recently discovered cell death caused by intracellular overload of copper ions. There have been no reports about the cuproptosis-related prognostic model in COAD. First, we screened 30 differentially expressed genes (DEGs) from patients with COAD using The Cancer Genome Atlas (TCGA) database. Gene Expression Omnibus (GEO) database was used as a validation set to establish a risk model of five cuproptosis-related genes (CKDN2A, SDHB, CCS, ULK1, and CMC1) by least absolute shrinkage and selection operator (LASSO) Cox regression analysis. In both TCGA and GEO cohorts, we could see that overall survival of COAD patients of the low-risk group was longer. Combined with the clinical characteristics, the risk score was found to be an independent prognostic factor. Furthermore, single-sample Gene Set Enrichment Analysis (ssGSEA) showed that the levels of Th1 and Treg immune cells changed both in TCGA and GEO databases. Finally, clinical samples were used to verify the mRNA and protein levels of five risk-model genes. In conclusion, this model could predict the prognosis of COAD patients, and the mechanism may be related to the changes in immune cells in the tumor microenvironment (TME).

Background: Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) has been proposed as a predictive biomarker to predict response to immunotherapy. Given the limitations of IHC test in PD-L1 detection, this study aimed to investigate the technical feasibility of using quantitative RT-PCR (qRT-PCR) to replace IHC in PD-L1 detection in gastrointestinal tumors.Materials and methods: The Cancer Genome Atlas database was used to evaluate the relationship between PD-L1 expression in tumor tissue and the patient prognosis. In addition, 52 patients with gastrointestinal cancer were enrolled and divided into the stomach (STAD), colon (COAD), and rectum (READ) adenocarcinoma cohorts. IHC test was used to determine the PD-L1 level of the tissue specimens, and the qRT-PCR test was used to analyze the mRNA expression in both blood and tissue specimens. Moreover, the correlation between blood PD-L1 mRNA expression and immunotherapy efficacy was investigated in additional 15 patients with gastric cancer that further enrolled.Results: The expression level of PD-L1 in tumor tissue is related to the tumor stage of COAD (p-value = 0.001) and primary therapy outcomes in patients with READ (p-value = 0.003) but not significantly correlated to the overall survival (OS) time of patients with gastrointestinal cancer. Moreover, the concordance of PD-L1 mRNA expression level of tissue and paired blood samples is low, despite a weak linear relationship that was found in the STAD cohort (r = 0.43, p-value = 0.049). We further demonstrated that qRT-PCR results in both tissue and blood specimens were numerically but not statistically significant consistent with IHC results (corresponding to a p-value of 0.84 and 0.55, respectively). Remarkably, high PD-L1 expression in blood of patients with STAD shows a better response to immunotherapy (p-value = 0.04), which could be well identified at the relative expression cutoff of 1.5 (sensitivity of 85.7%, specificity of 75.0%, and AUC of 0.82).Conclusions: Our study established a novel strategy for rapidly distinguishing patients with gastrointestinal cancer with the response to immunotherapy and has potential clinical benefits.