Zhongchuan Wang

The dynamic assembly and regulation of the IκB kinase (IKK) complex in the NF-κB pathway are central to the pathogenesis and progression of inflammatory bowel disease (IBD). We recently reported that the transcription factor hematopoietically expressed homeobox (HHEX) promotes colitis-associated colorectal cancer, but the potential role of HHEX in intestinal inflammation remains uncharacterized. Here, we found that HHEX is upregulated in inflamed colons in a colitis mouse model and in clinical IBD samples. HHEX overexpression increased inflammatory cytokine expression, and HHEX loss largely abrogated the inflammatory response in vitro and intestinal inflammation in vivo. Mechanistically, IKKα phosphorylates HHEX at S213 to stabilize HHEX in response to TNF-α by inhibiting the interaction of HHEX with the E3 ubiquitin ligase MID2 and subsequent K48-linked ubiquitination and protein degradation. Importantly, HHEX interacted with and stabilized the IKKα/IKKβ complex via its N-terminal domain, thereby activating the NF-κB pathway and establishing a positive feedback loop that exacerbates intestinal inflammation. Our study reveals a transcription-independent function of HHEX in promoting IKK complex assembly and colitis, identifying HHEX as an IBD susceptibility gene and a potential target for IBD treatment. HHEX as a positive regulator of the IKK complex that activates the NF-κB pathway. Inflammatory stimuli can stabilize HHEX by IKKα-dependent phosphorylation.

Objective: The plastic role of regulatory factor X1 (RFX1) in colon cancer progression and its impact on the tumor microenvironment remain poorly understood. The study aimed to clarify the molecular and clinical role of RFX1 in colon cancer. Methods: We classified colon cancers into subgroups with high and low RFX1 expression and characterized their immune profiles, mutational profiles, cancer immunotherapy and drug sensitivity. By combining RFX1 expression with persistent tumor mutational burden, we proposed a novel nomogram clinical prediction model and validated its predictive performance, and the correlation between high expression and poor prognosis. Results: Compared to tumor mutational burden (TMB), persistent tumor mutational burden (pTMB) is an independent predictor of prognosis in patients with colon cancer. The predictive efficacy of the combination of RFX1 expression and pTMB was superior to and sensitive than the combination of RFX1 expression with TMB. Among them, patients in the RFX1high/pTMBhigh subgroup had the worst quality of survival and prognosis, whereas those in the RFX1low/pTMBlow subgroup had a relatively better prognosis (p < 0.0001). Univariate Cox regression revealed a significant association between high RFX1 expression and increased risk in colon cancer patients (Hazard Ratio [HR] = 1.58, 95% Confidence Interval [CI]: 1.10–2.25, p = 0.012), which remained independently predictive in multivariate analysis after covariate adjustment (HR = 1.52, 95% CI: 1.04–2.22, p = 0.031). Conclusion: A nomogram model based on RFX1 combined with pTMB provides an alternative approach for the diagnosis and treatment of colon cancer.

Background: Nudix Hydrolase 21 (NUDT21) is crucial for the regulation of alternative polyadenylation, with its reduced expression frequently resulting in a shortened mRNA 3′ untranslated region (UTR), thereby enhancing the protein levels of downstream genes. Although NUDT21 is widely recognized for its tumor-suppressive function in various cancers, its involvement in colorectal cancer (CRC) remains poorly understood. Methods: The expression of NUDT21 in CRC and adjacent normal tissues was analyzed through qPCR, Western blot, and immunohistochemistry (IHC). Additionally, we investigated the correlation between NUDT21 expression and patient prognosis. With Cell Counting Kit-8 assay and Transwell assay, we detected the exact role of NUDT21 in the development and progress of CRC. We used RNA-Seq analysis to explore the downstream target gene of NUDT21. Meanwhile, DaPars analysis revealed the Hippo pathway as a critical pathway in CRC progress. Western blotting and luciferase assays were used to investigate the specific mechanisms by which NUDT21 regulates the Hippo pathway. In addition, the interaction between NUDT21 and TAZ was confirmed by co-immunoprecipitation and the correlation between the two expressions was verified by IHC. Results: In CRC, elevated NUDT21 expression has been identified as a predictor of poor prognosis, as well as a promoter of CRC cell proliferation and migration. Combined with RNA-seq and DaPars analyses, we identified the Hippo pathway as an important downstream target of NUDT21 in CRC. NUDT21 knockdown significantly downregulated the YAP, TAZ, and TEAD1 protein levels. Mechanistically, shortening of the 3′UTR of TAZ by NUDT21 knockdown suppressed TAZ protein expression. A positive correlation was observed between NUDT21 and TAZ proteins in CRC. Conclusion: NUDT21 plays a pro-tumorigenic role in CRC by upregulating TAZ protein expression, revealing a new regulatory mechanism of the Hippo pathway and providing insight into the biological effect of mRNA 3′UTR shortening.

Introduction: Over the past few years, multiple observational studies have speculated a potential association between inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), and osteoporosis. However, no consensus has been reached regarding their interdependence and pathogenesis. Herein, we sought to further explore the causal associations between them.Methods: We validated the association between IBD and reduced bone mineral density in humans based on genome-wide association studies (GWAS) data. To investigate the causal relationship between IBD and osteoporosis, we performed a two-sample Mendelian randomization study using training and validation sets. Genetic variation data for IBD, CD, UC, and osteoporosis were derived from published genome-wide association studies in individuals of European ancestry. After a series of robust quality control steps, we included eligible instrumental variables (SNPs) significantly associated with exposure (IBD/CD/UC). We adopted five algorithms, including MR Egger, Weighted median, Inverse variance weighted, Simple mode, and Weighted mode, to infer the causal association between IBD and osteoporosis. In addition, we evaluated the robustness of Mendelian randomization analysis by heterogeneity test, pleiotropy test, leave-one-out sensitivity test, and multivariate Mendelian randomization.Results: Genetically predicted CD was positively associated with osteoporosis risk, with ORs of 1.060 (95% CIs 1.016, 1.106; p = 0.007) and 1.044 (95% CIs 1.002, 1.088; p = 0.039) for CD in the training and validation sets, respectively. However, Mendelian randomization analysis did not reveal a significant causal relationship between UC and osteoporosis (p > 0.05). Furthermore, we found that overall IBD was associated with osteoporosis prediction, with ORs of 1.050 (95% CIs 0.999, 1.103; p = 0.055) and 1.063 (95% CIs 1.019, 1.109; p = 0.005) in the training and validation sets, respectively.Conclusion: We demonstrated the causal association between CD and osteoporosis, complementing the framework for genetic variants that predispose to autoimmune disease.

Crohn’s disease is a highly heterogeneous autoimmune disease with a unique inflammatory phenotype of T cells at the lesion site. We aim to further explore the diagnosis of Crohn’s disease and drug prediction of T cell marker gene expression. We obtained single-cell expression profile data from 22 CDs or normal samples and performed cell annotation and cellular communication analysis. Through the intersection of T cell marker genes, differential genes, and WGCNA results, we identified T cell-specific key genes and their immune landscapes and potential pathogenesis, and validated them across multiple datasets and patient tissue samples. We also explored the differentiation characteristics of genes by pseudo-temporal analysis and assessed their diagnostic performance and drug sensitivity by molecular docking. Finally, we extended this study to the prognosis of IBD-associated colon cancer. TNF-centered 5-gene diagnostic model not only has excellent diagnostic efficacy, but is also closely associated with KRAS, P53, and IL6/JAK/STAT3 pathways and physiological processes, such as EMT, coagulation, and apoptosis. In addition, this diagnostic model may have potential synergistic immunotherapeutic effects, with positive correlations with immune checkpoints such as CTLA4, CD86, PDCD1LG2, and CD40. Molecular docking demonstrated that BIRC3 and ANXA1 have strong binding properties to Azathioprine and Glucoocorticoid. Furthermore, the 5-gene model may suggest antagonism to IFX and prognosis for colon cancer associated with inflammatory bowel disease. Single-cell sequencing targeting T cell-related features in patients with Crohn’s disease may aid in new diagnostic decisions, as well as the initial exploration of high-potential therapies.

Pathogenic inflammation mediated by overactive type 1 helper T cell (Th1) responses could exacerbate and perpetuate Crohn's disease. Programmed death (PD)‐1 and its ligand PD‐L1 pathway could be upregulated to suppress inflammation. We wondered why this pathway is ineffective at suppressing pathogenic Th1 inflammation in Crohn's disease patients. Here, we found that overexpression of T‐bet via transfection significantly reduced the expression of PD‐1. PD‐L1 was capable of suppression proinflammatory CD4⁺ T cells, but T‐bet transfection significantly reduced the susceptibility of CD4⁺ T cells toward PD‐L1‐mediated suppression, evidenced by the observations that at low PD‐L1 concentration T‐bet transfected and mock transfected CD4⁺ T cells presented comparable IL‐2 production, but at high PD‐L1 concentration, T‐bet transfected CD4⁺ T cells presented significantly higher IL‐2 than mock transfected CD4⁺ T cells. PD‐L1 could significantly reduce the survival of CD4⁺ T cells from Crohn's disease patients, but interestingly, in the absence of PD‐L1, the survival was better in mock transfected CD4⁺ T cells, while in the presence of PD‐L1, the survival was better in T‐bet transfected CD4⁺ T cells. Crohn's disease patients with greater severity presented higher T‐bet expression and lower PD‐1 expression in CD4⁺ T cells, demonstrating an association between T‐bet expression and disease progression. We also discovered that stimulation with bacterial antigens could upregulate the expression of T‐bet. Together, this study demonstrated that T‐bet overexpression could interfere with PD‐1/PD‐L1‐mediated suppression of CD4⁺ T cell inflammation and survival, and potentially contributed to the development and persistence of Crohn's disease.