The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

The secreted protein collagen and calcium-binding EGF domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFß-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in patients with CRC. YAP/TAZ-TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts, which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells in vitro and lymphangiogenesis in a CRC cell-derived xenograft model in vivo. In addition, the bromodomain and extraterminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis, and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ-TEAD4-BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Clostridium difficile toxin A and toxin B inhibit YAP in the colonic epithelial cells.

Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of Clostridium difficile, are main causal agents for the colonic epithelium damage in Clostridium difficile infection (CDI). The Hippo pathway is crucial for the control of tissue homeostasis and regeneration of intestines. However, the dysregulation of Hippo pathway in CDI is unclear. Here we show that YAP and TAZ, the transcriptional coactivators downstream of the Hippo pathway, are sequestered in the cytoplasm, degraded, and inactivated by treatment with TcdA and TcdB in colonic epithelial cells. The overexpression of YAP restores the messenger RNA expressions of YAP target genes, attenuates the disruption of cytoskeleton and cell rounding, and rescues the cell proliferation of colonic epithelial cells under exposure of the two toxins. Our results demonstrate that inhibition of YAP and TAZ is involved in the pathogenesis of CDI, implicating that increasing YAP activity could be a potential therapeutic strategy for the CDI treatment.

N-glycosylation of Siglec-15 decreases its lysosome-dependent degradation and promotes its transportation to the cell membrane.

Siglec-15 was recently reported to be an immunosuppressive molecule that is expressed by tumor-associated macrophages and upregulated in some solid tumors. Targeting Siglec-15 is a potential strategy for normalization cancer immunotherapy. Here, we identified the important post-translational modification, N-glycosylation of Siglec-15, which is regulated by glucose uptake. Using a series of glycosidase and glycosylation inhibitors, we demonstrated that Siglec-15 was completely N-glycosylated in vitro and in vivo. The precise glycosylation site was determined. N-glycosylation stabilized Siglec-15 by decreasing its lysosome-dependent degradation. Siglec-15 subcellular distribution detected by immunofluorescence indicated that N-glycosylation promoted Siglec-15 transportation to the cell membrane. The collective observations indicate that targeting the N-glycosylation of Siglec-15 may be an effective supplement to immunotherapy.

P4HA2 promotes tumor progression and is transcriptionally regulated by SP1 in colorectal cancer.

P4HA2 has been implicated in various malignant tumors; however, its expression and functional role in colorectal cancer (CRC) remain poorly elucidated. This study aims to investigate the involvement of P4HA2 in CRC metastasis and progression, uncovering the underlying mechanisms. In colorectal cancer (CRC), P4HA2 exhibited overexpression, and elevated levels of P4HA2 expression were associated with an unfavorable prognosis. Functional assays demonstrated P4HA2's regulation of cell proliferation, and epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Additionally, the AGO1 expression was correlated with P4HA2, and depletion of AGO1 reversed the proliferation and EMT function induced by P4HA2. Chromatin immunoprecipitation (ChIP) and luciferase assays suggested that the transcription factor SP1 binds to the promoter sequence of P4HA2, activating its expression in CRC. This study unveiled SP1 as a transcriptional regulator of P4HA2 in CRC and AGO1 is a probable target of P4HA2. In conclusion, P4HA2 emerges as a potential prognostic biomarker and promising therapeutic target in colorectal 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.

Two-sample Mendelian randomization analysis evaluates causal associations between inflammatory bowel disease and osteoporosis.

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.

Intestinal Microbiota: The Driving Force behind Advances in Cancer Immunotherapy.

In recent years, cancer immunotherapy has become a breakthrough method to solve solid tumors. It uses immune checkpoint inhibitors to interfere with tumor immune escape to coordinate anti-tumor therapy. However, immunotherapy has an individualized response rate. Moreover, immune-related adverse events and drug resistance are still urgent issues that need to be resolved, which may be attributed to the immune imbalance caused by immune checkpoint inhibitors. Microbiome research has fully revealed the metabolic-immune interaction relationship between the microbiome and the host. Surprisingly, sequencing technology further proved that intestinal microbiota could effectively intervene in tumor immunotherapy and reduce the incidence of adverse events. Therefore, cancer immunotherapy under the intervention of intestinal microbiota has innovatively broadened the anti-tumor landscape and is expected to become an active strategy to enhance individualized responses.

Nuclear TEAD4 with SIX1 Overexpression is an Independent Prognostic Marker in the Stage I-III Colorectal Cancer.

INTRODUCTION: Stage I-III colorectal cancer patients are under risk of tumor recurrence and metachronous metastasis after radical surgery. An increased expression of transcription factor TEAD4 is associated with epithelial-mesenchymal transition, metastasis and poor prognosis in colorectal cancer. However, the mechanistic role of TEAD4 in driving colon cancer progression and its prognostic value in early stage of CRC remains unclear. METHODS: In this study, the regulation, function and prognostic significance of TEAD4 and its new direct target gene SIX1 in CRC progression were evaluated using human tissues, molecular and cell biology. RESULTS: We show that TEAD4 directly upregulates the expression of SIX1 at transcriptional level in CRC cells, establishing that SIX1 is a new direct target gene of TEAD4. TEAD4 promotes EMT and cell migration of CRC cells, while SIX1 knockdown attenuates this effect and SIX1 overexpression enhances this effect, indicating that SIX1 mediates the function of TEAD4 in promoting cell migration in CRC cells. Clinically, nuclear TEAD4, overexpression of SIX1 and nuclear TEAD4 with SIX1 overexpression predict poor prognosis in CRC patients. DISCUSSION: Our study identifies TEAD4-SIX1-CDH1 form a novel signaling axis, which contributes to CRC progression, and its aberrant expression and activation predicts poor prognostic for CRC patients in stage I-III.

CCBE1 promotes tumor lymphangiogenesis and is negatively regulated by TGFß signaling in colorectal cancer.

Collagen and calcium-binding EGF domain-1 (CCBE1) is essential for lymphatic vascular development as it promotes vascular endothelial growth factor C (VEGFC) proteolysis. A recent study reported that CCBE1 was overexpressed in epithelial colorectal cancer (CRC) cells; however, the role of CCBE1 in tumor lymphangiogenesis and the mechanism underlying dysregulated CCBE1 expression in CRC remain undefined. Methods: The role of CCBE1 in tumor lymphangiogenesis and lymphatic metastasis was investigated using human lymphatic endothelial cells (HLECs) model in vitro, and a hindfoot lymphatic metastasis model in vivo. Immunochemistry analysis was performed to assess CCBE1 expression, prognostic value and correlation with clinicopathological characteristics in CRC. The biochemical function and transcriptional regulatory mechanism of CCBE1 were explored by western blot, qPCR, and chromatin immunoprecipitation. Results: Cancer cell-derived CCBE1 enhances VEGFC proteolysis in vitro, facilitates tube formation and migration of HLECs in vitro, and promotes tumor lymphangiogenesis and lymphatic metastasis in vivo. In addition to CRC cells, tumor stroma within CRC tissue shows high CCBE1 expression, which is associated with high lymphatic vessel density, increased lymph node metastasis and poor prognosis. Cancer-associated fibroblasts (CAFs) express and secret CCBE1, thereby contributing to VEGFC maturation and tumor lymphangiogenesis in CRC. Transforming growth factor beta (TGF-ß) downregulates the transcription and lymphangiogenic function of CCBE1 in CAFs and CRC cells through direct binding of SMADs to CCBE1 gene locus. Inactivation of the TGF-ß pathway correlates with increased CCBE1 expression in CRC. Conclusion: Our results demonstrate the protumorigenic role of CCBE1 in promoting lymphangiogenesis and lymphatic metastasis in CRC, revealing a new mechanism by which loss of TGF-ß signaling promotes CRC metastasis.

Small-molecule activating SIRT6 elicits therapeutic effects and synergistically promotes anti-tumor activity of vitamin D3 in colorectal cancer.

Colorectal cancer (CRC) is the leading cause of cancer death; however, targets with broad anti-CRC effects are limited. Sirtuin6 (SIRT6) is a conserved nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that is widely pathologically downregulated in CRC, but its pharmacological effect in CRC remains undefined due to the lack of small-molecule SIRT6 activators. We searched for a compound activating SIRT6 and investigated its anti-CRC effect in various models. Methods: We identified an allosteric SIRT6 activator, MDL-811. Its ability to enhance SIRT6 deacetylation at protein and cellular levels was evaluated by Fluor de Lys (FDL) and western blots. We assessed the proliferation of 26 CRC cell lines and patient-derived organoids (PDOs) treated with MDL-811. In vivo efficacy of MDL-811 was evaluated in HCT116 cell line- and patient-derived xenografts as well as a spontaneous CRC model. RNA sequencing and real-time quantitative PCR assays were performed to analyze gene expression changes in MDL-811-treated HCT116 cells. Along with controls in SIRT6-overexpressing HCT116 cells, the SIRT6-mediated histone H3 deacetylation at the Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene locus was assessed by chromatin immunoprecipitation (ChIP) in MDL-811-treated HCT116 cells. A combination therapy against CRC based on the downstream gene of SIRT6 activation was evaluated in cells and mouse models. Results: MDL-811 significantly activated SIRT6 histone H3 deacetylation (H3K9Ac, H3K18Ac, and H3K56Ac) in vitro and had broad antiproliferative effects on diverse CRC cell lines and PDOs. More importantly, the in vivo anti-tumor efficacy of MDL-811 was demonstrated across cell line- and patient-derived xenografts and in the APCmin/+ spontaneous CRC model. Mechanically, we identified a new downstream target gene of SIRT6 in CRC, CYP24A1. Based on these findings, a combination drug strategy with MDL-811 to synergistically enhance the anti-CRC effect of vitamin D3 was validated in vitro and in vivo. Conclusions: Our data provide proof of concept that targeting SIRT6 using a small-molecule activator is an attractive therapeutic strategy for CRC and that MDL-811 could be a promising lead compound for further preclinical and clinical studies of treatments for CRC.

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.

Reduced Frequency and Prognostic Significance of Ring Finger Protein 43 Nucleotide Polymorphisms in a Chinese Colorectal Cancer Cohort.

The aim of this research was to verify the mutation rate of the ring finger protein 43 (RNF43) of the transmembrane E3 ubiquitin ligase and investigate the influence of single nucleotide polymorphisms (SNPs) rs2257205 in RNF43 in a Chinese colorectal cancer (CRC) cohort. DNA from 177 diagnosed CRC patients' tissues or blood samples were extracted, amplified, and sequenced. Clinicopathological features, including age, gender, tumor location, tumor-lymph node-metastasis stage, and survival information, were analyzed. Four novel RNF43 mutations (including G659 and R117 sites) were validated in 177 CRC patients; all events were somatic frameshift mutations. Furthermore, we also found that an SNP (rs2257205) of the RNF43 X117 site was associated with overall survival (OS) instead of disease-free survival. G homozygote subjects were significantly correlated with poor OS compared with another group. Then we rescued RNF43 R117R (encoded by nucleotide CGC) and R117H (encoded by nucleotide CAC) expression in the HCT116 cells and analyzed the targets of the Wnt/ß-catenin pathway. The expression of CCND1 and c-Myc were decreased. The prognosis of CRC patients could be affected by the different functions of SNPs of RNF43.

Publisher Correction: Deacetylation of serine hydroxymethyl-transferase 2 by SIRT3 promotes colorectal carcinogenesis.

The original version of this Article contained an error in the Data Availability Statement. The accession code indicated '53V' and should have read '5X3V'. This has been corrected in both PDF and HTML versions of the Article.

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.

Deacetylation of serine hydroxymethyl-transferase 2 by SIRT3 promotes colorectal carcinogenesis.

The conversion of serine and glycine that is accomplished by serine hydroxymethyltransferase 2 (SHMT2) in mitochondria is significantly upregulated in various cancers to support cancer cell proliferation. In this study, we observed that SHMT2 is acetylated at K95 in colorectal cancer (CRC) cells. SIRT3, the major deacetylase in mitochondria, is responsible for SHMT2 deacetylation. SHMT2-K95-Ac disrupts its functional tetramer structure and inhibits its enzymatic activity. SHMT2-K95-Ac also promotes its degradation via the K63-ubiquitin-lysosome pathway in a glucose-dependent manner. TRIM21 acts as an E3 ubiquitin ligase for SHMT2. SHMT2-K95-Ac decreases CRC cell proliferation and tumor growth in vivo through attenuation of serine consumption and reduction in NADPH levels. Finally, SHMT2-K95-Ac is significantly decreased in human CRC samples and is inversely associated with increased SIRT3 expression, which is correlated with poorer postoperative overall survival. Our study reveals the unknown mechanism of SHMT2 regulation by acetylation which is involved in colorectal carcinogenesis.

T-614 Promotes Osteoblastic Cell Differentiation by Increasing Dlx5 Expression and Regulating the Activation of p38 and NF-κB.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by bone loss. Degree of inflammation has been identified as an important initiator of skeletal damage in RA. Iguratimod (T-614) is an anti-inflammatory agent which has been reported to show the inhibitory effect of bone destruction in RA. However, the role of T-614 in osteoblast differentiation is still not clear. In this study, we intended to find the effect of T-614 on the osteogenesis process. We detected osteogenesis markers and transcription factors associated with osteoblastic lineage and bone formation in the culture of mesenchymal stem cells which differentiate osteoblast. The contents and activity of alkaline phosphatase, levels of collagen type I and bone gla protein, and calcium nodule formation were increased significantly after T-614 treated. Meanwhile, the mRNAs expressions of Osterix and Dlx5 were also found to be increased significantly by real-time PCR. The changes of levels of phosphorylation of p38 and NF-κB were also detected by Western blot. The results showed that T-614 promotes osteoblastic differentiation by increasing the expression of Osterix and Dlx5 and increasing the activation of P38. T-614 could advance the ectopic expression of NF-κB to suppress inflammation, which indirectly inhibits the damage of the osteoblasts.

Phosphorylase kinase ß affects colorectal cancer cell growth and represents a novel prognostic biomarker.

PURPOSE: To study the expression and intracellular localization of phosphorylase kinase ß (PHKß) protein in colorectal cancers (CRCs), analyze its correlation with clinicopathological features and prognosis, and study the biological roles and mechanism-of-action of PHKß in CRC cell lines. METHODS: Quantitative polymerase chain reaction (qPCR) and western blot assays were performed to compare the expressions of PHKß mRNA and protein in CRC tissues and matched normal mucosa. Tissue microarrays and immunohistochemical staining were performed to detect the expression and intracellular location of PHKß protein and analyze its correlation with the clinicopathological characteristics and prognosis in CRC patients. Proliferation, cell cycle, wound healing, and xenograft models were used to elucidate the potential role of PHKß in vitro and in vivo. RESULTS: PHKß mRNA and protein were found to be overexpressed in CRC tissue compared to the levels in normal mucosa. Positive expression of PHKß was significantly correlated with TNM stage and distal metastasis, and elevated expression of PHKß was an independent prognostic factor in patients with CRC. PHKß knockdown impaired proliferation of CRC in vitro and in vivo and induced cell cycle arrest. CONCLUSIONS: PHKß affects CRC cell growth and represents a novel prognostic biomarker.

Role of transcription factor TEAD4 in carcinogenesis, development and therapy of tumors / 中国肿瘤生物治疗杂志

@# TEA转录因子4（TEAD4）是Hippo信号通路中转录共激活子YAP/TAZ下游最重要的转录因子TEAD家族的成员之 一。近年来，TEAD4的促癌作用被逐渐发现，其可与YAP形成转录复合体或不依赖于YAP独立调控下游相关靶基因的表达， 在 胃肠道肿瘤、肝癌、肺癌、乳腺癌等多种人类实体肿瘤中发挥促癌作用，导致肿瘤的发生和进展，并且是多种肿瘤不良预后的标 志。此外，靶向TEAD4及以阻断YAP-TEAD4结合为靶点的药物在多种肿瘤的体外实验及动物模型中取得显著的治疗效果， 提 示TEAD4可能是肿瘤治疗的一个理想靶点。本文就目前TEAD4在肿瘤发生、发展及治疗中的研究现状作一总结及展望，以期 为TEAD4的后续研究及以其为靶点的肿瘤治疗提供新思路。