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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. Serine hydroxymethyltransferase 2 (SHMT2) converts serine to glycine in mitochondria and is upregulated in a variety of cancers. Here the authors show that acetylation of the lysine-95 (K95) residue negatively regulates SHMT2 expression and activity and is deacetylated by SIRT3 in colorectal cancer.

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. Hippo signalling is often deregulated in cancers. Here the authors show that CK2 enhances the cooperation of HHEX with YAP-TEAD complex to promote colorectal tumorigenesis.

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.

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 β3 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.

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. The role of CCBE1 in tumor lymphangiogenesis and lymphatic metastasis was investigated using human lymphatic endothelial cells (HLECs) model , and a hindfoot lymphatic metastasis model . 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. : Cancer cell-derived CCBE1 enhances VEGFC proteolysis , facilitates tube formation and migration of HLECs , and promotes tumor lymphangiogenesis and lymphatic metastasis . 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-β) 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-β pathway correlates with increased CCBE1 expression in CRC. : 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-β signaling promotes CRC metastasis.

The role of processing bodies (P-bodies) in tumorigenesis and tumor progression is not well understood. Here, we showed that the oncogenes YAP/TAZ promote P-body formation in a series of cancer cell lines. Mechanistically, both transcriptional activation of the P-body-related genes SAMD4A, AJUBA , and WTIP and transcriptional suppression of the tumor suppressor gene PNRC1 are involved in enhancing the effects of YAP/TAZ on P-body formation in colorectal cancer (CRC) cells. By reexpression of PNRC1 or knockdown of P-body core genes ( DDX6, DCP1A, and LSM14A ), we determined that disruption of P-bodies attenuates cell proliferation, cell migration, and tumor growth induced by overexpression of YAP 5SA in CRC. Analysis of a pancancer CRISPR screen database (DepMap) revealed co-dependencies between YAP/TEAD and the P-body core genes and correlations between the mRNA levels of SAMD4A, AJUBA, WTIP, PNRC1, and YAP target genes. Our study suggests that the P-body is a new downstream effector of YAP/TAZ, which implies that reexpression of PNRC1 or disruption of P-bodies is a potential therapeutic strategy for tumors with active YAP.

The folate-coupled metabolic enzyme MTHFD2 (the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase) confers redox homeostasis and drives cancer cell proliferation and migration. Here, we show that MTHFD2 is hyperacetylated and lysine 88 is the critical acetylated site. SIRT3, the major deacetylase in mitochondria, is responsible for MTHFD2 deacetylation. Interestingly, chemotherapeutic agent cisplatin inhibits expression of SIRT3 to induce acetylation of MTHFD2 in colorectal cancer cells. Cisplatin-induced acetylated K88 MTHFD2 is sufficient to inhibit its enzymatic activity and downregulate NADPH levels in colorectal cancer cells. Ac-K88-MTHFD2 is significantly decreased in human colorectal cancer samples and is inversely correlated with the upregulated expression of SIRT3. Our findings reveal an unknown regulation axis of cisplatin-SIRT3-MTHFD2 in redox homeostasis and suggest a potential therapeutic strategy for cancer treatments by targeting MTHFD2.

Several studies suggest that metformin has the potential effect of reducing cancer risk. However, its survival benefit in patients with colorectal cancer (CRC) and diabetes is unknown. The aim of our study is to address the effect of metformin on outcomes for CRC based on a systematic review and meta-analysis. We searched EMBASE and MEDLINE databases from inception through August, 2013, using search terms related to metformin, diabetes, colorectal cancer, and prognostic outcome. The outcome measures were hazard ratios (HRs) with 95% CIs comparing CRC survival in diabetic patients using metformin and without using metformin. The primary end points were overall survival (OS) and CRC specific survival (CS). A total of six cohort studies including 2,461 patients met full eligibility criteria. The pooled HR favoring metformin users was 0.56 for OS (95% CI, 0.41 to 0.77) and 0.66 for CRC-specific survival (95% CI, 0.50 to 0.87). Thus metformin therapy reduced the risk of all cause of death by 44% and the risk of CRC specific death by 34% in CRC patients compared to those in non-users. However, evidence of heterogeneity and possible publication bias was noted for OS. Patients with CRC and diabetes treated with metformin appear to have an improved survival outcome. Prospective study should be warranted to examine the association between metformin exposure intensity as well as some other confounding variables and survival outcome in diabetic CRC patients.

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.

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 ( K d  = 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 IC 50 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. The adenomatous polyposis coli (APC)–Asef protein interaction is essential for colorectal cancer metastasis. Here, the authors present the rational design of a sensitivity-enhanced tracer for fluorescence polarization assays, enabling them to discover more efficient APC–Asef interaction inhibitors.