TRIP6 disrupts tight junctions to promote metastasis and drug resistance and is a therapeutic target in colorectal cancer.

Metastasis is the primary cause of death in colorectal cancer (CRC). Thyroid hormone receptor interacting protein 6 (TRIP6) is an adaptor protein that regulates cell motility. Here, we aim to elucidate the role of TRIP6 in driving CRC tumorigenesis and metastasis and evaluate its potential as a therapeutic target. TRIP6 mRNA is up-regulated in CRC compared to adjacent normal tissues in three independent cohorts (all P < 0.0001), especially in liver metastases (P < 0.001). High TRIP6 expression predicts poor prognosis of CRC patients in our cohort (P = 0.01) and TCGA cohort (P = 0.02). Colon-specific TRIP6 overexpression (Trip6KIVillin-Cre) in mice accelerated azoxymethane (AOM)-induced CRC (P < 0.05) and submucosal invasion (P < 0.0001). In contrast, TRIP6 knockout (Trip6+/- mice) slowed tumorigenesis (P < 0.05). Consistently, TRIP6 overexpression in CRC cells promoted epithelial-mesenchymal transition (EMT), cell migration/invasion in vitro, and metastases in vivo (all P < 0.05), whereas knockdown of TRIP6 exerted opposite phenotypes. Mechanistically, TRIP6 interacted PDZ domain-containing proteins such as PARD3 to impair tight junctions, evidenced by decreased tight junction markers and gut permeability dysfunction, inhibit PTEN, and activate oncogenic Akt signaling. TRIP6-induced pro-metastatic phenotypes and Akt activation depends on PARD3. Targeting TRIP6 by VNP-encapsulated TRIP6-siRNA synergized with Oxaliplatin and 5-Fluorouracil to suppress CRC liver metastases. In conclusion, TRIP6 promotes CRC metastasis by directly interacting with PARD3 to disrupt tight junctions and activating Akt signaling. Targeting of TRIP6 in combination with chemotherapy is a promising strategy for the treatment of metastatic CRC.

METTL3 drives NAFLD-related hepatocellular carcinoma and is a therapeutic target for boosting immunotherapy.

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor of hepatocellular carcinoma (HCC). However, the mechanism and target therapy of NAFLD-HCC are still unclear. Here, we identify that the N6-methyladenosine (m6A) methyltransferase METTL3 promotes NAFLD-HCC. Hepatocyte-specific Mettl3 knockin exacerbated NAFLD-HCC formation, while Mettl3 knockout exerted the opposite effect in mice. Single-cell RNA sequencing revealed that METTL3 suppressed antitumor immune response by reducing granzyme B (GZMB+) and interferon gamma-positive (IFN-γ+) CD8+ T cell infiltration, thereby facilitating immune escape. Mechanistically, METTL3 mediates sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) mRNA m6A to promote its translation, leading to the activation of cholesterol biosynthesis. This enhanced secretion of cholesterol and cholesteryl esters that impair CD8+ T cell function in the tumor microenvironment. Targeting METTL3 by single-guide RNA, nanoparticle small interfering RNA (siRNA), or pharmacological inhibitor (STM2457) in combination with anti-programmed cell death protein 1 (PD-1) synergized to reinvigorate cytotoxic CD8+ T cells and mediate tumor regression. Together, METTL3 is a therapeutic target in NAFLD-HCC, especially in conjunction with immune checkpoint blockade (ICB) therapy.

ALKBH5 Drives Immune Suppression Via Targeting AXIN2 to Promote Colorectal Cancer and Is a Target for Boosting Immunotherapy.

BACKGROUND & AIMS: Immune checkpoint blockade therapy benefits only a small subset of patients with colorectal cancer (CRC), and identification of CRC-intrinsic events modulating immune checkpoint blockade efficacy is an unmet need. We found that AlkB homolog 5 (ALKBH5), an RNA N6-methyladenosine eraser, drives immunosuppression and is a molecular target to boost immune checkpoint blockade therapy in CRC. METHODS: Clinical significance of ALKBH5 was evaluated in human samples (n = 205). Function of ALKBH5 was investigated in allografts, CD34+ humanized mice, and Alkbh5 knockin mice. Immunity change was determined by means of flow cytometry, immunofluorescence, and functional investigation. Methylated RNA immunoprecipitation sequencing and RNA sequencing were used to identify ALKBH5 targets. Vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA was constructed for targeting ALKBH5 in vivo. RESULTS: High ALKBH5 expression predicts poor prognosis in CRC. ALKBH5 induced myeloid-derived suppressor cell accumulation but reduced natural killer cells and cytotoxic CD8+ T cells to induce colorectal tumorigenesis in allografts, CD34+ humanized mice, and intestine-specific Alkbh5 knockin mice. Mechanistically, AXIN2, a Wnt suppressor, was identified as a target of ALKBH5. ALKBH5 binds and demethylates AXIN2 messenger RNA, which caused its dissociation from N6-methyladenosine reader IGF2BP1 and degradation, resulting in hyperactivated Wnt/ß-catenin. Subsequently, Wnt/ß-catenin targets, including Dickkopf-related protein 1 (DKK1) were induced by ALKBH5. ALKBH5-induced DKK1 recruited myeloid-derived suppressor cells to drive immunosuppression in CRC, and this effect was abolished by anti-DKK1 in vitro and in vivo. Finally, vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA, or anti-DKK1 potentiated anti-PD1 treatment in suppressing CRC growth by enhancing antitumor immunity. CONCLUSIONS: This study identified an ALKBH5-N6-methyladenosine-AXIN2-Wnt-DKK1 axis in CRC, which drives immune suppression to facilitate tumorigenesis. Targeting of ALKBH5 is a promising strategy for sensitizing CRC to immunotherapy.

Targeting m6A reader YTHDF1 augments antitumour immunity and boosts anti-PD-1 efficacy in colorectal cancer.

OBJECTIVE: The role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME. DESIGN: Clinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts. YTHDF1 function was determined in syngeneic tumours, intestine-specific Ythdf1 knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulated YTHDF1-siRNA was used for YTHDF1 silencing in vivo. RESULTS: YTHDF1 expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8+ T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion of Ythdf1 augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, while Ythdf1 knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium or ApcMin/+ models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells in Ythdf1 knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8+ T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-siYTHDF1 boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC. CONCLUSION: YTHDF1 impairs antitumour immunity via an m6A-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.

METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer.

BACKGROUND & AIMS: N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS: Mettl3 knockout mice, CD34+ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS: We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells, and eventually suppressed CRC in ApcMin/+Mettl3+/- mice, CD34+ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS: Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

Loss of YTHDF1 in gastric tumors restores sensitivity to antitumor immunity by recruiting mature dendritic cells.

BACKGROUND: Gastric cancer (GC) is one of the most common cancer worldwide. We analyzed the expression of m6A regulatory genes in GC cohorts and revealed that YTHDF1 was uniquely upregulated in GC as compared with adjacent normal tissues. In this study, we analyzed the role of YTHDF1 in GC cells and modulation of the tumor immune microenvironment. METHODS: Three GC cohorts (cohort 1, n=101; cohort 2, n=278, and the Cancer Genome Atlas cohort, n=375) were analyzed for YTHDF1 expression. Function of YTHDF1 in GC was determined in GC cell lines. Role of YTHDF1 in antitumor immunity was investigated in allograft models. RESULTS: YTHDF1 is upregulated in GC compared with adjacent normal tissues, and high YTHDF1 expression was correlated with poor survival of patients with GC at mRNA (p=0.016) and protein levels (p=0.039). Loss of YTHDF1 in human (AGS, BGC823, MKN74) or mouse (YTN16) GC cell lines inhibited cell growth and colony formation in vitro. Strikingly, syngeneic YTN16 tumors with loss of YTHDF1 underwent complete remission in immunocompetent mice, while a lesser effect was found in immunodeficient mice. Consistently, YTHDF1 loss in GC tumors led to recruitment of mature dendritic cells (DCs) with increased MHCII expression and interleukin-12 (IL-12) secretion, which in turn, promoted CD4+ and CD8+ T cells infiltration with increased interferon-γ (IFN-γ) secretion. Loss of YTHDF1 mediated the overexpression of IFN-γ receptor 1 and JAK/STAT1 signaling pathway in tumor cells, which might contribute to restored sensitivity to antitumor immunity. In addition, pre-emptive exposure of YTN16 tumors with YTHDF1 loss triggered a potent antitumor immune response on rechallenge with wild-type YTN16 cells, implying that YTHDF1 loss induced a lasting systemic antitumor immunity. CONCLUSIONS: YTHDF1 is overexpressed in GC and promotes GC by inducing cell proliferation and repression of DCs-mediated antitumor immune response. YTHDF1 is a promising therapeutic target for GC treatment.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

Integrative metabolomic characterisation identifies altered portal vein serum metabolome contributing to human hepatocellular carcinoma.

OBJECTIVE: Altered metabolites are important for the tumourigenicity of hepatocellular carcinoma (HCC). We performed integrative metabolomics analysis of the metabolites changes in portal venous blood and in comparison with the metabolites changes in liver tissues and stool samples of HCC patients and healthy liver donors. DESIGN: Serum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines. RESULTS: Detailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (p<0.001). HCC patients had significantly higher levels of portal vein serum and HCC tissue metabolites of DL-3-phenyllactic acid, L-tryptophan, glycocholic acid and 1-methylnicotinamide than healthy controls, which were associated with impaired liver function and poor survival. On the other hand, HCC patients had lower levels of linoleic acid and phenol in portal vein and stool samples than healthy controls. Linoleic acid and phenol significantly inhibited HCC proliferation, inferring their anti-HCC function as protective metabolites. CONCLUSIONS: The integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development.

A novel amplification gene PCI domain containing 2 (PCID2) promotes colorectal cancer through directly degrading a tumor suppressor promyelocytic leukemia (PML).

Using whole genome sequencing, PCI Domain Containing 2 (PCID2) was identified to be amplified in colorectal cancer (CRC). In this study, we investigated the expression, biological function, molecular mechanism, and clinical implication of PCID2 in CRC. PCID2 mRNA and protein expression were higher in CRC cells and tumor tissues compared to healthy colonic tissues. The copy number of PCID2 was positively correlated with its mRNA expression. Multivariate analysis revealed that PCID2 is an independent prognostic factor for CRC recurrence. Functional studies showed that PCID2 promoted cell growth, cell cycle progression, and cell migration/invasion, while apoptosis was suppressed. Moreover, PCID2 promoted xenograft growth and lung metastasis in nude mice. Using co-immunoprecipitation and mass spectroscopy, we showed that PCID2 binds to promyelocytic leukemia (PML), a tumor suppressor involved in non-canonical ß-catenin signaling. PCID2 promoted the degradation of PML via poly-ubiquitination, which in turn, induced Wnt/ß-catenin signaling while simultaneously repressing ARF-p53 pathway. Thus, these results demonstrated that PCID2 functions as an oncogene in CRC by enhancing canonical Wnt/ß-catenin signaling and inhibition of CTNNB1-ARF-p53 axis. PCID2 promoted canonical Wnt/ß-catenin signaling in CRC via degradation of PML. PCID2 may serve as an independent prediction marker for CRC recurrence.

ZNF545 loss promotes ribosome biogenesis and protein translation to initiate colorectal tumorigenesis in mice.

Ribosome biogenesis plays a pivotal role in tumorigenesis by supporting robust protein translation. We investigate the functional and molecular mechanism of Zinc finger protein 545 (ZNF545), a transcriptional repressor for ribosomal RNA (rRNA), in colorectal cancer (CRC). ZNF545 was silenced in CRC compared to adjacent normal tissues (P < 0.0001), implying a tumor-suppressive role. Colon-specific Znf545 knockout in mice accelerated CRC in ApcMin/+ and azoxymethane/dextran sulfate sodium-induced CRC. Mechanistically, we demonstrated that ZNF545 uses its two zinc finger clusters to bind to minimal rDNA promoter, where it assembled transcriptional repressor complex by interacting with KAP1. Znf545 deletion in mouse embryonic fibroblasts not only increased rRNA transcription rate and the nucleolar size and number but also altered the nucleolar composition and architecture with an increased number of fibrillar centers surrounded by net-like dense fibrillar components. Consequently, Znf545 deletion promoted the gene expression of translation machinery, protein translation, and cell growth. Consistent with its tumor-suppressive role, ZNF545 overexpression in CRC cells induced growth arrest and apoptosis. Finally, administration of rRNA synthesis inhibitor, CX-5461, inhibited CRC development in Znf545Δ/ΔApcMin/+ mice. In conclusion, ZNF545 suppresses CRC through repressing rRNA transcription and protein translation. Targeting rRNA biosynthesis in ZNF545-silenced tumors is a potential therapeutic strategy for CRC.

RNA N6-Methyladenosine Methyltransferase METTL3 Facilitates Colorectal Cancer by Activating the m6A-GLUT1-mTORC1 Axis and Is a Therapeutic Target.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) modification has recently emerged as a new regulatory mechanism in cancer progression. We aimed to explore the role of the m6A regulatory enzyme METTL3 in colorectal cancer (CRC) pathogenesis and its potential as a therapeutic target. METHODS: The expression and clinical implication of METTL3 were investigated in multiple human CRC cohorts. The underlying mechanisms of METTL3 in CRC were investigated by integrative m6A sequencing, RNA sequencing, and ribosome profiling analyses. The efficacy of targeting METTL3 in CRC treatment was elucidated in CRC cell lines, patient-derived CRC organoids, and Mettl3-knockout mouse models. RESULTS: Using targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 dropout screening, we identified METTL3 as the top essential m6A regulatory enzyme in CRC. METTL3 was overexpressed in 62.2% (79/127) and 88.0% (44/50) of primary CRCs from 2 independent cohorts. High METTL3 expression predicted poor survival in patients with CRC (n = 374, P < .01). Functionally, silencing METTL3 suppressed tumorigenesis in CRC cells, human-derived primary CRC organoids, and Mettl3-knockout mouse models. We discovered the novel functional m6A methyltransferase domain of METTL3 in CRC cells by domain-focused CRISPR screening and mutagenesis assays. Mechanistically, METTL3 directly induced the m6A-GLUT1-mTORC1 axis as identified by integrated m6A sequencing, RNA sequencing, ribosome sequencing, and functional validation. METTL3 induced GLUT1 translation in an m6A-dependent manner, which subsequently promoted glucose uptake and lactate production, leading to the activation of mTORC1 signaling and CRC development. Furthermore, inhibition of mTORC1 potentiated the anticancer effect of METTL3 silencing in CRC patient-derived organoids and METTL3 transgenic mouse models. CONCLUSIONS: METTL3 promotes CRC by activating the m6A-GLUT1-mTORC1 axis. METTL3 is a promising therapeutic target for the treatment of CRC.

MAP9 Loss Triggers Chromosomal Instability, Initiates Colorectal Tumorigenesis, and Is Associated with Poor Survival of Patients with Colorectal Cancer.

PURPOSE: Chromosomal instability (CIN) is a common phenomenon in colorectal cancer, but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) is a critical regulator of CIN in colorectal cancer. We thus studied the effect of MAP9 loss on colorectal cancer in Map9-knockout mice and in cell lines. EXPERIMENTAL DESIGN: We generated colon epithelial-specific Map9-knockout mice and evaluated colorectal cancer development. Effect of Map9 knockout on colorectal cancer progression was determined in chemical or ApcMin /+ -induced colorectal cancer. Molecular mechanism of MAP9 was determined using spectral karyotyping, microtubule assays, and whole-genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 patients with CRC. RESULTS: Spontaneous colonic tumors (9.1%) were developed in colon epithelium-specific Map9-knockout mice at 17 months, but none was observed in wild-type littermates. Map9 deletion accelerated colorectal cancer formation both in ApcMin /+ mice and azoxymethane-treated mice, and reduced survival in ApcMin /+ mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fiber from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors, and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from Map9 knockout ApcMin /+ mice. In patients with colorectal cancer, MAP9 was frequently silenced and its downregulation was associated with poor survival. CONCLUSIONS: MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in colorectal cancer.

TRIM67 Activates p53 to Suppress Colorectal Cancer Initiation and Progression.

Tripartite motif (TRIM) family proteins participate in a variety of important cellular processes, including apoptosis, cell-cycle arrest, DNA repair, and senescence. In this study, we demonstrated that a novel TRIM family member, TRIM67, was commonly silenced in colorectal cancer and its downregulation was associated with poor survival. Trim67 knockout in ApcMin/+ mice increased the incidence, multiplicity, and burden of colorectal tumors. Similarly, colon-specific knockout of Trim67 significantly accelerated azoxymethane-induced colorectal cancer in mice. RNA sequencing revealed that the antitumor effect of TRIM67 was mediated by activation of the p53 signaling pathway. TRIM67 interacted directly with the C-terminus of p53, inhibiting p53 degradation by its ubiquitin ligase MDM2. TRIM67 was also a transcriptional target of p53; upon cellular stress, p53 bound to the TRIM67 promoter and induced significant upregulation of TRIM67, thereby forming a TRIM67/p53 self-amplifying loop that boosts p53-induced cell growth inhibition and apoptosis. Consequently, loss of this p53-positive regulatory program profoundly compromised p53-mediated responses to chemotherapy-induced DNA damage. Dampened p53 response was also observed in tumors of Trim67 knockout mice and Trim67 knockout embryonic fibroblasts. TRIM67 reactivation restored p53 activation and sensitized colorectal cancer cells to chemotherapy in vitro and in vivo. TRIM67 thus functions as a pivotal tumor suppressor in colorectal cancer and is a potential target for improving chemotherapy responsiveness. SIGNIFICANCE: The TRIM67/p53 axis represents a novel therapeutic target that could be harnessed to improve chemotherapy efficacy in colorectal cancer expressing wild-type p53 but with repressed p53 signaling.