The Effect And Molecular Mechanisms Of ABHD5 On The Sensitivity Of C-Met Inhibitors For Targeted Therapy And Immunotherapy In Colorectal Cancer

BackgroundColorectal cancer（CRC）is a highly prevalent malignancy worldwide,and metastatic colorectal cancer（m CRC）has a poor prognosis and is the leading cause of death in patients.In the past 20 years,with the leading role of systemic therapies including chemotherapy,radiotherapy,targeted therapy and immunotherapy,the prognosis of m CRC patients has improved significantly.With the advent of the era of precision medicine,targeted therapies are gradually showing a better prospect by virtue of their safety and efficacy,but then the problems such as drug resistance and non-response gradually appear,which seriously restrict their efficacy and prognosis.Research on targeted therapies has been a recent research hotspot,and breakthroughs have been made.Current targeted treatment for colorectal cancer is commonly used is represented by bevacizumab for antiangiogenic drugs and ceuximab to inhibit epidermal growth factor receptor drugs,as well as for KRAS,BRAF mutations,in addition,there is a series of molecular targeted drugs for other targets have been developed and gradually applied in clinical practice,but it is unclear which molecules can be used to predict the targeted sensitivity of the therapy.We have previously identified triglyceride（TG）deposition as an important metabolic phenotype in colorectal cancer,and metabolomic analysis has identified a classical lipolytic factor containing autolytic enzyme domain protein 5（ABHD5,also known as CGI58）deficiency in expression as an important cause of TG deposition in colorectal cancer.Our group has previously demonstrated that ABHD5 is an oncogene with metabolic activity.Moreover,it has been reported that ABHD5can play an important role in various biological behaviors of colorectal cancer,such as malignant progression and 5-Fu chemosensitivity.Our previous work showed that loss of ABHD5expression promotes Wnt signaling pathway withβ-Catenin-dependent pathway in colorectal cancer and regulates the self-renewal capacity of tumor stem cells.But whether ABHD5 is associated with targeted therapy for CRC,and whether it has the potential as a synergistic target for CRC therapy,is currently unknown.Therefore,exploring whether ABHD5 plays a regulatory role in the targeted treatment of CRC and exploring its mechanism of action will help to optimize small-molecule drugs and open up a new direction for CRC treatment targeting metabolic-related targets.c-Mesenchymal epithelial transition factor（c-Met）is a receptor-type tyrosine protein kinase,also known as hepatocyte growth factor（HGF）.As one of the downstream target genes of Wnt,c-Met is an important molecular therapeutic target.Abnormal expression of c-Met promotes the malignant progression of colorectal cancer and is a well-studied and important molecular therapeutic target,with several clinical trials of small molecule inhibitors targeting c-Met underway,but whether ABHD5 affects the sensitivity of colorectal cancer cells to c-Met inhibitors remains unclear.It is known that c-Met is the target gene of classical Wnt pathway,and YAP（Yes-Associated protein）is an essential alternative transcriptional regulator of Wnt signaling and EMT in many tumors.However,as the core molecule of Hippo signaling pathway,YAP protein is involved in the regulation of intracellular signal transduction and gene transcription,and is closely related to the malignant progression of tumors.Previous studies have established that Wnt pathway-driven tumors require YAP transcription complexes to promote proliferation and tumorigenesis,but whether ABHD5 deficiency in colorectal cancer affects the sensitivity to c-Met inhibitors in a YAP-dependent manner remains elusive.Therefore,further screening and identification of relevant molecules that play an important role in regulating the development of colorectal cancer and the efficacy of c-Met inhibitors,as well as exploring their mechanisms of function,will help optimize small molecule drugs to inhibit the development of colorectal cancer.Based on the foundation of the previous study,this project used a genome-wide library for CRISPR knockdown screening to identify ABHD5 as a key effector that determines the response of colorectal cancer cells to c-Met inhibitors,and further investigated in depth to reveal that ABHD5 expression deletion promotes c-Met expression and activation by regulating SET1A-induced YAP methylation,thereby improving the sensitivity of CRCs to c-Met inhibitors.Meanwhile,significant breakthroughs in tumor immunotherapy have been achieved.Subsequently,the problem of treatment non-response emergedin recent years.Studies have confirmed that the immunosuppressive microenvironment is the main cause of the development of colorectal cancer and the resistance to immunotherapy.The mechanisms affecting the response to immunotherapy mainly include tumor mutation load（Tumor Mutation Burden,TMB）,microsatellite instability（MSI）,PD-1/PD-L1 expression levels,IFN-γsignaling pathway,and MHC-I expression.Immunotherapy has made great progress in the treatment of melanoma and lung cancer,But in colorectal cancer,The alteration of colorectal cancer tumor microenvironment and what factors are unknown,Whether ABHD5 has an effect on the immunosuppressive microenvironment of colorectal cancer has not been reported,So we performed infiltrating immune cell analysis using mouse ABHD5 knockdown and control human colorectal cancer cell lines knockdown HCT 116 and ABHD5,Combined with TCGA database analysis,mass spectrometry flow detection and analysis data,ABHD5 deficiency was found to increase regulatory T cells（Treg）in colorectal cancer expression,It is closely related to the suppressive immune microenvironment in colorectal cancer.Intet microbiota is an important microenvironment related to human health.As an environmental molecule in close contact with the intestinal tract,many studies have confirmed that intestinal microbiosis is a key factor in the development and development of colorectal cancer.As is well known,under normal circumstances,the homeostatic intestinal flora can help the digestion and absorption of nutrients and maintain the normal physiological function of the intestinal tract.Later studies also reported that the intestinal flora plays an important role in regulating the immune process of the body.However,the impact of intestinal microbiota on the CRC immune microenvironment in the absence of ABHD5 is not clear.Therefore,clarifying whether ABHD5 deficiency changes the CRC immune microenvironment by affecting the intestinal flora,and deeply exploring the molecular mechanisms,is expected to open up a new direction for CRC treatment targeting metabolic-related targets.Therefore,we carried out a series of biological experiments through proteomics,proteome microarray,mass spectrometry flow and 16S r DNA,combining ABHD5-knockdown HCT 116 human colon cancer cell line and ABHD5 gut-specific knockout Apc^Min/+mice,and confirmed that ABHD5 deficiency significantly changed the composition of intestinal flora,and inhibited A.muciniphila by promoting Reg3A secretion,but the specific mechanism needs to be further studied.Objectives1.To expolore the effect of ABHD5 on the efficacy of molecular targeted therapy in the treatment for colorectal cancer.2.To elucidate the effect of ABHD5 on the on the sensitivity of c-Met inhibitors in the treatment for colorectal cancer and the underlying molecular mechanism.3.To investigate the molecular mechanism of the oncogenic effect of ABHD5 in colorectal cancer and to provide new targets for the treatment of colorectal cancer by targeting metabolism-related targets.4.To explore the role and molecular mechanisms of ABHD5 in the immune microenvironment and immunotherapy of colorectal cancer,and further explore the effects of molecular isoforms characterized by high and low expression of ABHD5 on the metabolic reprogramming and immune microenvironment of colorectal cancer.5.To clarify the role and mechanism of ABHD5 in regulating intestinal microflora-mediated immune microenvironment changes in colorectal cancer.Methods1.Using the sg RNA genome-wide compound library and ABHD5 knockdown and control human colon cancer cell line HCT 116,the compounds with the greatest effect on cell viability were screened.2.Based on colorectal cancer cell lines and animal models,cell viability assays,and subcutaneous transplantation tumors in mice based on cell lines and animal modelswere performed to verify the effect of ABHD 5 deficiency on c-Met inhibitor sensitivity.3.TCGA database,mouse colorectal cancer cells with high and low ABHD5 expression and their established transplant tumors,mouse models of primary intestinal cancer with intestinal Abhd5 specific knockout and knock-in Apc^Min/+mice were combined with immunohistochemistry,Western Blotting,adeno-associated virus,and mouse subcutaneous transplant tumors and metastases to further clarify ABHD5 as an important gene regulating the sensitivity of colorectal cancer cells to c-Met inhibitors.4.Using control and ABHD5 knockdown intestinal cancer cells HCT 116,Western Blotting and spatial transcriptomic assays were performed to elucidate the molecular mechanism of ABHD5 synergistically controlling yap-induced c-Met transcription.5.Western Blotting,immunohistochemistry,immunofluorescence,and clone formation assays were performed using mouse colorectal cancer cells with high and low ABHD5expression combined with LPA1-3 inhibitors to explore the effect of LPA signaling pathway on YAP methylation induced by ABHD5 deletion.6.Using TCGA database analysis,combined with mass spectrometry flow analysis and immunofluorescence,we proved the impact of ABHD5 on the immune microenvironment and the possible impact on immunotherapy in colorectal cancer.7.Based on the Apc^Min/+and Apc^Min/+Abhd ^f/f/Cre+mouse models,using the results of 16S r DNA assay combination with Co-IP,Duolink PLA,immunofluorescence,WB are used to further clarify Reg3A-A.muciniphila alterations caused by loss of ABHD5.Results1.sg RNA genome-wide compound library screening analysis identified seven c-Met inhibitors that significantly reduced the viability of ABHD5 knockdown cells（>90%）and had little effect on control cells,the most efficient of which was voritinib.2.Voritinib significantly reduced subcutaneous tumor formation and lung metastasis in ABHD5-knockdown cells with diminished cell viability and colony formation in ABHD5-knockdown HCT 116 cells.3.TCGA database analysis showed that ABHD5 and c-Met were negatively correlated in CRCs and that c-Met protein levels were significantly elevated in ABHD5 knockout HCT116colorectal cancer cells.In Apc^Min/+mice with intestine-specific ABHD5 knockout(Apc^Min/+/Abhd5^f/f/Cre+),c-Met expression was significantly increased in intestinal tumors and intestinal mucosa compared with control mice,while AAV-induced c-Met silencing significantly inhibited intestinal tumorigenesis and malignant transformation.The critical role of c-Met downstream of ABHD5 in maintaining the viability and metastatic ability of colorectal cancer cells was confirmed.4.The results of mass spectrometry analysis confirmed that phosphorylation of YAP was reduced in ABHD5 knockdown cells,whereas methylation at YAPK342 was increased in ABHD5 knockdown HCT 116 cells.the level of YAP K342-methylated was significantly higher in ABHD5 knockdown cells than in control cells.In addition,mutation of K342 significantly reduced ABHD5 deficiency-mediated YAP K342 methylation.5.SET1A silencing in ABHD5 knockdown cells significantly reduced YAP nuclear localization and TEAD expression,and Co-IP assays confirmed the interaction between SET1A and YAP,with increased interaction between SET1A and YAP in ABHD5 knockdown cells compared to controls.6.H3K4me were significantly increased in ABHD5 knockdown HCT 116 cells relative to control cells,and high-throughput sequencing and multi-component submapping analysis revealed increased H3K4me3 levels,chromatin accessibility and SET1A binding,accompanied by increased c-Met transcription.7.Silencing ABHD5 in HCT 116 cells significantly increased LPA secretion and LPA1expression,but the LPA1-3 inhibitor Ki16425 had less effect on YAP K342 methylation,localization and activation.Moreover,Ki16425 failed to reverse the c-Met expression,cell growth and invasion ability of ABHD5 knockdown HCT116 cells,suggesting that the LPA signaling pathway is not the main mechanism of ABHD5 deficient[induced YAP methylation and activation.8.TCGA database analysis revealed significantly increase of inhibitory immune cellsless in the tumor microenvironment of colorectal cancers with low ABHD5 expression compared to those with high ABHD5 expression,and the mass spectrometry flow analysis results also found increased Treg in mice with loss of ABHD5 expression.9.Loss of ABHD5 expression was associated with molecules（tumor burden that did not respond to colorectal cancer immunotherapy,tumor mutation load,MSI,MHC-I deletion）.10.ABHD5 deficiency changed the abundance,diversity,similarity,and proportion of dominant bacteria in mice.11.16S r DNA assays for analysis of ABHD5 deficiency caused reduced A.muciniphila bacteria in the intestinal microenvironment.12.ABHD5 deficiency inhibits A.muciniphila by promoting Reg3A secretion.Conclusions1.ABHD5 is an important gene that regulates the sensitivity of colorectal cancer cells to c-Met inhibitors.2.Loss of ABHD5 activates YAP signaling pathway-mediated c-Met expression to promotes colorectal cancer sensitivity to c-Met inhibitors.3.ABHD5 deletion segregates YAP in the nucleus and increases chromatin accessibility through SET1A-mediated methylation of YAP and histone H3.4.ABHD5 is strongly associated with immunotherapy resistance in colorectal cancer.5.ABHD5 deficiency affecting the homeostasis of intestinal microbiota is an important reason for suppressing anti-tumor immunity in intestinal cancer.6.ABHD5 binds to REG3A to maintain its release homeostasis,and ABHD5 deletion promotes the abnormal release activation of Reg3A is an important molecular mechanism for its inhibition of A.muciniphila.