HDAC7 Controls ATF3 Self-Directed Transcription In Colorectal Cancer Tumorigenesis

Colorectal cancer(CRC)is third globally as the most prevalent tumor,following breast and lung cancer,and second as the deadliest.Despite significant breakthroughs in diagnosis and treatments that have positively impacted the average survival rate of CRC patients in recent years,the worldwide incidence and mortality continue to rise annually,along with poor clinical outcomes and grim long-term prognoses.Despite the fact that CRC arises predominantly from the assembling of hereditary and epigenetic changes in standard colon tissue,the definite fundamental molecular systems in charge of its pathogenesis still unclear.Therefore,comprehensive research into the molecular mechanisms of CRC is paramount in identifying therapeutic targets and enabling effective diagnosis and treatment.Acetylation and deacetylation of histones are dynamic and reversible process catalyzed by HATs and HDACs,respectively.Hyperacetylation of histones has been correlated to oncogene activation,while hypoacetylation with tumor suppressor repression.A total of 18 different HDACs exist in human,which can be further subdivided into four classes: Class I HDACs(HDAC1,2,3,and 8),Class IIa(HDAC4,5,7,and 9),Class IIb(HDAC6 and 10),and Class IV(HDAC11).HDAC7,one of class IIa HDACs,plays an important role in the transcriptional regulation of genes.Although it has low activity of deacetylase,its function largely dependent on binding to class I HDACs.But their expression is significantly increased in a variety of human malignant tumors,indicating that they may play a role in the regulation of gene transcription in tumor cells through a deacetylase-independent pathway.However,the detailed molecular mechanism is still poorly understood.ATF3 is a stress response transcription factor in the ATF/cyclic AMP response element-binding family that is usually expressed at low background levels but is rapidly activated under cellular stress response conditions.ATF3 may have a dual function in mediating gene transcription.Previous reports have shown that although ATF3 expression is lower in colorectal cancer tumor specimens than in surrounding non-cancerous tissues,but promotes tumor growth and migration in HT29 colon cancer models.These results suggest that the potential dual effects of ATF3 may depend on the cell type,environment of tumor,and stage of tumorigenesis of colorectal cancer,and further studies may be needed to elucidate the mechanisms involved.Studies have reported that treatment with HDACi can increase histone acetylation,thereby increasing the expression of ATF3,and ATF3 overexpression combined with HDACi can synergistically induce apoptosis and migration of CRC cells.These results suggest that ATF3 may be regulated by HDAC proteins to play an antitumor synergistic role in CRC.The specific mechanism is still unclear and needs to be clarified.Objective:Exploring the role of HDAC7 in CRC and evaluating its epigenetic impact on the self-directed transcription of ATF3 suggests that HDAC7 has potential as a molecular therapeutic target,paving the way for innovative approaches to CRC treatment.Methods:Research on the regulatory role of HDAC7 in CRC: Utilizing TCGA database analysis to investigate the expression of HDAC7 in CRC and its impact on overall survival,disease-free survival,and different stages of CRC.HDAC7 expression was detected in human CRC tissue samples using RT-q PCR,Western Blot,and immunohistochemistry.Stable HDAC7 knockdown CRC cell lines were constructed,and the effects of HDAC7 knockdown on cell proliferation,viability,cycle,apoptosis,and migration of CRC cell lines were texted using Edu experiments,flow cytometry,colony formation assays,Transwell experiments,and xenograft models in nude mice.RNA-seq analysis was conducted to identify key genes and downstream signaling pathways changes regulated by HDAC7 in SW480 cell lines,and changes in the expression of relevant genes and downstream signaling pathway molecules were validated using RT-q PCR and Western blot.Nude mice xenograft tumor model was established to confirm that HDAC7 knockdown reduces tumor proliferation.Study on HDAC7 regulation of ATF3 self-directed transcription: The relationship between HDAC7 and ATF3,as well as the relationship between ATF3 and CRC staging and prognosis,was analyzed using GEO and GEPIA databases.Validation of HDAC7 upregulation of ATF3 expression using RT-q PCR and Western blot.Study on the dual role mechanism of self-directed transcriptional inhibition/activation of ATF3: The relationship between ATF3,HDAC7,and HDAC3 was examined through Co-IP experiments,and chromatin immunoprecipitation assays(Ch IP)were performed to determine the enrichment of ATF3,H3K27 ac,BRD4,RNA polymerase II(RNA Pol II),on ATF3 promoters or enhancers before and after HDAC7 knockdown or HDACi treatment,to determine the reasons for changes in ATF3 gene transcriptional activation and inhibition.Overexpression(plasmid)or knockdown of ATF3(si RNA method)was performed,and changes in related genes were detected using RT-PCR and Western Blot.Inhibition study: SW480 cell lines were treated with pan-HDACi SAHA and selective IIa class HDACi TMP269,and changes in related genes regulated by HDAC7 and downstream oncogenic signaling pathways were validated using RTq PCR and Western blot.Finally,the effects of these treatments on CRC cell viability,cycle,apoptosis,and migration were explored by MTT assays,flow cytometry,colony formation assays,and Transwell experiments.Results:1.HDAC7 is highly expressed in CRC tissues and correlates with poor prognosis in patients.High expression of HDAC7 in CRC is positively correlated with stages on CRC and poor prognosis;compared to adjacent normal tissues,III/IV stage human CRC tissue samples exhibit higher HDAC7 expression,Effects of HDAC7 on the proliferation,apoptosis and migration of CRC.Nevertheless,the TCGA database and human CRC tissue samples compared to adjacent tissues showed no statistic difference in the expression levels of other class IIa HDACs(HDAC4,HDAC5,HDAC9).It was also determined that there exists no obvious correlation between tumor stages and patient prognosis.2.HDAC7 promotes proliferation,survival,and migration of CRC cells in vitro and in vivo.Knockdown of HDAC7 inhibits CRC cell proliferation and migration while promoting apoptosis.Xenograft tumor models in nude mice demonstrate that knockdown of HDAC7 significantly reduces tumor size and weight in nude mice,and the tumor growth marker Ki67 also significantly decreases.3.HDAC7 inhibits ATF3 expression and activates oncogenic signaling pathways.Low expression of ATF3 normalized by HDAC7 restoration correlates with better prognosis in CRC,and ATF3 is negatively correlated with HDAC7 in III/IV stage of CRC.Knockdown of HDAC7 in SW480 cells leads to decreased levels of pPI3K(Tyr458),p-Akt(Ser473),p53 ac,p-SRC(Tyr416),and CCND1,while levels of ATF3,p21,p-YAP(Ser127),and Cleaved Caspase-3(c-Caspase-3)increased.4.HDAC7 regulates the dual role conversion of ATF3 and its self-directed gene transcription.Overexpression of ATF3 in SW480 cells increases CDKN1 A and decreases Bcl-2m RNA and protein levels,while knockdown of ATF3 reduces CDKN1 A and moderately increases Bcl-2 m RNA levels;overexpression of ATF3 inhibits the PI3K/AKT pathway.Formation of a repressive complex involving ATF3,HDAC7,and HDAC3 synergistically inhibits ATF3 transcription.Knockdown or chemical inhibition of HDAC7 leads to recruitment of transcriptional activation factors(BRD4,RNA Pol II)by ATF3,activating its own transcription.5.Inhibition of HDAC7 promotes ATF3-directed cancer cell survival suppression and cell cycle arrest.Treatment of CRC cells with HDACi inhibits their proliferation and migration,and promotes apoptosis of CRC cells.The molecular mechanisms underlying phenotypic changes were validated using RT-q PCR and Western blot.Conclusions:1.HDAC7 is highly expressed in CRC,regulating various genes and signaling pathways related to cell proliferation and apoptosis in CRC,HDAC7 promotes the proliferation,survival,and migration of CRC tumor cells were shown in vivo and in vitro.2.HDAC7 forms a repressive complex with HDAC3 and ATF3 in CRC,inhibiting ATF3 transcription,knockdown or chemical inhibition of HDAC7 did not affect ATF3 enrichment at its loci,but led to ATF3 recruitment of transcriptional activation factors at its loci,promoting its expression level,and downstream gene/protein and cell signaling pathway changes.3.ATF3 is a cellular stress factor that can accumulate in its own promoter and enhancer regions to regulate its own transcription.After stress,ATF3 protein decreases through degradation,while m RNA may decrease through self-regulated transcription.4.ATF3 can recruit different transcription factors to switch between self-directed transcription,thereby acquiring a dual transcriptional mechanism of activation or inhibition.5.HDACi can inhibit ATF3-mediated cell proliferation,block the cell cycle,promote apoptosis,and regulate the levels of a series of oncogenic pathway proteins.