1.Tumor Subtyping Based On DNA Damage Repair Pathways Uncovers Prognostic Biomarkers And Immunotherapeutic Targets In Locoregional Esophageal Squamous Cell Carcinoma 2.EN1 Promotes Esophageal Squamous Cell Carcinoma Cell Proliferation And Migration Via He

The prognosis of ESCC is generally poor.Specially,a fraction of patients with locoregional ESCC often relapse rapidly and have poor prognoses,yet accurate molecular biomarkers and treatments are lacking.Here,we identify the molecular subtypes and precise prognostic biomarkers based on DNA damage repair pathways and propose new treatment strategies for locoregional ESCC patients.A total of 377 ESCC transcriptome and clinical data from our institute and other public cohorts were used.We leveraged DDR pathway gene expression profiles to delineate the major DDR subtypes using consensus clustering and identified DDR-active and DDR-silent subtypes with independent prognostic value in locoregional ESCC but not in metastatic ESCC.Cox regression revealed that BRCA1 and HFM1 alterations were significantly associated with the prognosis of locoregional ESCC and their DDR-related functions were validated by Western blotting and immunofluorescence in ESCC cell lines.Afterward,the biological features and tumor microenvironment properties underlying DDR subtyping were analyzed using ssGSEA,which demonstrated that the DDR-silent subtype was associated with an inflamed but immune-suppressed microenvironment characterized by relatively high immune cell infiltration,abnormal immune checkpoint expression and T-cell exhaustion,and enrichment of cancer-related pathways.Importantly,we proposed that concomitant GITR triggering or BTLA blockade with PD-1 blockade is an effective immunotherapy combination strategy for high-risk locoregional ESCC tumors based on TME characterization of DDR subtypes.Finally,we experimentally validated the therapeutic efficacy of treatments using syngeneic mouse models of ESCC.In conclusion,Some DDR-silent locoregional ESCCs may be identified by the expression of BRCA1 and HFM1 and responsive to anti-GITR/anti-BTLA combined with anti-PD-1 therapy.EN1 plays an important role in regulating early embryogenesis.Abnormal expression of EN1 is associated with tumorigenesis and tumor development.However,the function of EN1 in ESCC remains unclear.The purpose of this study aims to investigate the function and mechanism of EN1 in affecting ESCC cell proliferation and migration.By analyzing TCGA pan-cancer data,we found that patients with high EN1 expression had a shorter overall survival and progression-free survival than those with low EN1 expression.The GEO and GSA of ESCC data showed that EN1 was significantly overexpressed in ESCC tissues compared with paired adjacent normal tissues.These results suggest that EN1 may be involved in the tumorigenesis and development of ESCC.To clarify the functional role of EN1 in ESCC,we firstly established the stable EN1 knockdown KYSE180 and KYSE450 cells mediated by shRNA lentivirus,then examined the effect of EN1 knockdown on ESCC cell proliferation and migration using CCK-8,colony formation and Transwell assays.The results showed that knockdown of EN1 significantly inhibited the proliferation,colony formation and migration of ESCC cells.And xenograft assays showed that EN1 knockdown inhibited tumor growth in vivo.Mechanistic study demonstrated that EN1 expression was positively correlated with the major regulatory factors in Hedgehog signaling pathway,which were upregulated in ESCC tissues.RT-qPCR analysis further verified that the expression of GLI1 was obviously decreased in KYSE180 and KYSE450 cells after EN1 knockdown.Finally,the rescue results showed that the exogenous GLI1 overexpression at least partially reversed the inhibitory effect of EN1 knockdown on cell proliferation,suggesting that GLI1 is a functional downstream target of EN1 to promote ESCC cell proliferation and migration.In conclusion,EN1 activates Hedgehog signaling pathway by upregulating the expression of GLI1 to promote the proliferation and migration of ESCC cells.Thus,EN1 has been shown to exert an oncogenic function and may serve as a new potential therapeutic target for targeted therapy of ESCC.