Mechanistic Study Of Transcriptional Factors TFAP2C And ZNF280 Promoting Chemoresistance And Stemness Of Colorectal Cancer Via Inactivating Hippo Signaling

Background:Colorectal cancer(CRC)is the most common malignant cancer in digestive tract worldwide.Despite great progress in treatment of CRC,including 5-fluorouracil(5FU)-based chemotherapy,the combination strategies of 5-FU and oxaliplatin or irinotecan,the five-year survival rate is still dismal.In addition,the prognosis remains bleak due to late detection and diagnosis at an advanced stage of CRC.Therefore,studying the molecular mechanism of colorectal cancer will provide new strategies for improving the survival rate and quality of life of patients.The Hippo signaling pathway has been reported to be dysregulated in several biological processes and tumorigenesis of multiple human cancers.In the mammal,the Hippo pathway constitutes of four core kinase cassette components,including kinases MST1/2 and LATS1/2,as well as the adaptor proteins SAV1 and MOB1.The Hippo signaling is active via tightly balancing the activity of YAP and TAZ,to low levels through phosphorylation– ubiquitination mechanisms.While Hippo signaling is absent,unphosphorylated YAP1/TAZ enters the nucleus and induce the transcriptional activity of TEA domain(TEAD)family members(TEAD1–TEAD4)as the transcriptional co-activators,which further transcriptionally upregulate multiple downstream effectors to exert a pleiotropic role in tumor progression and metastasis.Moreover,accumulating studies has shown that the inactivation of Hippo signaling rendered resistance of cancer cells to chemotherapeutic drugs in various types of cancers.Chen et al.reported that simultaneous downregulation of MST1,LATS2,MOB1 and SAV1 by upregulation of mi R-183 c contributed to chemoresistance in pancreatic cancer;in addition,the study by Touil and colleagues showed that hyperactivation of YAP promoted resistace of colon cancer cells to 5-FU.Accordingly,the inactivation of Hippo pathway is considered as a crucial mediator in development of cancer chemoresistance.Several lines of evidence have shown that the inactivation of Hippo signaling was implicated in various processes of CRC.For example,REGγ contributed to the growth of CRC cells via directly interacting with LATS1,leading to the degradation of LATS1 and inactivation of Hippo signaling;in addition,SCC-S2 overexpression was found to promote the invasion and metastasis of CRC via depressing Hippo signaling.Thus,these studies support the notion that the inactivation of Hippo pathway plays a crucial role in the progression of CRC.Better understanding of the mechanisms underlying the inactivation of Hippo pathway may provide new insights for the development of more effective cancer therapy.The existence of cancer stem cells(CSCs)that are the minority population of cells characterized by the capabilities of self-renewal,unlimited proliferation and differentiation into the multiple lineages of cancer cells has been regarded to be responsible for the failure of chemotherapy in CRC patients,which contributes to the poor prognosis of CRC patients.Indeed,multiple studies have shown that CSCs play crucial roles in the induction and maintenance of chemotherapeutic resistance in several human cancers.Thus,improved understanding of the mechanisms that maintain CSCs properties will improve the efficacy of chemotherapy in patients with CRC via eradicating the CSC population.Activator protein-2(AP-2)factors are a conserved family of DNA-binding transcription factors in different species and five members have been identified in mammals,including AP-2α-?.AP-2 factors play important roles in embryogenesis,and interestingly AP-2 factors expression is hardly detectable in most adult tissues.However,overexpression of AP-2 factors has been observed in multiple human cancers.For example,TFAP2 A expression was elevated in nasopharyngeal carcinoma,which promoted nasopharyngeal carcinoma proliferation and growth via inducing cyclooxygenase-2 expression;in addition,TFAP2 B was reported to be overexpressed in non-small cell lung cancer(NSCLC)tissues and cell lines.TFAP2 B knockdown by si RNA significantly attenuated cell growth and induced apoptosis in NSCLC cells in vitro and in a lung cancer subcutaneous xenograft model;conversely,upregulation of TFAP2 B yielded an opposite effect.Furthermore,accumulating literatures have shown that AP-2 proteins play crucial roles in the development of therapeutic resistance in the treatment of cancers.TFAP2 A was elevated in breast cancer tissue and cell lines and more highly expressed in tamoxifen resistant tumor tissues and cell lines.High expression of TFAP2 C in breast cancer contributed to hormone resistance,which positively correlated with poor survival in breast cancer patients.Moreover,low expression of TFAP2 E due to hypermethylation was significantly associated with nonresponse to chemotherapy in colorectal cancer.Therefore,these studies have indicated that different members of AP-2 proteins have stimulatory or inhibitory affect on chemotherapeutic response in cancer treatment,which may be environment and tumor type dependent.ZNF280A,first identified through an integrative high-resolution whole-genome profiling in mantle cell lymphoma,is a member of the zinc-finger protein family,carrying a unique zinc-finger motif composed of two contiguous Cys2His2-type fingers.This Cys2His2-like fold group(C2H2)is by far the best-characterized class of zinc fingers,and it is extremely common in mammalian transcription factors.Although functioning in diverse biological roles by binding DNA or RNA and mediating protein-protein interactions,this class of zinc fingers is best known for its role in sequence-specific DNAbinding proteins via its typical tandem repeats,with two or more fingers comprising the DNA-binding domain of the protein that occupies the major groove of DNA.Numerous studies have demonstrated that different members of the zinc-finger proteins,such as ZNF746,PISA,and Snail1,have been reported to be involved in the development,progression,and metastasis of CRC,indicating that zinc-finger proteins may play an important role in CRC.Compared with these well-documented zinc-finger proteins members,reports on the biological function and role of ZNF280 A in cancers are scanty.Part Ⅰ: TFAP2 C promotes stemness and chemotherapeutic resistance in colorectal cancer via inactivating Hippo signaling Pathway Methods:TFAP2C expression was evaluated by real-time PCR,Western blot and immunohistochemistry(IHC)respectively in clinical CRC tissues.Statistical analysis was performed to explore the correlation between TFAP2 C expression and clinicopathological features,and overall and progression-free survival in CRC patients.In vitro and in vivo assays were performed to assess the biological roles of TFAP2 C in CRC cells.Western blot,luciferase and Chromatin immunoprecipitation(Ch IP)assays were used to identify the underlying pathway mediating the biological roles of TFAP2 C in CRC.Results:TFAP2C is robustly upregulated in CRC tissues and cells,and high expression of TFAP2 C correlates with advanced clinicopathological features,poor prognosis and disease progression in CRC patients.Furthermore,upregulating TFAP2 C enhances spheroids formation ability,the fraction of SP cells,expression of stem cell factors and the mitochondrial potential,and reduces the apoptosis induced by 5-fluorouracil in colorectal cancer cells in vitro,and promotes stemness and chemoresistance of CRC cells in vivo;while silencing TFAP2 C yields an opposite effect.Importantly,downregulation of TFAP2 C dramatically restores chemotherapeutic sensitivity of CRC cells to 5-FU in vivo.Our results further demonstrate that TFAP2 C promotes stemness and chemoresistance of CRC cells to 5-FU by inhibiting Hippo signaling via transcriptionally upregulating ROCK1 and ROCK2 in CRC cells.Conclusion:(1)TFAP2C promotes stemness and chemotherapeutic resistance in colorectal cancer via inactivating Hippo signaling Pathway.(2)TFAP2C may serve as a novel prognostic factor in CRC patients,and a therapeutic target for the treatment of CRC,suggesting that silencing TFAP2 C in combination with 5-FU may be an effective therapeutic strategy to improve survival in CRC patients.Part Ⅱ: ZNF280 A Promotes Proliferation and Tumorigenicity via Inactivating the Hippo-Signaling Pathway in Colorectal Cancer Methods:TFAP2C expression was evaluated by real-time PCR,Western blot and immunohistochemistry(IHC)respectively in clinical CRC tissues.Statistical analysis was performed to explore the correlation between TFAP2 C expression and clinicopathological features,and overall and progression-free survival in CRC patients.In vitro and in vivo assays were performed to assess the biological roles of TFAP2 C in CRC cells.Western blot,luciferase and Chromatin immunoprecipitation(Ch IP)assays were used to identify the underlying pathway mediating the biological roles of TFAP2 C in CRC.Results:Here we report that ZNF280 A was upregulated in CRC tissues and cells and a high expression of ZNF280 A correlated with tumor,lymph node,and metastasis(TNM)classifications,clinical stage,and predicted poor prognosis and disease progression in CRC patients.Moreover,silencing ZNF280 A repressed proliferation and induced G0 and/or G1 arrest in vitro,and it inhibited tumorigenesis of CRC cells in vivo.Our results further demonstrate that silencing ZNF280 A inhibited the proliferation of CRC cells by activating Hippo signaling.Conclusion:(1)ZNF280A Promotes Proliferation and Tumorigenicity via Inactivating the Hippo-Signaling Pathway in Colorectal Cancer(2)ZNF280A may serve as a novel prognostic factor in CRC patients,and a potential therapeutic target for the treatment of CRC.