Transcriptional Regulation Of Notch1 And Its Relationship With Chemosensitivity And Progression In Head And Neck Squamous Cell Carcinoma

Background:Head and neck squamous cell carcinoma（HNSCC）,accounting for about 95%of head and neck cancers,is the sixth most common type of cancer presently.Over 60%of patients with HNSCC present with advanced-stage disease（ie,stage Ⅲ or Ⅳ）.Surgery,radiotherapy and chemotherapy are the main means for curative management of advanced HNSCC patients,but its 5-year survival rate is still less than 50%.Chemotherapy is beneficial to preserve organ function,improve locoregional control,reduce incidence of distant metastasis and improve overall survival time.Paclitaxel,cisplatin and 5-Fu are main chemotherapeutic drugs used in HNSCC.However,chemotherapy often fails to eliminate all tumor cells due to intrinsic or acquired drug-resistance,which is the most common cause of tumor progression.Moreover,HNSCC is heterogeneous.Therefore,predicting the sensitivity of antitumor drugs before chemotherapy is helpful to individualized treatment and reduce the burden of patients.In addition,studies about antitumor drug resistance can be helpful to improve the efficacy of chemotherapy by reversing drug resistance.Aberrant Notch1 plays an essential role in cancer promotion in a variety of human tumors.The up-regulated expression of Notch1 is significantly correlated with the unfavorable prognosis of patients.Moreover,Notch1 is related to chemoresistance by regulating EMT and cancer stem cell phenotype in the process of tumor occurrence and development.However,the relationship between Notch1 and chemosensitivity and progression in HNSCC is still unclear.Our previous studies showed that the Notch1 expression in HNSCC was significantly higher than in normal squamous epithelium,and was negatively correlated with chemotherapeutic response to cisplatin.Therefore,it was necessary to study the relationship between Notchl and HNSCC chemosensitivity in vitro and in vivo,to study the relationship between highly expressed Notch1 and progression and prognosis in HNSCC.The transcriptional regulation mechanism of Notch1 should be studied in order to improve the chemosensitivity of HNSCC and inhibit tumor progression by regulating the expression of Notch1,and provide a theoretical basis for the targeted therapy of Notch1.Objective:1.To evaluate the association between Notchl and HNSCC chemosensitivity in vitro and in vivo.2.To study the relationship between Notch1 and the progression and prognosis in HNSCC.3.To analyze the promoter structure of Notch1 and explore the mechanism by which Notch1 is regulated.Methods:1.Study on the relationship between Notch1 expression and HNSCC chemosensitivity in vitro（1）Fresh HNSCC tissues were collected for primary cell culture.Then we used CD-DST to assess HNSCC chemosensitivity to paclitaxel and 5-Fu.（2）The expression of Notchl protein in HNSCC tissues was detected by immunohistochemistry.And then we evaluated the relationship between Notchl expression and chemosensitivity in vitro.（3）Human tongue cancer cell lines Tb and laryngeal squamous cell cancer primary cells（LSCC-PC）were employed for in vitro experiments.The expression of Notchl was detected by immunocytochemistry.We treated Tb cells and LSCC-PC cells with Notchl inhibitor DAPT,and then we used CD-DST to assess the changes of chemosensitivity to paclitaxel and cisplatin in these two cells.2.Study on the relationship between Notchl expression and HNSCC chemosensitivity in vivo（1）We retrospectively analyzed the clinical and follow-up data of HNSCC patients receiving induction chemotherapy.According to the inclusion criteria,patients receiving induction chemotherapy with TPF（paclitaxel+cisplatin+5-Fu）and PF（cisplatin+5-Fu）were selected respectively.The chemotherapeutic response was evaluated according to the response evaluation criteria in solid tumors（RECIST）（2）We used immunohistochemistry to assess the expression of Notch1 protein in HNSCC pre-chemotherapy tissues.And then we evaluated the relationship between Notchl expression and chemotherapeutic response.We used the Log-rank method to assess the difference in the prognosis of patients with different Notch1 expression after induction chemotherapy with TPF or PF.3.The clinical significance of Notchl expression in HNSCC（1）To evaluate the association of Notchl expression with clinicopathological parameters according to the score of immunohistochemical staining.（2）The relationship between Notchl expression and progression-free survival or overall survival was statistically analyzed to study the prognosis of HNSCC patients affected by Notchl expression.4.The effects of Notchl on the proliferation,migration and invasion of HNSCC cells in vitro（1）Knockdown of Notchl in HNSCC cells was carried out by the lentivirus infection technique,and the Notchl knockout effect was detected by qRT-PCR.（2）Cell proliferation capacity was determined by CCK-8 assay;Cell migration and invasion potential were detected by Transwell assay.5.Analysis of Notchl promoter Structure and identification of transcription factor（1）The promoter-reporter and several deleted mutants of the promoter-reporter were constructed,and the activity of the reporters was measured by firefly luciferase activity experiment.（2）The potential transcription factors that might bind to the core promoter of Notchl were predicted by the PROMO software.（3）Overexpression of Spl was carried out by the lentivirus infection technique,and then Notch1 mRNA was measured by qRT-PCR and Notchl core promoter activity was determined by firefly luciferase activity assay.Results:1.Notchl was correlated with HNSCC chemosensitivity in vitro（1）HNSCC has different chemosensitivity to paclitaxel and 5-Fu.The expression of Notch1 was negatively correlated with the HNSCC sensitivity to paclitaxel.But there was no correlation between Notchl expression and HNSCC chemosensitivity to 5-Fu.（2）CD-DST results showed that Tb cells were sensitive to paclitaxel and cisplatin,and DAPT could significantly increase Tb cells’ sensitivity to paclitaxel and cisplatin.Because Tb cells were sensitive to paclitaxel and cisplatin,we repeated the experiment using LSCC-PC cells which were resistant to paclitaxel and cisplatin,After DAPT treatment,LSCC-PC cells were turned into sensitive to paclitaxel and cisplatin detected by CD-DST.2.Notchl was correlated with induction chemotherapeutic response in vivo（1）The statistical results of induction chemotherapy cases showed that the chemotherapeutic response rate of the TPF group was significantly higher than that of the PF group.Notch1 expression in patients who did not respond to treatment was significantly higher than that in patients who responded to treatment in both the PF group and TPF group.（2）TPF group received more survival benefits than those from the PF group among all patients.After we stratified by Notch1 expression,we found that the TPF patients had significantly better survival than the PF patients did among patients without Notch1 expression,while no such similarly significant association was found among the patients with Notch1 expression.3.Notchl was associated with the malignant progression in HNSCC,and the positive expression of Notch1 suggested a poor prognosis（1）Immunohistochemistry results showed that the positive expression rate of Notch1 in HNSCC was 67%.（2）Notchl expression was negatively correlated with both T classification and N classification.There was no difference in Notch1 expression among different age,gender,T stage or differentiation degree of HNSCC.（3）The Log-rank analysis showed that patients with Notch1 expression had significantly shorter PFS and OS than those without Notch1 expression in both the PF group and TPF group.（4）The progression-free survival and overall survival were significantly shortened in patients with lymph node metastasis,advanced T stage or Notchl positive expression.Multivariable analysis showed that Notch1 expression was significantly associated with progression-free survival and overall survival.4.Knockdown Notch1 inhibited cell proliferation,migration and invasion of FaDu and CAL27 cells（1）In order to investigate whether knockdown Notch1 affected tumor progression.We first generated stably knockdown Notch1 FaDu and CAL27cells by lentivirus infection technique.Notch1 knockdown efficiency was confirmed by qRT-PCR.（2）The results of the CCK-8 assay showed that knockdown Notch1 decreased the proliferation activity of FaDu cells and CAL27 cells significantly.（3）The results of the transwell assay showed that knockdown Notch1 decreased the number of migration and invasion of FaDu cells and CAL27 cells significantly.5.Identification of the core promoter of Notch1 and transcription factors（1）The Notch1 promoter-reporter gene pGL3-notch1 and several deletion mutants were constructed.By comparing the activity of each deletion mutant,the mutant（-70/+20）possessed about 66.34%of the full-lenth promoter activity.（2）Three possible transcription factors were predicted by PROMO software,namely WT1,Pax-5 and Sp1.（3）We generated stably overexpressed Sp1 CAL27 cells by lentivirus-mediated transduction.Sp1 overexpression was validated.（4）qRT-PCR results showed that overexpression of Sp1 could upregulate Notch1 mRNA expression.（5）The results of promoter activity showed that overexpression of Sp1 could upregulate Notch1 promoter activity.Conclusion:1.Notch1 expression was negatively correlated with HNSCC response to paclitaxel.Notch1 signaling pathway inhibitor DAPT could improve the sensitivity of HNSCC cells to paclitaxel and cisplatin and reverse resistance to paclitaxel and cisplatin.2.Notch1 was related to the malignant progression of HNSCC,and the progression-free survival and overall survival of patients with Notch1 positive expression were significantly shortened.Notch1 could be used as an independent index to predict the prognosis of HNSCC patients.3.TPF induction chemotherapy was superior to PF induction chemotherapy in chemotherapy response and prognosis,but for patients with Notch1 positive expression,TPF induction chemotherapy was not superior to PF in OS.4.Notch 1 promoted the proliferation,migration and invasion of HNSCC cells in vitro.5.The core promoter of Notchl was located in the-70/+20 region.Transcription factor Spl was an important regulator of Notch1,which regulated its expression at the transcriptional level.