PDK1 Promotes Tumor Growth And Metastasis Via The Notch1 Signaling Pathway In Hypophaiyngeal Carcinoma

BackgroundHypopharyngeal squamous cell carcinoma(HSCC)accounts for approximately 3%-5%of all cases of head and neck squamous cell carcinoma(HNSCC).These tumors are characterized by advanced disease with early submucosal spread and high risk of cervical metastases.Squamous cell carcinoma accounts for 95%of hypopharyngeal pathology.Within the hypopharynx are three anatomic subsites,and the pyriform sinus is the most commonly involved site.Hypopharyngeal carcinoma show a tendency to spread submucosally and are always asymptomatic until they have reached clinical advanced stage,contributing to higher rates of delayed and distant metastatic disease.Therapeutic options include chemotherapy,radiotherapy,surgery,or their combination,Notably,the five-year overall survival rate for patients with HSCC is only approximately 30%-35%and has not improved in the past few decades,mostly because patients eventually succumb to regional and distant metastasis at presentation or early in the course of the disease.Therefore,further studies on the molecular mechanism underlying HSCC progression and the identification of new effective therapeutic targets are urgently needed.3-Phosphoinositide-dependent protein kinase 1(PDK1 or PDPK1)is a pivotal transducer of phosphatidylinositol 3-kinase(PI3K)signaling,which phosphorylates protein kinase B(Akt)interacting with phosphatidylinositol 3,4,5-phosphate(PIP3,)and has been implicated in many altered signaling pathways in cancer.PDK1 is also a master regulator of many other AGC kinases,including serum/glucocorticoid regulated kinase,p70 ribosomal protein S6 kinase,p90 ribosomal protein S6 kinase and the protein kinase C family.Thus,PDK1 plays crucial roles in the regulation of cell growth,apoptosis,metastasis and survival,PDK1 is abnormally expressed and activated in various tumors and is an ideal target in multiple myeloma,prostate cancer,esophageal carcinoma and gastric carcinoma.For instance,PDK1 is overexpressed in over 40%of acute leukemia patients and is associated with poor treatment outcomes.However,the exact biological roles and underlying mechanism of PDK1 in HSCC remain undeterminedEpithelial-mesenchymal transition(EMT)is gaining considerable attention for elucidating invasive and metastatic characteristics during cancer progression.Metastasis accounts for the vast majority of cancer-associated death,but this complex process remains the least understood aspect of tumorigenic process.The dissemination of cancer cells from primary neoplasm and their subsequent seeding of distant sites involve a multi-step process known as the invasion-metastasis cascade.PDK1 modulates cancer cell migration and invasion by inducing EMT in gall-bladder cancer.Nevertheless,whether PDK1 promotes EMT and metastasis in HSCC remains unclear.On the binding of Notch ligands,Notch receptors were cleaved by a y-secretase activity The process triggers the expression of target genes,such as those of the Hairy enhancer of split(Hes)family.An increasing number of studies have implied the role of aberrant Notch1 signaling pathway in the tumorigenesis of many malignancies,including endometrial,colon and breast cancers,as well as head neck and lung carcinomas.In our previous study,the downregulation of Notch1 suppressed hypopharyngeal cancer cell migration and invasion.Moreover,Notchl regulated the metastasis of head and neck squamous cell carcinoma by inducing EMT,as well as breast cancerPDK1 regulated the basal-to-suprabasal switch in developing epidermis by acting the Notch-dependent differentiation program.PDK1 was also essential for Notch-mediated trophic and proliferative responses in thymocytes.Therefore,the potential connection between PDK1 and Notch1 in inducing EMT should be determinedConsidering the emerging importance of PDK1 in tumors,in this report,we first showed that PDK1 promoted HSCC progression through the Notch1 signaling pathway.Therefore,the combined targeting of PDK1 and Notchl signaling pathways may be a novel therapeutic strategy for treating HSCC patients.PurposeThis study aims to explore the biological function of PDK1 and to investigate the possible molecular mechanisms by which PDK1 promotes tumor metastasis.Methods1.The expression of PDK1 in HSCC tissues and clinical significance of PDK1 expression in HSCC patients(1)The protein expression of PDK1 in HSCC tissues were detected by immunohistochemistry and Western blot.Based on the immunostaining score,HSCC patients were divided into two groups:the patient with low expression and the patient with high expression.(2)To evaluate the association of PDK1 expression with clinicopathological characteristics and survival data.2.The effects of PDK1 on the proliferation,migration and invasion of HSCC in vitro(1)The FaDu cells were transfected with lentiviruses to knockdown PDK1 and/or overexpress PDK1,all these stable transfected cells were tested regularly by Western blot to confirm the efficiency of down-regulation or up-regulation.(2)CCK-8 assay and Soft Agar assay were performed to determine cancer cell viability and growth,(3)The effects of PDK1 on migration and invasion in HSCC cells were demonstrated using the Transwell migration and invasion assays.3.The effects of PDK1 on tumorigenesis and metastasis in vivo(1)For the tumor growth model,cancer cells were subcutaneously injected into the right flank of the nude mice.Tumor volume was measured using calipers and the weights were measured.(2)To check the effect of PDK1 on tumor metastasis,cancer cells were injected into the tail vein of nude mice.The lung tissues were performed using HE staining and the number of pulmonary metastasis nodules was counted under a microscope4.Study on molecular mechanism of PDK1 promoting metastasis of hypopharyngeal carcinoma(1)To investigate whether Notch1 mediated the influence of PDK1 on the biological behavior of FaDu cells,we performed rescue experiments by treating PDK1 overexpressed FaDu cells(Lv-PDK1 group)with small interfering RNA targeting Notch1(Si-Notchl)and/or y-secretase inhibitor DAPT.Western blot analysis tested the expression of EMT related factor,NICD1 and Hes1.Furthermore,migration and invasion assays were performed using Transwell assays(2)To investagate whether PDK1 activated Notch1 signaling via the Akt-dependent signaling pathway,we first treated FaDu cells with MK2206,Western blot analysis detected the expression of NICD1 and Hes1.We further treated MK2206 in PDK1-overexperssing cells,Western blot tested the expression of NICD1 and Hesl(3)To determine whether the regulation of Notch1 by PDK1 occurred at transcriptional level,real-time PCR was preformed.We next explore the relationship between PDK1 and NICD1 by Co-IP experiment.To determine whether PDK1 affected the NICD1 level by affecting NICD1 ubiquitination/degration,we examined the ubiquitination and protein level of NICD1.Furthermore,we detected the expression and intracellular distribution of PDK1 and NICD1 by immunofluorescence in HSCC tumor tissues.Results1.PDK1 expression significantly increased in HSCC tissues and was associated with tumor metastasis and prognosis among patients(1)To investigate the expression status of PDK1 in HSCC tissues,immunohistochemical(IHC)analysis showed that the expression of PDK1 was upregulated in tumors tissues.Western blot analysis further verified the results of IHC analysis.(2)We then evaluated the association of PDK1 overexpression with clinicopathological characteristics.Results showed that PDK1 overexpression was associated with lymph node metastasis,advanced clinical stage,and distant metastasis.No substantial relationship was found between PDK1 expression and other clinical features,including gender,age and T status of tumor.(3)The HSCC patients with high PDK1 expression had significantly poor clinical outcome.Further analysis showed that PDK1 overexpression was an independent prognostic factor of poor survival of patients with HSCC.2.Inhibition of PDK1 decreased HSCC cell proliferation,migration and invasion(1)To study the role of PDK1 in HSCC carcinogenesis,we generated PDK1-depleted FaDu cell line by using the lentiviral approach.PDK1 knockdown efficiency was confirmed by Western blot analysis.(2)We found that PDK1 knockdown(Lv-PDK1-RNAi group)suppressed cell viability and the colony formation frequency in soft agar.(3)Transwell assay showed that PDK1 depletion markedly reduced the migration and invasion capacity of FaDu cells.Hence,PDK1 suppressed HSCC cell proliferation,migration,and invasion.3.PDK1 overexpression promoted proliferation,migration and invasion of FaDu cells(1)We further investigated whether PDK1 overexpression affected tumor growth and metastasis.We first generated stably overexpressed PDK1 FaDu cells by lentivirus-mediated transduction.PDK1 overexpression was validated at the proteomic levels.(2)Results showed that the ectopic expression of PDK1 in the FaDu cells remarkably enhanced cell viability and anchorage-independent growth.(3)Transwell assay demonstrated that PDK1 remarkably enhanced the migration and invasion of FaDu cells.4.Knockdown of PDK1 suppressed xenograft tumor growth and tumor metastasis in mouse models(1)The xenograft mouse model was established by subcutaneously injecting stably Lv-PDK1-RNAi group cells or Lv-CON group cells into the right flanks of nude mice.During the course of six weeks,tumor volume was measured using a ruler,and tumor growth was slowed with PDK1 depletion.The expression of PDK1 in xenograft tumor decreased in the Lv-PDK1-RNAi group.The tumor weight in the Lv-PDK1-RNAi group was remarkably lower than that in the control group.(2)We injected Lv-PDK1-RNAi and Lv-CON FaDu cells into the tail vein of nude mice(five nude mice per group).After eight weeks,all mice were sacrificed and the lungs were analyzed.The mice injected with Lv-PDK1-RNAi FaDu cells formed fewer metastatic nodules than the mice injected with control cells.5.PDK1 promoted EMT and metastasis through the Notch1 signaling pathway(1)We hypothesized that PDK1 induces EMT by activating the Notch1 signaling pathway.When PDK1 was silenced,the E-cadherin was upregulated,whereas N-cadherin and Vimentin were downregulated in FaDu cells.As expected,the NICD1 and the Hesl were downregulated in Lv-PDK1-RNAi group.(2)To confirm that PDK1 affected HSCC progression through Notchl signaling,we performed rescue experiments by treating PDK1 overexpressed FaDu cells(Lv-PDK1 group)with small interfering RNA targeting Notchl(Si-Notch1)and/or?-secretase inhibitor DAPT.Western blot analysis revealed that the downregulation of Notch1 notably inhibited PDK1-induced EMT and the expression of NICD1 and Hes1.(3)Transwell assay also showed that the knockdown of Notch1 remarkably reversed the increased migration and invasion capacity induced by PDK1 overexpression.Thus,PDK1 promoted EMT and metastasis by activating the Notch1 signaling pathway.6.PDK1 activated the Notch1 signaling pathwy via Akt-independent manner(1)To further explore the molecular mechanisms of PDK1,we evaluated the possibility of PDK1 activating Notchl signaling via the Akt-dependent signaling pathway.The expression levels NICD1 and Hesl were downregulated in Lv-PDK1-RNAi group.As expected,the Lv-PDK1 group showed opposite results.To confirm that the aggressive effect of PDK1 was mediated by the activation of the Akt signaling pathway,we used the Akt inhibitor MK-2206 in FaDu cells.Unexpectedly,we did not detect a significant change in NICD1 and Hesl expression in FaDu cells upon Akt inhibitor.(2)In addition,we used the Akt inhibitor MK-2206 in Lv-PDK1 FaDu cells,we did not detect a significant decrease in NICD1 and Hes1 expression in PDK1-overexpressing FaDu cells upon Akt inhibitor.These data indicate that Notch1 signaling is not strongly regulated by PDK1/Akt axis.7.PDK1 upregulated the level of NICD1 by reducing the ubiquitination/degradation of NICD1 in cancer cells(1)We performed co-immunoprecipitation(Co-IP)analysis and observed that PDK1 can bind to NICD1,confirming the physical interaction between PDK1 and NICD1.(2)To determine whether PDK1 increased the NICD1 level by affecting NICD1 ubiquitination/degration,PDK1 decreased the ubiquitination level of NICD1(anti-NICD1 IP product),while both PDK1 expression and MG132 treatment increased the protein level of NICD1.(3)To determine the clinical relevance,HSCC specimens were subjected to IHC staining for PDK1 and Notch1.Results showed that the protein expression of Notch1 was also high in tumors,and PDK1 expression level was positively correlated with the expression levels of Notch1.We next examined the expression and intracellular distribution of PDK1 and NICD1 by immunofluorescence in HSCC tumor tissues.Confocal assays demonstrated the co-localization of PDK1 and NICD1 in the cytoplasm of the HSCC tumor cells.Conclusions1.High level of PDK1 correlated positively with tumor metastasis and indicated poor prognosis in patients with HSCC.2.PDK1 promoted growth and metastasis of HSCC cells in vitro3.Knockdown of PDK1 suppressed xenograft tumor growth and tumor metastasis in mouse models.4.PDK1 promoted EMT and metastasis through the Notchl signaling pathway.5.PDK1 interacted with NICD1.More importantly,upregulation of NICD1 by PDK1 was mediated by reducing the ubiquitination/degradation of NICD1 in cancer cells.