The Role And Mechanism Of PKD3 In Regulating The Expression Of PD-L1 And Invasion And Metastasis Of Oral Squamous Cell Carcinoma

Background:Oral squamous cell carcinoma(OSCC)is the most common malignant tumor of the oral mucosa,and more than 550,000 cases occur every year worldwide.About 90% of oral malignant tumors belong to squamous cell carcinoma or one of its variants.It is characterized by rapid growth,strong invasiveness,early cervical lymph node metastasis and a high rate of metastasis.Although great progress has been made in treatment methods such as surgery,chemotherapy,and radiation therapy in recent years,the prognosis of OSCC is still poor,and the overall mortality rate of patients with OSCC has remained constant over the past few decades,at approximately 50%.PD-L1(programmed cell death 1 ligand 1),also known as CD274(cluster of differentiation 274)or B7-H1(B7 homolog 1),is an important target of tumor biotherapy.The PD-L1/PD-1 signaling pathway is an important component of tumor immunosuppression,which can inhibit the activation of T lymphocytes,induce the depletion of tumor reactive T cells,promote the immune tolerance of tumor cells,and thus achieve tumor immune escape.Moreover,it is closely related to the development and prognosis of tumor.PD-L1 is widely expressed on tumor cells and their stromal cells.Although inflammatory factors and chemoradiotherapy have been known to induce its expression,the regulatory mechanism of its expression in tumor cells is not completely clear.Although tumor biotherapeutics targeting PD-L1 has achieved remarkable results,it still doesn’t have significant and effective effects on some patients.Protein kinase D(PKD)is a serine / threonine kinase discovered in recent years.It includes three family members: PKDμ/PKD1,PKD2,and PKDν/PKD3.They are involved in cell proliferation,invasion,differentiation,apoptosis,angiogenesis,tumorigenesis,protein secretion,epithelial mesenchymal transition(EMT)and immune escape.Among them,PKD3 participates in multiple oncogenic signaling pathways,such as Erk1/2,NF-κB,STAT1,and STAT3.PKD3 is abnormally expressed and accumulated in the nucleus in various tumors,which is closely related to tumor classification,immune escape,invasion and metastasis,and EMT.This project explores the influence of PKD3 on OSCC cell growth,invasion and metastasis,the expression of immune escape-related protein PD-L1,and its regulatory mechanism through clinical tissue samples,UALCAN database,UCSC Xena and TCGA database analysis,and in vitro and in vivo experiments.Moreover,this project explored the antitumor activity of small molecule PKD inhibitor CRT0066101 in vivo,providing new ideas and new targets for breaking the bottleneck of clinical targeted PD-L1 biotherapy.Objective:1.To explore the role and mechanism of PKD3 in regulating the expression of PD-L1 in OSCC2.Role and mechanism of PKD3 in regulating OSCC growth,invasion and migration,and EMT3.To verify the function of PKD3 in promoting OSCC growth,EMT,invasion and metastasis in vivo4.To verify the anti-tumor activity of CRT0066101 in OSCC tumor-bearing modelMethod:1.The expression levels of PKD3,PD-L1 and EMT markers and their correlation were analyzed by Western blot,immunofluorescence and immunohistochemistry(IHC),and further validated using the TCGA(The Cancer Genome Atlas)database.2.Western blot was used to detect the expression of PD-L1 induced by inflammatory factors IL-1β,IL-6,TNF-α and IFN-γ in OSCC.3.Western blot,flow cytometry,and Q-PCR were used to study the molecular mechanism of PKD3 for regulating PD-L1 expression.4.The effects of PKD3 on the growth,invasion and migration of OSCC cells were observed by CCK-8,cell clone formation,wound healing assays and Transwell assays,respectively.5.Western blot was used to study the influence of PKD3 on tumor EMT marker protein and its related molecular mechanism.6.PKD3 deficient OSCC cell line was inoculated subcutaneously on the foot pad of nude mice to establish the animal model of transplanted tumor.To observe the role of PKD3 in the expression of PD-L1 and the invasion and metastasis of OSCC.7.The mice oral squamous cell carcinoma cell SCC-7 was used to establish the subcutaneous tumor model of the foot pad to explore whether the PKD inhibitor CRT0066101 can affect tumor growth,metastasis and the expression of PD-L1 and EMT marker proteins like silencing PKD3.Result:1.The expression of PKD3 is closely related to PD-L1 and EMT markers in OSCC.Western blot,immunofluorescence,immunohistochemistry(IHC),and TCGA database analysis showed that the expression levels of PKD3 and PD-L1 in oral squamous cell carcinoma were increased,and there was a significant positive correlation.PKD3 and PD-L1 were positively correlated with mesenchymal markers and negatively correlated with epithelial markers.PKD3 expression level,nuclear distribution and Vimentin expression level were significantly positively correlated with tumor pathological grade.2.The level of PD-L1 expression decreased following the silencing of PKD3 and that the ability of IFN-γ to induce PD-L1 expression was also decreased in OSCC.The opposite phenomenon occurred following the overexpression of PKD3.Control sh RNA and PKD3 sh RNA plasmids were used to construct the model of PKD3 deficient OSCC cells.The level of PD-L1 expression decreased,and the ability of IFN-γ to induce PD-L1 expression decreased in OSCC cells.DOK cells were transfected with PKD3 eukaryotic expression plasmid to establish a PKD3over-expressing cell model.The level of PD-L1 expression increased slightly,but the ability of IFN-γ to induce PD-L1 expression was significantly increased.3.PKD3 regulates the expression of PD-L1 by regulating the phosphorylation level of STAT1/STAT3 in OSCC cells.Western blot was used to detect key node proteins that regulate PD-L1 signaling pathway.It was found that silencing PKD3 can reduce the phosphorylation of STAT1/STAT3 in OSCC cells.After further silencing STAT1 and STAT3 with si RNA,the expression level of PD-L1 was significantly reduced,and the ability of IFN-γ to induce PD-L1 expression was decreased.4.PKD3 promotes OSCC cell growth,invasion and migration.The cell growth,invasion and migration ability were weakened when PKD3 was silenced by sh RNA in OSCC cells.Over expression of PKD3 enhanced cell growth,invasion and migration.5.PKD3 regulates the expression of EMT-related proteins.Western blot was used to detect the expression of EMT-related proteins.After silencing of PKD3,it was found that the expression level of E-cadherin in OSCC cells was significantly increased,and the expression levels of Snail,N-cadherin and Vimentin were significantly reduced.After overexpression of PKD3,Snail,N-cadherin And Vimentin expression levels increased significantly,while E-cadherin expression levels decreased.6.PKD3/PD-L1 positive feedback loop drives EMT through ERK/STAT1/3signals to promote OSCC cell growth and metastasis.Silencing PKD3,the phosphorylation levels of ERK1/2,STAT1,and STAT3 were significantly reduced,and the expression level of PD-L1 was reduced.After silencing PD-L1,the expression level of PKD3 was reduced,and the phosphorylation levels of ERK1/2,STAT1,and STAT3 were also decreased.Using si-RNA to silence ERK1/2,STAT1,and STAT3,respectively,we found that they could block EMT induced by PD-1/PD-L1 interaction.7.The mouse footpad xenograft model was used to study the promoting effects of PKD3 on tumor growth,EMT and lymph node metastasis in vivo.In order to validate the biological function of PKD3 in vivo,we first established footpad xenograft model in nude mouse.We found that silencing PKD3 inhibited the expression of PD-L1 and mesenchymal markers(Snail,Vimentin and N-cadherin),and promoted E-cadherin expression in subcutaneous xenografts of the footpad.Moreover,silencing PKD3 significantly reduced the proliferation capacity and lymph node metastasis rate of OSCC.8.The subcutaneous tumor-bearing model in the footpad of C57 mouse was used to study the inhibitory effect of PKD inhibitor CRT0066101 on the growth,metastasis and EMT of OSCC cells.We first established subcutaneous tumor-bearing model in the mouse footpad.We found that the growth ability and lymph node metastasis rate of OSCC cells treated with CRT0066101 were significantly decreased compared with the Con group,as well as the expression of PD-L1 and mesenchymal markers(Vimentin,Snail and N-cadherin)was reduced,and the expression of E-cadherin was elevated.the results of using CRT0066101 were similar to silencing PKD3.Conclusion:We discovered and reported that the expression of PKD3 was evaluated in OSCC,and its overexpression could promote the growth,EMT,invasion and metastasis of OSCC in vivo and in vitro.The detailed regulation mechanism is as follows: PKD3 can regulate PD-L1 expression through ERK/STAT1/3,and there is a positive feedback loop between PKD3 and PD-L1 to drive EMT,thereby promoting the growth,invasion and metastasis of OSCC.