Hypoxia-induced Serine/Threonine Kinase 33 Promotes Growth And Progression Of Pancreatic Cancer As A Critical Downstream Mediator Of HIF-1?

Tumor microenvironment is the"soil"of tumor development,which is closely related to the initiation and progression of tumor.Hypoxia is one of the most represented physical and chemical characteristics of tumor microenvironment,especially in pancreatic cancer?PC?.PC is particularly lack of blood supply,and the great proportion of the stromal component?more than 90%?also acts as drug transport barriers,which leads to the formation of hypoxic environment.Hypoxia microenvironment plays a vital role in the regulation of proliferation,invasion and metastasis of pancreatic cancer cells.Hypoxia-inducible factor 1??HIF-1??is one of the most important regulators of hypoxia microenvironment.HIF-1?and its downstream targets have been shown to be involved in the whole process of pancreatic cancer initiation,invasion,metastasis,neovascularization and drug resistance,and are closely related to the poor prognosis.Therefore,it is important to explore the target gene of HIF-1?that regulate the invasion and metastasis of pancreatic cancer,and it is clear that the identification of functional mechanism of activated signal pathways in hypoxic microenvironment is important for finding new clinical therapeutic target of pancreatic cancer.Serine/Threonine Kinase 33?STK33?belongs to the calcium/calmodulin-dependent kinase family,located on chromosome 11 of the human body.STK33 is highly expressed in normal human like testis,fetal lung and heart,and weakly expressed in human liver,stomach and panceas.STK33 has been shown to be widely and highly expressed in various cancerous tissues such as hepatocellular carcinoma,nasopharyngeal carcinoma,and lung cancer.Mechanism studies have shown that STK33 could self-phosphorylated or change the molecular function of downstream genes by phosphorylation,such as Vimentin protein.Recent studies have shown that STK33 can affect the transcriptional activity of C-myc gene in a non-kinase-dependent pathway,suggesting that STK33 contributes carcinogenesis and progression in a variety of ways.A number of studies have shown that STK33 is highly expressed in KRAS mutant cell lines and that inhibition of STK33expression significantly attenues the growth and invasion of tumor cells with the context of KRAS mutations.Therefore,STK33 is treated as an important potential target for treatment of PC which harbors high frequency of KRAS mutation.Previous studies have shown that HIF-1?triggers the activation of KRAS signaling pathway.Taking advantage of bioinformatics analysis,we found that there were multiple HIF-1?binding hypoxia responsive elements?HRE?in the promoter region of STK33.Therefore,we hypothesized that hypoxia induced STK33 promotes the malignant progression of pancreatic cancer as a critical mediator of HIF-1?.The aim of this study was to investigate the specific role of STK33 in hypoxia microenvironment of PC and to identify the contribution of STK33 to the malignant biological function of PC cells.It was found that hypoxia treatment could significantly increase the expression level of STK33 in PC cells,while STK33 could be transcriptionally regulated by HIF-1?in hypoxic environment.Functional study found that overexpression of STK33 significantly improved the proliferation and invasion of PC cells in vitro,while knockdown of STK33 significantly inhibited the proliferation and invasion of PC cells.Analysis of clinical tissue samples revealed that STK33 was significantly overexpressed in patients with PC.Clinicopathological correlation analysis confirmed a close relation of high level of STK33 expression with pathological differentiation and poor prognosis,respectively.Increased expression of STK33 in the nucleus was associated with poor prognosis.STK33 exhibit significant cancer-promoting effect and close clinical relevance,suggesting that STK33 may become a potential clinical target for PC treatment.Part I The Expression of STK33 in Pancreatic Cancer Cells was Regulated by HIF-1?under HypoxiaOBJECTIVE:To investigate the expression of HIF-1?and STK33 protein in pancreatic cancer cells under hypoxia?1%O2?,and to clarify the mutual fuctional mechanism of HIF-1?and STK33.METHODS:Real-time PCR was used to analyse STK33 mRNA levels in PC cell lines exposed to hypoxic condition?1%O₂?at 0h,6h,12h,24h and 48h in hypoxic incubator.The expression of STK33 and HIF-1?protein of PC cell lines were examinated by Western blot assay under hypoxia?1%O₂?from 0 to 48h.The mRNA levels were also detected after incubation with different concentrations of CoCl₂ after 24h nomoxic incubation following by a Western blot detection of HIF-1?and STK33.We transfected the PC cells with HIF-1?overexpression plasmid,control vector and Mock or siRNA-HIF-1?,control siRNA and Mock.Western blot assay was used to analyze the expression levels of STK33 after up-regulating or silencing HIF-1?.Double luciferase reporter assay and chromatin immunoprecipitation were utilized to determine the responsive HRE of STK33promoter mediating the transcriptional activity of HIF-1?RESULTS:After treatment of pancreatic cancer cells with hypoxia?1%O₂?,the mRNA and protein expression STK33 was significantly increased in a time dependent manner.Meanwhile,the mRNA and protein expression of HIF-1?and STK33 were increased rapidly in a dose-dependent manner in CoCl₂-treated pancreatic cancer cells.Overexpression of HIF-1?significantly up-regulated STK33,whereas knockdown of HIF-1?silenced the expression of STK33.The results of double fluorescent fluorescein reporter gene and chromatin immunoprecipitation suggest that HIF-1?is regulated by HRE2 is the critical binding site which mediated the transcriptional activation of HIF-1?on the STK33 promoter region.CONCLUSION:The expression of STK33 was regulated by HIF-1?under hypoxia environment.Part II The Biofunction of Hypoxia-induced Expression of STK33 in Pancreatic Cancer cellsOBJECTIVE:To study multiple biological functions of of STK33 in PC cells.METHODS:The relative expression of STK33 in different PC cell lines were screened by Western blot assay.The cell lines with relative high expression of STK33 were selected to be transfected with a knockdown siRNA,whereas the low STK33 PC cell lines were interfered with an overexpression plasmid.CCK8,transwell invasion and metastasis experiments and wound healing experiment were applied to observe the pancreatic cellular proliferation,migration and invasion in vitro.Under hypoxia?1%O₂?condition,the invasiveness of PC cells were observed after inhibition of intracellular STK33.Nude mice were inoculated with PC cells to observe the effect of STK33 on tumor growth.RESULTS:Overexpression of STK33 could significantly improve the proliferation,migration and invasion of PC cells in vitro.On the contrary,silencing STK33 expression could significantly inhibit the proliferation,migration and invasion of PC cells.Hypoxia treatment improved the invasive ability of pancreatic cancer cells,and blockade of STK33in hypoxic environment partially attenued the hypoxia-induced invasiveness.In vivo,the nude mice injected with STK33-overexpressed cells showed higher tumor volume and weight compared to the control and mock group,on the contrary,the tumor volume and weight in mice with down-regulated STK33 were significantly lower that the control and mock group.CONCLUSION:STK33 is a carcinogenic factor that promotes the malignant biological behavior of PC cells.Part III Expression of STK33 in Pancreatic Cancer Patients and Its Clinical SignificanceOBJECTIVE:To detect the expression of STK33 in pancreatic cancer tissues and to analyze its clinical value in judging pathological grade and evaluating prognosis.METHODS:Western blot was used to detect the expression of STK33 protein in 5pairs of pancreatic cancer and adjacent tissues,with an internal reference of?-actin to detect the differential expression.The expression levels of STK33 and HIF-1?in 98 cases of pancreatic cancer tissue samples?pancreatic cancer tissue microarray?were detected by immunohistochemical staining.The indivudal expression of HIF-1?and STK33,as well as the combined expression of STK33 and HIF-1?utilized by immunohistochemical staining to evaluate the clinical relavance of STK33 with clinicopathological parameters of PC patients.Furthermore,we will evaluate the subcellular localization of STK33 and its predictive value for clinical prognosis.RESULTS:The expression level of STK33 in malignant tissues was significantly higher than that in adjacent tissues in the tissue array.Relevance anaylysis showed that the expression of STK33 was positively correlated with the expression of HIF in 98 cases of PC tissues.Correlation analysis of STK33 with clinical prognostic parameters suggests that the expression level of STK33 is positively correlated with the pathological differentiation of PC.The overall survival of patients with high expression of either STK33 or HIF-1?were much lower than that with low expression of STK33 or HIF-1?.Furthermore,patients with both high expression of HIF-1 and STK33 had a shorter survival time than those with a single highly-expressed indicator.STK33 expressed in both the nucleus and cytoplasm,but an accumulation of STK33 in the nuleus is associated with a shorter survival.CONCLUSION:STK33 and HIF-1?protein expression were positively correlated.High expression of STK33 is closely related to the high grade pathological lesions and poor prognosis of the PC patients.STK33 subcellular localization is also an important predictor of the prognosis of PC patients.