The Role And Mechanism Of HSPA2 In The Invasion And Metastasis Of Pancreatic Cancer

Background:Pancreatic carcinoma is one of the deadliest solid malignancies in human beings,which is extremely aggressive,rapidly progressive and has a very poor prognosis.Global malignancy epidemiology data indicate that more than 490,000 people worldwide have developed pancreatic carcinoma and over 460,000 have died from it in 2020,ranking it seventh in mortality among all malignancies.Cancer epidemiology data for the United States indicate that more than 60,000 new cases of pancreatic cancer and nearly 50,000 deaths from pancreatic cancer are estimated to occur in the United States in 2022,making it the fourth deadliest of all cancers in the country.The incidence of pancreatic cancer varies widely around the world,with the highest incidence in Western Europe and North America,and the lowest in Central Africa and South-Central Asia.Compared to developing countries,the incidence of pancreatic cancer is generally higher in developed countries and is on a gradual increase.The mortality rate of pancreatic cancer remains stable or on the rise in most countries,and the disease is expected to rank third in mortality among all cancer types in European countries by 2025,and is expected to rank second in mortality among all cancer types in the United States by 2030.Currently,radical surgical procedures remain the most effective therapeutic option for pancreatic cancer.However,due to the characteristics of high malignancy,rapid progression and insidious onset,pancreatic cancer is often in an intermediate to advanced stage when it is first detected,and the majority of patients have missed the chance to receive radical surgical procedures.In addition,pancreatic cancer cells are not sensitive to conventional radiotherapy and chemotherapy,and there are currently no targeted and immunotherapeutic drugs with significant efficacy,which is also an important reason for the poor prognosis of pancreatic cancer.Despite the fact that some advancements have been achieved in the diagnosis and management of pancreatic carcinoma,the long-term survival rate of patients has not yet been substantially improved.Therefore,identifying specific biomarkers for pancreatic cancer and exploring the molecular mechanisms of pancreatic cancer progression are of great importance for developing new therapeutic approaches and improving patient prognosis.Heat shock proteins(HSPs)are a group of proteins produced by cells in response to stress factors and often function as molecular chaperones.HSPs are intimately involved in tumorigenesis and tumor progression,and play a very prominent role in the proliferation,apoptosis,invasion,angiogenesis and metastasis of tumor cells.The HSPs family is further subdivided into several subfamilies based on the molecular weight of their proteins and the homology of their amino acids.HSP70s is the most important one,with a molecular weight of about 70-kDa.HSPA2,also known as HSP70-2,is one of the important members of the HSP70s family.Its coding gene is located on chromosome 14(14q24.1),and the amino acid sequence of its protein shares high similarity with those of others in the family.HSPA2 was originally found in male germ cells and is closely associated with sperm production.Reduced expression of HSPA2 leads to meiotic arrest of primary spermatocytes,resulting in impaired spermatogenesis and male sterility.Recent studies have found that HSPA2 has an intimate correlation with the genesis and progression of malignant tumors.In cervical cancer,bladder cancer,esophageal squamous cancer,non-small cell lung cancer,hepatocellular carcinoma,colorectal cancer and breast cancer,the expression level of HSPA2 was significantly increased in tumor specimens compared with corresponding non-tumor specimens.Increased HSPA2 expression is strongly associated with reduced survival of tumor patients and is a risk factor for poor prognosis.Studies have shown that inhibition of HSPA2 expression in tumor cells reduces the proliferation,migration,and invasive ability of tumor cells,promotes apoptosis,and leads to cell cycle arrest.Previous studies have reported that HSPA2 expression levels were significantly upregulated in pancreatic cancer tissues relative to normal tissues of the pancreas,and that HSPA2 upregulation was significantly associated with shorter survival times in pancreatic cancer patients.However,the role and mechanism of HSPA2 in the progression of pancreatic cancer remains unclear.Therefore,in this study,we investigated the role and molecular mechanisms of HSPA2 in the invasion and metastasis of pancreatic cancer.The current study includes the following three components.Part Ⅰ Expression and clinical value of HSPA2 in pancreatic cancer tissuesObjective:To assess the expression of HSPA2 in pancreatic carcinoma tissues and its relevance to clinicopathologic characteristics,early postoperative relapse and survival.Methods:The mRNA expression profile data were downloaded from the Cancer Genome Atlas(TCGA)database,followed by the extraction of HSPA2 mRNA expression data by Perl software,and the differences in HSPA2 mRNA expression between pancreatic cancer specimens and normal pancreatic specimens were analyzed by R software.Fresh specimens from 20 pancreatic cancer patients were collected,and the expression levels of HSPA2 mRNA in 20 pairs of pancreatic cancer specimens and matched paracancer specimens were detected by real-time quantitative polymerase chain reaction(RT-qPCR)assay.Paraffin specimens and clinical data of 85 pancreatic cancer patients were collected,and the expression of HSPA2 protein in pancreatic cancer and adjacent paraneoplastic tissues was detected by immunohi stochemi stry.The link between HSPA2 expression and clinicopathologic characteristics of pancreatic cancer was assessed using chi-square test.The predictive value of HSPA2 expression on early postoperative relapse of pancreatic cancer was estimated through logistic regression analysis,receiver operating characteristic(ROC)curve analysis and decision curve analysis(DCA).The effect of HSPA2 expression on recurrence-free survival(RFS)and overall survival(OS)of pancreatic cancer patients was estimated by Kaplan-Meier method and log-rank test.Risk factors affecting survival of pancreatic cancer patients were determined by Cox regression model.Results:Analytical data from both the TCGA dataset and RT-qPCR assay showed that HSPA2 mRNA was markedly upregulated in pancreatic cancer specimens relative to the corresponding normal pancreas specimens.Immunohi stochemi cal analysis demonstrated that HSPA2 expression was strongly enhanced in pancreatic cancer tissues relative to adjacent normal pancreas tissues.HSPA2 expression was markedly linked to aggressive clinicopathological features such as high serum CA19-9 levels,poor tumor differentiation,lymph node metastasis,peripheral nerve invasion and advanced tumor stage.Logistic regression analysis showed that HSPA2 upregulation was an independent predictor of early postoperative relapse of pancreatic cancer.ROC curve analysis and DCA results showed that the combination of HSPA2 expression and aggressive clinicopathological features was more reliable for the prediction of early postoperative recurrence and had greater application value.Survival curves revealed that RFS and OS were significantly shorter in pancreatic cancer patients with high-expressed HSPA2 relative to those with low-expressed HSPA2.Cox regression analysis indicated that highly expressed HSPA2 was an independent predictor of shortened RFS and OS.Survival curves indicated thatConclusion:Taken together,HSPA2 was significantly highly expressed in pancreatic cancer tissues and significantly correlated with malignant clinicopathological features.Upregulated HSPA2 was an independent predictor of early recurrence after surgery for pancreatic cancer.The combination of HSPA2 expression and aggressive clinicopathological characteristics showed better predictive performance and higher potential clinical value in predicting early postoperative recurrence.Furthermore,high HSPA2 expression was associated with both reduced RFS and OS,and was an independent risk marker for shorter RFS and OS.Our results suggest that HSPA2 is a promising prognostic marker for pancreatic cancer and plays an important role in pancreatic cancer progression.Part Ⅱ Effect of HSPA2 expression on the invasion and metastasis of pancreatic cancerObjective:To investigate the effects of HSPA2 on the proliferation,apoptosis,invasion and migration of pancreatic carcinoma cells in vitro,and the effects of HSPA2 on the growth and metastasis of pancreatic carcinoma in vivo.Methods:The expression levels of HSPA2 protein in human pancreatic cancer cells BxPC-3,PANC-1 and SW1990 were detected by Western Blotting.Specific siRNA against HSPA2 sequence and control siRNA were transfected into BxPC-3 cells to silence HSPA2 expression,and HSPA2 overexpression plasmid and control null plasmid were transfected into PANC-1 cells to upregulate HSPA2 expression.The effects of altered HSPA2 expression on the proliferative,apoptotic,migratory and invasive capacities of pancreatic carcinoma cells were then assessed through CCK-8 assay,flow cytometry and Transwell assay,respectively.The effect of altered HSPA2 expression on the expression levels of epithelial-mesenchymal transition(EMT)-related molecular markers in pancreatic cancer cells was assessed by Western Blotting.The HSPA2 overexpressing lentivirus(Lenti-HSPA2)and the control lentivirus(Lenti-control)were transferred into PANC-1 cells to construct HSPA2 stable overexpressing pancreatic cancer cells.The successfully constructed HSPA2 stably overexpressed PANC-1 cells and control cells were then inoculated subcutaneously in nude mice to generate transplanted tumour models for pancreatic cancer in nude mice,and the volume and weight of transplanted tumors in the two groups were compared.Lung metastasis model of pancreatic cancer was established by tail vein injection of HSPA2 stably overexpressing PANC-1 cells and control PANC-1 cells in nude mice,and the counts of lung metastatic lesions was compared between the two groups.Results:The altered expression of HSPA2 had no significant influence on the proliferation and apoptosis of pancreatic cancer cells.Silencing of HSPA2 significantly impaired the migration and invasion capabilities of BxPC-3 cells,and upregulation of HSPA2 prominently increased the migration and invasion capacities of PANC-1 cells.In addition,reduction of HSPA2 expression resulted in significant upregulation of E-cadherin and significant downregulation of ZEB1,N-cadherin,Vimentin,Slug,Snail and Twistl in BxPC-3 cells.Overexpression of HSPA2 caused significant downregulation of E-cadherin,and significant upregulation of ZEB1,N-cadherin,Vimentin,Slug,Snail and Twistl in PANC-1 cells.In vivo experiments demonstrated that the volume and weight of subcutaneous transplanted tumours were observably larger in the HSPA2 overexpression group than in the control group in nude mice.The number of metastatic tumor lesions in the lungs of nude mice in the HSPA2 overexpression group was remarkably greater than that in the control group.Conclusion:In summary,HSPA2 expression potentiated the migratory and invasive capabilities of pancreatic carcinoma cells in vitro and promoted the growth and metastasis of pancreatic cancer in vivo.Furthermore,inhibition of HSPA2 expression caused elevated expression of E-cadherin and diminished expression of ZEB1,N-cadherin,Vimentin,Slug,Snail and Twist1.Upregulation of HSPA2 resulted in a reduction in E-cadherin expression and an enhancement in ZEB1,N-cadherin,Vimentin,Slug,Snail and Twistl expression.Our findings suggest that HSPA2 has a significant role in the infiltration and metastasis of pancreatic cancer and may confer potent migratory and invasive abilities to pancreatic cancer cells through EMT.Part Ⅲ The role and mechanism of HSPA2/ANGPT2/ERK pathway in the invasion and metastasis of pancreatic cancerObjective:To investigate the role and mechanism of HSPA2 in the invasion and metastasis of pancreatic cancer via the regulation of ANGPT2-mediated ERK signaling pathway.Methods:After transfecting PANC-1 cells with HSPA2 overexpression plasmid and control null plasmid,total RNA was extracted,2/3 samples were used for mRNA sequencing analysis,and 1/3 samples were used for subsequent RT-qPCR experiments to verify the mRNA sequencing results.Differential genes between the PANC-1 cells with overexpressed HSPA2 and the control PANC-1 cells were screened by mRNA sequencing analysis,and genes that were significantly differentially expressed were selected for enrichment analysis.Then the 10 genes with the most significant differential expression were selected and validated by RT-qPCR assay.After identifying ANGPT2 as the target gene of HSPA2,the expression difference of ANGPT2 mRNA between pancreatic cancer tissues and normal pancreas tissues and its correlation with HSPA2 expression were analyzed by GEPIA2(Gene Expression Profiling Interactive Analysis 2)database.The relationship between ANGPT2 expression and survival of pancreatic cancer patients was estimated by Kaplan-Meier Plotter,UALCAN and DriverDBv3 databases.The expression levels of ANGPT2 in PANC-1 cells with HSPA2 overexpression and in BxPC-3 cells with HSPA2 silencing were detected by Western Blotting.We performed rescue experiments by transfecting BxPC-3 cells with HSPA2-specific siRNA along with ANGPT2 overexpression plasmid and PANC-1 cells with HSPA2 overexpression plasmid along with ANGPT2-specific siRNA.Then the expression levels of ANGPT2 and EMT-related indicators(three essential markers)as well as the phosphorylation of ERK were detected by Western Blotting,and the migration and invasion capabilities of the cells were detected by Transwell assay.Results:The mRNA sequencing results showed that there were 115 upregulated genes and 80 downregulated genes in the HSPA2 overexpression group compared to the control group.Combined with the validation results of RT-qPCR for the 10 most significantly differentially expressed genes and with reference to the results of enrichment analysis,ANGPT2 was finally identified as the target gene of HSPA2.Data from the GEPIA2 server showed that ANGPT2 mRNA was significantly upregulated in pancreatic cancer specimens relative to normal pancreas specimens and correlated significantly positively with HSPA2 expression.Survival analysis indicated that high ANGPT2 expression was significantly associated with reduced survival.Western Blotting showed that upregulating HSPA2 increased ANGPT2 expression in PANC-1 cells and downregulating HSPA2 decreased ANGPT2 expression in BxPC-3 cells.Rescue experiments demonstrated that upregulation of ANGPT2 partially reversed the inhibitory effect of HSPA2 silencing on EMT in BxPC-3 cells,and downregulation of ANGPT2 partially reversed the promotional effect of HSPA2 overexpression on EMT in PANC-1 cells.Moreover,immunohistochemistry results showed that HSPA2 overexpression increased ANGPT2 expression in subcutaneous transplanted tumors of nude mice.Conclusion:In conclusion,HSPA2 influences the invasion and metastasis of pancreatic cancer by regulating the expression of ANGPT2.Mechanistically,HSPA2 activates the ERK signaling pathway via upregulation of ANGPT2 expression,which contributes to the EMT of pancreatic cancer cells.Our findings suggest that the HSPA2/ANGPT2/ERK pathway plays a significant role in the progression of pancreatic cancer.