Role And Mechanism Study Of Heat Shock Factor 1(HSF1)on Malignant Biological Behavior Of Osteosarcoma

Background:Osteosarcoma is one of the most common malignant tumors in the bones,mainly occurs in children and adolescents.There is a slightly higher incidence for men than women.It accounts for 2.4% of all malignant tumors in pediatric patients.Osteosarcoma usually occurs in the extremities,including the distal femur(30%),the proximal tibia(15%),and the proximal tibia(15%).The standard treatments for osteosarcoma are neoadjuvant chemotherapy,surgical resection and postoperative radiotherapy or chemotherapy.Although this comprehensive treatment strategy improves the prognosis of patients with local osteosarcoma,there is still a lack of effective treatment strategies for patients with metastatic or recurrent osteosarcoma,and their expected survival rate is still low.Heat shock factor 1(HSF1)is a protein encoded by the hsf1 gene.HSF1 is highly conserved in eukaryotes and is a major mediator of stress transcriptional responses.Additionally,HSF1 also plays an important role in non-stress regulation such as development and metabolism.The heat shock response protects cells by regulating the correct folding and distribution of proteins within the cell.HSF1 can trans-activate many genes involving in DNA damage repair and metabolism.This suggests that HSF1 has multiple effects,not only in heat shock response,but also in aging and diseases.Recent studies have shown that HSF1 is activated and highly expressed in many malignant tumors,and high expression of HSF1 has been shown to be associated with poor prognosis of various solid tumors.Therefore,the expression of HSF1 in tumors has important clinical significance with the prognosis of tumors,but the expression of HSF1 in osteosarcoma and its correlation with prognosis have not been studied previously.Protein kinase A(PKA)plays an important role in many aspects of signal transduction pathways involving in cell growth,proliferation,metabolism and disease.PKA can interact with a variety of protein substrates to cause phosphorylation regulation on serine or threonine residues.The main research method on PKA function is the use of pharmacological inhibitors.One of them,H89,is a potent and specific PKA inhibitor with the ability to easily cross cell membranes.Previous studies have shown that after the formation of trimers,HSF1 proteins are regulated by phosphorylation to mediate their nucleation and function.Among them,PKA plays an important role in phosphorylation of 320-serine(S320)of HSF1 protein.Phosphorylation of S320 of HSF1 is important for the nuclear distribution and transcriptional functions of HSF1.However,in osteosarcoma cells,the roles of S320 phosphorylation regulation in nuclear translocation and transcriptional function of HSF1 are unclear.Objective:To explore the relationship between HSF1 expression in osteosarcoma and prognosis,and to investigate the effects of HSF1 on the biological behaviors of osteosarcoma at molecular,cellular and animal levels.This study is trying to explore prognostic-related biomarkers and possible therapeutic targets for osteosarcoma.Methods:1,Firstly,we selected 20 pairs of tumor specimens and paraneoplastic tissue samples from osteosarcoma patients in our department.The expression of HSF1 in these samples was detected by qRT-PCR and immunohistochemical(IHC)staining.65 Osteosarcoma tissues were assessed by IHC and were classified into two groups.Log-rank analysis and COX analysis were conducted to evaluate the correlation between HSF1 level and clinical prognosis of patients with osteosarcoma;2,We interfered with the expression of HSF1 by lenti virus or siRNA on MNNG and U-2OS osteosarcoma cell lines,and detected the biological effects of HSF1 on osteosarcoma cells through cell functional experiments such as proliferation,migration,invasion and apoptosis.We further examined the effect of HSF1 on the tumorigenic ability of osteosarcoma cell lines in nude mice;3,Finally,in order to explore the mechanism of HSF1 affecting the biological behavior of osteosarcoma,we used the small molecule inhibitor H89 of phosphokinase A(PKA)as a tool to interfere with the phosphorylation regulation of HSF1(S320).The nuclear translocation of HSF1 was detected by immunofluorescence(IF)staining and Western blotting.Further,the U2 OS cell line was used as the target cell,and the mutant strain of phosphorylation important site(S320A)was constructed by CRISP/CAS9 technique.We investigated the effects of nuclear translocation of HSF1 on the malignant biological behavior of osteosarcoma cells and did the analysis of the major signaling pathways and downstream genes of HSF1 affecting the biological behavior of osteosarcoma by gene expression profiling.Results:1,By qRT-PCR and tissue microarray IHC staining,we found that HSF1 mRNA and protein expression were significantly elevated in osteosarcoma tissues,and HSF1 protein was mainly accumulated in the nuclear region.We expanded the sample number and scored the IHC results of 65 patients with osteosarcoma.The high expression group was found to be associated with larger tumor diameter and higher recurrence and metastatic rates.2,By interfering with the expression of HSF1 in MNNG and U2 OS osteosarcoma cell lines,we found that up-regulation of HSF1 can significantly promote the proliferation,migration and invasion of osteosarcoma cells,while down-regulation of HSF1 expression could lead to opposite results and promote apoptosis.Through tumor spheroid formation experiments,we found that HSF1 had no direct effect on tumor stem cells of osteosarcoma;it was further confirmed by nude mice experiments that HSF1 overexpression could significantly promote osteosarcoma tumor formation.3,The expression of S320 phosphorylated HSF1 was significantly increased in osteosarcoma;Through IF staining and WB experiments,it was suggested that the expression of phosphorylated S320 HSF1 protein was decreased,and the nuclear localization of HSF1 was weakened after H89 treatment.We found that H89 treatment or S320 A mutation could significantly inhibit the proliferation and invasion of osteosarcoma cells and promote apoptosis,while,treatment of S320 A mutant cell line with H89 had no significant effect,suggesting that phosphorylation regulation of S320 is an important mechanism for the function of HSF1 in osteosarcoma.By sequencing the expression profiles of S320 A mutant cell line and normal U2 OS cell line,it was found that after S320 A mutation,the differentially expressed genes were mainly enriched in the cell cycle and adhesion-related pathways,and genes related to tumor migration and EMT,such as Vimentin.Expression of Mmp2,Snail1,Smad2,KRT14 and Mmp9 were significantly reduced.Conclusion:Based on the above results,we believe that the high expression of HSF1 indicates a poor clinical prognosis;HSF1 can enhance the proliferation,migration and invasion of osteosarcoma cells,and reduce the apoptosis ability of osteosarcoma cells,thereby promoting the development and metastasis of osteosarcoma;Phosphorylation of S320 is an important site to regulate HSF1 tumorigenesis and may be an important target for future research on osteosarcoma drug therapy.