The Role Of Human Dermal Fibroblasts With High Expression Of ATF3 In Inhibiting The Growth And Migration Of Skin Melanoma Cells

Background and ObjectivesDerived from melanocytes in the basal layer of skin epidermis,cutaneous melanoma is the most lethal one in three of the major malignant cancers of the skin due to its characteristics of high rate metastasis.Clinically,the treatment of malignant melanoma,especially metastatic malignant melanoma cases has been a big challenge.Melanoma cells are usually regulated by the activated extracellular signal-regulated kinase(ERK)pathway,which is induced by BRAF-activating mutations.BRAF,a downstream of RAS,is a protein kinase of the RAF family,and activates ERK through phosphorylation of MEK,play an important role in stimulating cell growth and survival.BRAF inhibitors have been used clinically to treat cutaneous malignant melanoma with high efficacy,but most patients will develop drug resistance after treatment.The TME is comprised of all the nonmalignant host cellular and non-cellular components of the tumor niche,including,but not limited to,the immune system,blood cells,endothelial cells,fat cells,and the stroma.The tumor stroma,the main component of TME,mainly consists of fibroblasts and extracellular matrix.In the past few decades,more and more studies have demostrated that TME plays an increasingly important role in determining tumor progression and treatment outcome.The interaction mechanism between tumor cells and microenvironment is very complex,but it can be divided into two categories:contact and non-contact pathways.The contact pathway involves the contact dependent mechanism of cell-cell and extracellular matrix adhesion molecules.In the non-contact pathway,many soluble molecules,such as growth factors,chemokines and cytokines,as well as soluble subcellular organelles(including microbubbles and exosomes),play an important role in the whole process.Finally,these interactions regulate tumor cells through the mechanism of secretion and paracrine pathway.Recently,more and more evidences showed that stroma invelves the development of tumor drug resistance.Therefore,the development of therapeutic methods for treatment of cancer should consider strategies of targeting and inhibiting tumor stroma.ATF3(activating transcription factor 3,ATF3)is one of the important members of ATF/CREB family.As an important transcription factor,ATF3 may have different functions in different tissues.ATF3 has been shown to play as either oncogene or cancer suppressor gene in tumor development.Our previous study showed that in human epidermis,up-regulation of ATF3 expression suppresses p53-dependent cellular senescence to promote keratinocyte tumorigenesis.ATF3 gene has been also found to be expressed in human dermal fibroblasts,but its function has not been well studied and especilly,its role in skin melanoma development has not been reported.So far,only one report indicates that the fibroblasts with low ATF3 expression can be transformed into CAFs form,and ultimately promoted the development of skin squamous cell carcinoma.In order to fulfill this gap,this study first was going to check the ATF3 expression level in the stroma of clinical skin melanoma samples.Then,genetically modified methods to either overexpress or delete ATF3 gene in human dermal fibroblasts were involved to examine the effect of primary human dermal fibroblasts with different expression of ATF3 on the growth and migration changes of skin melanoma through in vitro and in vivo experiments with application of co-culture system and conditioned medium.More importantly,the underlying molecular mechanism would be also explored.Finally,this study will try to verify the effect of human dermal fibroblast with induced expression of ATF3 by pretreatment of interested compounds on the growth of human skin melanoma cells.This research would provide new ideas and solutions for the treatment of skin malignant melanoma in the future.Methods1.Detection of ATF3 expression in clinical cutaneous melanoma tissuesFirst,the paraffin samples of clinical melanoma tissues were collected,and the paraffin sections were stained by immunohistochemistry staining,then the results were verified by RNAscope in situ hybridization.The expression of ATF3 in dermal fibroblasts,stromal fibroblasts of benign nevus and malignant melanoma were detected by immunofluorescent double staining.2.The effect of human dermal fibroblast ATF3 on the growth and migration of malignant melanoma cells in vitro and in vivoFirst,primary human dermal fibroblasts were isolated and cultured.Lentivirus and retrovirus expression systems were used to either overexpress or delete ATF3 gene in the human dermal fibroblasts,using qRT-PCR and Western Blot to verify the expression or deletion efficiency of ATF3.The co-culture system and conditioned medium collected from cultured dermal fibroblasts with different expression of ATF3,together with application of CCK-8,colony formation assay,transwell assay,and would healing assay to examine the effect of human dermal fibroblast with either overexpression or deletion of ATF3 medium on the growth and migration of malignant melanoma of the skin.Human dermal fibroblasts with overexpression of ATF3 mixed with melanoma cells were grafted onto nude mice to evaluate their effects on melanoma cell tumorigenicity in vivo.3.Detection of the molecular mechanism of human dermal fibroblast ATF3 on the growth and migration of malignant melanoma cellsqRT-PCR analysis was used to screen corresponding cytokines and other gene changes,and candidate downstream genes in human dermal fibroblasts with either overexpression or deletion of ATF3 ELISA was used to confirm the production of identified cytokines affected by ATF3 in human dermal fibroblasts by qRT-PCR results.Using the recombinant protein or inhibitor corresponding to the candidate gene,combined with the conditioned medium of human dermal fibroblasts edited by the ATF3 gene to test the effect on the growth and migration of melanoma cells.Western Blot was used to detect changes in protein phosphorylation levels of corresponding pathways in melanoma cells treated with conditioned medium and candidate gene recombinant proteins or inhibitors,to clarify specific signaling pathways.4.In vivo verification of the molecular mechanism of the effect of human dermal fibroblast ATF3 on the growth and migration of malignant melanoma cells8-week old nude/nude female nude mice were randomly divided into three groups,each group contains with three mice in each group.Human dermal fibroblasts overexpressed with ATF3 gene and melanoma cells were mixed in proportion and implanted subcutaneously in the back of nude mice.For one of the experimental groups,candidate gene recombinant protein or inhibitor will be involved as simulating factors.After 3 weeks,the tumor formation rate and tumor weight of the three groups of nude mice were analyzed.5.Detection the effect on the change of melanoma cell growth from drug-induced ATF3 gene expressed human dermal fibroblasts.First,we used cell culture,qRT-PCR and Western Blot and other technologies to clarify the effect of candidate compounds on the expression of ATF3 gene in human dermal fibroblasts.Secondly,the conditioned medium was collected from compound pretreated human dermal fibroblasts,by using CCK-8 and other technologies to detect its effect on melanoma cell proliferation,and qRT-PCR and ELISA were used to detect the expression level of corresponding genes.Finally,compound pretreated human dermal fibroblasts were mixed with cutaneous malignant melanoma,and the effect of nude/nude female nude mice on the tumorigenicity of melanoma cells in vivo was detected by xenograft assay.Results1.Results of ATF3 expression level in clinical specimens of cutaneous malignant melanomaImmunohistochemistry staining and RNAscope in situ hybridization showed low expression of ATF3 in stromal tissues of clinical melanoma tissues.Immunofluorescent double staining showed that compared with those of normal skin and human skin benign nevus,lower expression level ATF3 in stromal cells of malignant melanoma and the difference between groups has statistical significance.2.The effect of human dermal fibroblast ATF3 on the growth and migration of malignant melanoma cells in vitro and in vivoCompared with the control group,co-culture with ATF3 overexpressed human dermal fibroblasts can inhibit the growth and migration of melanoma cells.The difference between the experimental group and the control group was statistically significant.Compared with the control group,co-culture of the CRISPR/Cas9 mediated ATF3 knockout human dermal fibroblasts had no significant inhibition and promotion on melanoma cell growth and migration.Compared with the control group,the conditioned medium of ATF3 overexpressed human dermal fibroblasts inhibited the growth and migration of melanoma cells.The difference between the experimental group and the control group was statistically significant.Compared with the control group,the tumor formation rate and tumor weight of melanoma cells in nude mice grafted with mixture of melanoma cells with ATF3 over-expressed human dermal fibroblast were decreased.The difference between the experimental group and the control group was statistically significant.3.Detection of the molecular mechanism of the effect of ATF3 on the proliferation and migration of malignant melanoma cellsThe results of qRT-PCR showed that the mRNA levels of IL-6,IL-8,IL-1?,Cox 1-3 and other cytokines in human dermal fibroblasts with overexpression of ATF3 gene were significantly lower than those in the control group,while only the levels of IL-6 and IL-8 mRNAs in the ATF3 knock-out group were higher than those in the control group.The results of ELISA confirmed that the level of IL-6 and IL-8 protein in the conditioned medium of human dermal fibroblasts overexpressed with ATF3 gene was significantly lower than that in the corresponding control group,and the difference was statistically significant.Compared with the corresponding control group,the level of IL-6 and IL-8 protein in the conditioned medium of human dermal fibroblast group with deletion of ATF3 did not increase significantly.Human recombinant interleukin-6(rhIL-6)can increase the growth and migration of melanoma cells.Western blot results showed that the over expression of ATF3 gene in human dermal fibroblast conditioned medium inhibited the phosphorylation of STAT3 protein in melanoma cells,rhIL-6 could promote the phosphorylation of STAT3 protein in melanoma cells,and the over expression of ATF3 human dermal fibroblasts inhibited the growth and migration of melanoma IL-6/STAT3 signaling pathway.4.The effect of human dermal fibroblasts ATF3 on growth and migration of melanoma cells and its mechanism verificationCompared with the control group,the growth rate and weight of melanoma cells in nude mice were inhibited in the experimental mixed group,while the growth rate and weight of melanoma cells in nude mice were promoted in the experimental mixed group plus rhIL-6.5.Detection of proliferation of melanoma cells by human dermal fibroblasts expressing ATF3 gene induced by drugsThe results of qRT-PCR and Western blot showed that phenformin and cyclosporine A promoted the expression of ATF3 gene and protein,and inhibited the expression and secretion of IL-6 and IL-8 in human dermal fibroblasts.The growth of melanoma cells in vitro was inhibited by phenformin and cyclosporine A pretreated human skin fibroblasts conditioned medium.Compared with the control group,the growth rate and weight of melanoma cells in nude mice in drug pretreated group were inhibited.All above expriments were repeated at least three times.Conclusions:1.The low expression level of fibroblast ATF3 was found in stromal cells of clinical melanoma tissues.2.Overexpression of ATF3 gene inhibits the expression and secretion of inflammatory factors,especially IL-6,in human dermal fibroblast.3.Overexpression of human dermal fibroblasts inhibits the IL-6/STAT3 signaling pathway of melanoma cells and ultimately inhibits their growth and migration.4.Phenformin and cyclosporine A can induce ATF3 gene and protein expression and inhibit the expression and secretion inflammatory factors in human dermal fibroblasts.5.Pretreatment of human dermal fibroblasts with phenformin and cyclosporine A inhibits the growth of skin melanoma cells.