Effects Of Androgen Deprivation Therapy On The Prostate Cancer Immune Microenvironment And The Underlying Mechanisms

ObjectiveProstate cancer(PCa)is the most common cancer among men.In the last decade,the incidence of prostate cancer in China has increased rapidly.Androgen deprivation therapy(ADT)is a cornerstone for locally advanced or metastatic PCa treatment.Although the mechanisms of the direct antitumor effects of ADT have been widely studied,the potential profound effects on the PCa tumor immune microenvironment remain largely unclear.PCa shows high genomic variability among different ethnic populations.This leads to highly variable clinical features and large differences among individuals for therapeutic response.At present,most of the studies on ADT are based on the westerners,the effect of ADT on the immune microenvironment of prostate tissue of patients in China is not completely clear.The purpose of this study was to investigate the effect of ADT on the immune microenvironment of PCa in Chinese population and the possible mechanism behind it.Methods1.RNA sequencing was performed in PCa tissues and paracancerous normal tissues before and after ADT,and "DESeq2" package was used to calculate the differentially expressed genes for PCa tissues and paracancerous tissues before and after ADT treatment,and enrichment analysis was performed.2.The invasive activities of 22 immune cell types in PCa tissues and paracancerous tissues before and after ADT were calculated by single sample Gene Set Enrichment Analysis(ssGSEA)and verified by immunohistochemistry.3.Weighted Correlation Network Analysis(WGCNA)was used to explore the hub genes in the process of ADT-induced immune remodeling of prostate tissue.The relationship between the hub genes and the immune signature constructed by hub genes,and the prognosis of patients in the TCGA PCa cohort and our neoadjuvant ADT PCa cohort was explored.4.Furthermore according to the immune signature score,we classified patients into different immune subtypes,and the differences of immune infiltration,mutation landscape,and prognosis of different immune subtypes were compared.5.Androgen deprivation PCa model was established by using charcoal stripped FBS to continuous culture of PCa cell line LNCap.The expression of hub gene after continuous androgen deprivation was analyzed by RT-PCR and western blot.The hub gene SOCS3 gene was blocked by siRNA,and the expressions of various immune-related factors in PCa cell line were compared after androgen blockade and SOCS3 gene knock down combined with androgen blockade.Results1.ADT could up-regulate and change many immune-related genes in prostate tissues,and could stimulate immune activation and immunosuppression related functions and pathways.A variety of immune cells,including CD8+T cells,were significantly enriched after ADT.Compared with paracancerous tissues,ADT had a stronger effect on immune remodeling of PCa tissues.2.No significant difference was found in the number of fusion genes detected before and after ADT,compared with the study based on European and American population.In addition,some fusion genes such as the ERG family,which are common in Western population,have not been detected in the samples.3.WGCNA showed that SOCS3,ZFP36 and JUNB were hub genes in the process of prostate immune reconstruction induced by ADT.Both the TCGA prostate cancer cohort and our neoadjuvant ADT PCa cohort showed better PSA recurrence-free survival in patients with high expressions of SOCS3,ZFP36 and JUNB genes.4.The immune signature score based on SOCS3,ZFP36 and JUNB genes was related to the PSA recurrence-free survival.Immune signature score-based immune subtypes reveal the heterogeneity of the immune microenvironment of PCa and showed significant differences in patient prognosis,tumor immune infiltration,mutation burden,and landscape.5.In vitro experiment showed that continuous androgen blockade could significantly increase the mRNA and protein levels of SOCS3,ZFP36 and JUNB genes in prostate cancer cell line,in which the change of SOCS3 gene was the most significant one.RT-PCR found that the mRNA levels of various cytokines(IL-6,CCL2,CCR7 and TGFB1)related to the immune microenvironment of prostate cancer changed significantly after continuous androgen blockade,and that silencing SOCS3 gene with siRNA could significantly affect the changes of cytokines induced by continuous androgen blockade culture.Conclusion1.The study finds that ADT remodeling the prostate immune microenvironment.Immune-related genes SOCS3,ZFP36 and JUNB may have significant effects in the process of ADT immune remodeling.The immune subtypes of PCa based on the above three genes have great differences in PSA recurrence-free survival,immune infiltration,and mutation burden,and landscape.2.In vitro experiment shows that SOCS3 may have significant effects in the immune remodeling of PCa induced by ADT by regulating the expression and secretion of various immune-related factors in prostate cancer cells.