Infiltrating Mast Cells Promote Neuroendocrine Differentiation And Increase Docetaxel Resistance By Regulating P21 In Prostate Cancer

Background:Prostate cancer is one of the most common malignant tumors of the genitourinary system.Androgen deprivation therapy(ADT)is currently the first-line treatment for advanced prostate cancer.However,prostate cancer would develop into castrate-resistant prostate cancer(CRPC)2-3 years after ADT.Metastatic CRPC(mCRPC)has a very poor prognosis.Docetaxel has been widely used in clinical treatment of mCRPC and can provide patients with modest survival benefit.However,due to the abnormal intracellular pathways of cancer cells and the change of the structure of cancer tissue,many patients appear to become resistance to chemotherapeutic drugs during the course of treatment.Therefore,the effect of chemotherapy is very limited.Neuroendocrine(NE)tumor cells are found in all stages of prostate cancer.Neuroendocrine differentiation(NED)is a common feature of prostate cancer.NE cells secreting products,including neuron specific enolase(NSE),Chromogranin A(ChrA),synaptophysin(SYP),can be used as a molecular marker of prostate cancer NED.In addition,it has been found in vitro that ADT induced NED in androgen-sensitive prostate cancer cell lines and in vivo studies have shown that ADT in the human prostate cancer model resulted in differentiation of cancer cells into NE cells,demonstrating that ADT can cause NED of prostate cancer.A previous study showed that mast cells could participate in tumor progression by promoting angiogenesis,tissue remodeling and immune regulation.ADT can promote NED by recruiting mast cells to tumors and altering a series of downstream cellular signaling pathways.P21 is a cyclin-dependent kinase inhibitor(CKI)that inhibits cyclin/CDK complex and proliferating cell nuclear antigen(PCNA)activity.Although p21 plays a key role in regulating cell cycle,studies have shown that under certain conditions,p21 can promote cell proliferation and carcinogenesis.Our previous studies demonstrated that ADT recruited mast cells and promoted NED of prostate cancer through the AR/miRNA32 pathway.However,the relationship between p21 and NED of prostate cancer and mechanism of mast cell infiltration leading to NED of prostate cancer are not fully elucidated.This study is to investigate the role of p21 in mast cell infiltration and NED of prostate cancer in vitro,to further reveal the relationship between mast cell infiltration,p21,NED and chemoresistance in prostate cancer.Methods:In this study,0.4μm transwell chambers were used to co-culture PCa cells(LNCaP and C4-2)with HMC-1 cells,and the morphology changes of PCa cells were observed.Cell viability of co-cultured PCa cells was determined by MTT assay.The effect of mast cells on PCa cells proliferation was determined by colony formation assay.CCK8 assay was applied to test docetaxel sensitivity of co-cultured PCa cells.RT-qPCR assay and western blot assay were used to determine the expression levels of AR,p21,and NE markers of cultured/co-cultured PCa cells.Results:1.There was a morphology change of PCa cells(LNCap and C4-2)after being co-cultured with mast cells from spindle-shaped cells to longer cells that display dendrite-like processes extending between adjacent cells.The mRNA levels of NE markers,including neuron-specific enolase(NSE),Chromogranin(ChrA),synaptophysin(SYP),in PCa cells co-cultured with HMC-1 cells were significantly increased compared with untreated PCa cells.Also,Western Blot analysis revealed that NSE protein level was upregulated in co-cultured LNCaP and C4-2 cells.2.MTT assay showed that the co-cultured LNCaP and C4-2 cells presented a decrease in cell growth.Colony formation was remarkably inhibited by HMC-1 cells co-culture in LNCaP and C4-2 cells.We treated C4-2 or co-cultured C4-2 cells with different doses of docetaxel for 48 hours,or 6nM docetaxel for different times.Results showed that the co-cultured C4-2 cells had better cell viability after docetaxel treatment,indicating that mast cells co-culturing could enhance resistance to docetaxel of PCa cell in dose-and time-dependent manner.3.AR expression was significantly repressed in co-cultured PCa cells,which is in consistence with the result from our previous study.Moreover,there was a remarkable increase in the expression of p21.Our previous study shows that suppression of AR signals can enhance NED by upregulating miRNA 32 expression.So we next investigated whether AR suppression also promoted NED by upregulating p21 expression.Consequently,we did find that targeting AR with sh-AR lentivirus could increase NSE expression,but decrease the expression of p21,while targeting p21 with sh-RNA in turn did not affect AR expression,but led to a significant decrease of NSE expression in C4-2 cells.Furthermore,overexpressing p21 in C4-2 cells significantly increased NSE expression and induced the NE morphology changes.These results suggested that that p21 could positively regulate NED.P21 can regulate NED independent of AR signaling in PCa,and mast cells may function through p21 to promote NED in PCa.4.We further knocked down p21 in C4-2 cells,then co-cultured C4-2 cells with HMC-1 cells.NSE mRNA was remarkably overexpression in co-cultured C4-2 cells in the scramble groups.In p21-silenced C4-2 cells,by contrast,co-culture with HMC-1 cells did not cause a dramatic increase of NSE expression.Similar result was obtained with Western Blot assay.The protein level of NSE in co-cultured C4-2 cells was significantly elevated and the facilitative effect of mast cells on NSE expression was impaired by sh-p21.In addition,the NE-like morphologic changes in co-cultured c4-2 cells were inhibited by targeting p21.Knocking down p21 also partially reversed co-culture-induced docetaxel resistance in C4-2 cells.These results suggest that mast cells promote NED and enhance docetaxel resistance of PCa cells partly through altering p21 expression.Conclusion:Infiltrating mast cells can promote NED and inhibit the proliferation of PCa cells.Mast cells-induced NED reduces the docetaxel treatment sensitivity of PCa cells.Mast cells infiltration can lead to upregulation of p21 expression which modulates NED of PCa cells through a non-AR signaling pathway.Targeting p21 partially reverses the mast cell-mediated NED and docetaxel resistance of PCa cells,indicating that mast cells promote NED and docetaxel resistance partially by altering p21 expression,and targeting p21 may provide new insights into therapy for PCa.