The Mechanism Of ApoA-I And SLPI In Promoting Castration Resistance Of Prostate Cancer

Background:Prostate cancer(PCa)is one of the most common malignancies in older men and is the fifth leading cause of cancer death worldwide.In 2015,there were about 60300 new cases and 26600 deaths of PCa in China.Therefore,the study of prostate cancer is of universal significance and can benefit the majority of older men.At the time of initial diagnosis,most of the tumors rely on the androgen receptor(AR)signaling pathway to activate the expression of downstream target genes for tumor growth.Androgen deprivation therapy(ADT)can reduce serum testosterone levels by more than 90 percent.Therefore,even patients who have lost the opportunity of surgery can still obtain a reasonably good outcome through ADT treatment.However,after a response period of about two years,most PCa will progress to castration resistant prostate cancer(CRPC).After the further treatment of the new generation AR pathway inhibitors,some patients will progress to CRPC-adenocarcinoma(CRPC-adeno)with the tumor histology remaining luminal phenotype.Some other patients will progress to neuroendocrine prostate cancer(NEPC).As a highly aggressive subtype of CRPC,neuroendocrine PCa(NEPC)does not respond to hormonal therapy because of the extensive expression of the NE markers,rather than the luminal markers(AR,PSA),which are abundant in adenocarcinoma.Thus,treatment of NEPC is an unmet clinical challenge.At present,most of the PCa treatments are based on the inhibition of the AR pathway,and it is urgent to find new therapeutic targets for patients with castration resistant prostate cancer.The unique metabolic characteristics and lineage plasticity of PCa are essential mechanisms of tumor metastasis and progression.Specifically,the metabolism of prostate cancer is different from that of other malignant tumors.Glutamine and lipids can be energy sources utilized by the tricarboxylic acid cycle to promote cell growth and proliferation.Apolipoprotein A-I(ApoA-I)is the main component of high-density lipoprotein,which plays an important role in the regulation of lipid metabolism in PCa.Lineage plasticity,manifesting the ability of cells to transition from luminal phenotype to NE phenotype,can be mediated by transcription factor SOX2 and up-regulate the expression of secretory leukocyte peptidase inhibitor(SLPI).It has been proposed as a mechanism of tumor adaptation to avoid harsh microenvironment such as exposure to hormonal therapy.Here,we intend to elucidate the mechanism of progression and treatment strategies of castration resistant prostate cancer from metabolic reprogramming and lineage plasticity perspectives.Part-? ApoA-I promotes the progression of prostate cancer by metabolic reprogrammingObjective:This study is aimed to investigate the expression of Apolipoprotein A-I(ApoA-I)in PCa of different grades and stages,the relationship between its expression level and related metabolic reprogramming.We will explore the underlying mechanism of Apo A-I promoting the progression from hormone-dependent PCa to castration resistant PCa.Methods:1.We compared the expression of ApoA-I in prostate tissues of different grades and stages(normal prostate tissues,primary adenocarcinoma,CRPC-adeno,and NEPC)using bioinformatics,immunohistochemical staining,and Western blot.The correlation between APOA1 and canonical markers in CRPC and NEPC was analyzed.2.We overexpressed the expression of APOA1 in LNCaP cells and knocked down the expression of APOA1 in PC3,C4-2,and LNCaP cells.MTS assay,colony formation,and Transwell assays were used to investigate the effects of APOA1 on cell viability,colony formation,and invasion ability.?-galactosidase staining was used to detect the proportion of senile cells in LNCaP cells after the overexpression of ApoA-I,which were treated with ADT treatment.3.The concentration of high-density lipoprotein in the cell culture medium was determined by Enzyme-linked immunosorbent assay(ELISA).The contents of various metabolites in cell lines were analyzed by liquid chromatography-mass spectrometry(LC/MS)to explore the metabolic landscape differences under different cell lines and different ApoA-I expression levels.Gene set enrichment analysis(GSEA)was used to explore the public data sets to determine the activation of the lipoprotein metabolic pathway in the PCa metastases and NEPC compared with primary adenocarcinoma.4.GSEA was used to explore the effect of N-Myc on the lipoprotein metabolism pathway in PCa.The expression of c-Myc and N-Myc in LNCaP cells was overexpressed,and the expression c-Myc in PC3 cells was knocked down.The effect of gene modification was verified by Western blot,and the changes of APOA1 mRNA level were measured by RT-qPCR.Results:1.The expression of ApoA-I in PCa tissues is higher than that in normal prostate tissues.The expression of ApoA-I in PCa metastatic tissues and castration resistant PCa tissues is higher than that in primary PCa lesions.2.The expression of ApoA-I is low in normal prostate epithelial cell lines RWPE-1 and is slightly higher in hormone-sensitive PCa cells(LNCaP and LAPC4)and is significantly elevated in castration resistant PCa cells(C4-2,DU145,and PC3).3.The expression of APOA1 is negatively correlated with the expression of tumor suppressor genes and is positively correlated with canonical markers related to the progression of CRPC and NEPC.4.The overexpression of APOA1 can promote the survival,proliferation,colony formation and invasion ability of LNCaP cells,and enhance their resistance to androgen deprivation therapy.On the contrary,APOA1 knockdown can weaken the viability,proliferation,colony formation and invasion ability of PC3 cells,and the viability of C4-2 cells,but it has no effect on the viability of LNCap cells,which is considered to be related to its lower baseline expression.5.GSEA reveals that the lipoprotein metabolism pathways in PCa metastatic tissue and NEPC are significantly activated,which is conducive to tumor growth,when compared to the primary adenocarcinoma.6.The amount of high-density lipoprotein in the culture medium of PC3 and DU145 cells is significantly higher than that in the culture medium of LNCaP and LAPC4 cells,suggesting that PC3 and DU145 cells could utilize APOA1 to produce more lipid metabolites as the energy source for tumor growth.Pathways related to lipid metabolism(such as aerobic glycolysis,tricarboxylic acid cycle,fatty acid metabolism,fatty acid synthesis,and fatty acid oxidation,etc.)are generally activated in PC3 cells when compared to LNCaP cells.Overexpression of APOA1 in LNCaP cells and knockdown of APOA1 in PC3 cells can significantly affect lipid metabolism(such as fatty acid synthesis,fatty acid chain extension and fatty acid degradation,etc.).7.Genome enrichment analysis showed that N-Myc could activate the lipoprotein metabolism pathway in PCa cells.There is a significant positive correlation between the expression of APO A1 and MYCN and MYC in the public dataset.Overexpression of N-Myc and c-Myc in LNCaP cells can upregulate APOA1,while knockdown of c-Myc in PC3 cells can inhibit the transcription of APOA1.Conclusion:1.The stepwise elevation of ApoA-I expression across the spectrum of PCa progression was observed from the normal prostate tissue to the primary adenocarcinoma and then to the castration resistant PCa.Further,ApoA-I can promote the survival,proliferation,invasion,and castration resistance of PCa cells,indicating that ApoA-I is an important pro-cancer factor in the pathogenesis and progression of PCa.2.APOA1 is regulated by the MYC family,and the relatively high expression of APOA1 can significantly affect the lipid metabolism reprogramming of PCa,and promotes PCa to a lipid-producing phenotype,and promotes the progression from hormone-dependent PCa to castration resistant PCa.Part-? SLPI promotes neuroendocrine differentiation in prostate cancer via lineage plasticityObjective:Neuroendocrine(NE)differentiation often occurs in prostate cancer(PCa)after long-term androgen withdrawal,which leads to the therapeutic failure of hormonal therapy.Because of the extensive expression of the NE markers,rather than the luminal markers(AR,PSA),which are abundant in adenocarcinoma,neuroendocrine PCa(NEPC)does not respond to hormonal therapy.Thus,treatment of NEPC is an unmet clinical challenge.However,the underlying etiology has not been fully clarified.In this part,we will investigate the expression of Secretory Leukocyte Peptidase Inhibitor(SLPI)in PCa tissues of different grades and stages,interrogate the relationship between its expression level of luminal and NE phenotype,and explore the potential mechanism of SLPI promoting the progression of adenocarcinoma to NEPC.Methods:The data from TCGA(The Cancer Genome Atlas),GEO(Gene Expression Omnibus),Oncomine,cBioPortal(The cBio Cancer Genomics Portal),and CCLE(Cancer Cell Line Encyclopedia)databases were downloaded to analyze the expression of SLPI in normal prostate tissue,primary adenocarcinoma,CRPC and NEPC by bioinformatics.Then the expression pattern was verified by immunohistochemical staining and Western blot to understand the role of SLPI in the pathogenesis and progression of PCa,The correlations between SLPI expression,AR markers,and NE markers were analyzed to interrogate the different functions of SLPI in AR and NE pathways.Furthermore,we will investigate the expression of canonical NE markers after knocking down SLPI in PC3 cells,and the expression of SLPI,NE markers and AR markers after upregulating SOX2 in LNCaP cells.Results:1.The expression of SLPI is downregulated in primary adenocarcinoma with an active AR pathway when compared to normal prostate tissues,suggesting that SLPI is inhibited in primary adenocarcinoma.2.Compared with low-stage(T2c and below)PCa,the expression of SLPI in high-stage(T3a and above)PCa is upregulated,suggesting that the tumor suppressor role of SLPI may be switched to a carcinogenic role in advanced PC a.3.Compared with primary adenocarcinoma,the expression of SLPI is upregulated in metastatic PCa and NEPC,in which the AR pathway is suppressed.By immunohistochemical staining,we found that the expression of SLPI in high-grade PCa(Gleason>8)is higher than that in low-grade PCa(Gleason ?7).Compared with localized PCa,small cell neuroendocrine PCa(SCNC,a histological type of NEPC)contain more SLPI-positive cells.Consistent with the above results,the protein expression of SLPI in two different NE-specific cell lines(PC3 and C4-MDVR)is significantly higher than that in hormone-sensitive PCa cell lines(LNCap and LAPC4),which was shown by Western blot.Public dataset analysis showed that the expression of SLPI is significantly upregulated in metastatic and NEPC tissues compared to primary adenocarcinoma.4.Since SLPI is barely expressed in AR-positive adenocarcinoma and highly expressed in AR-negative NEPC,we further analyzed the relationship between SLPI and AR and NE pathways.Based on the analyses of TCGA,GEO,and CCLE datasets,we found that the expression of SLPI is negatively correlated with the expression of marker genes in the AR pathway(AR,KLK3,KLK2,NKX3-1,and TMPRSS2),and GSEA indicates that the expression of SLPI is negatively correlated with AR pathway.On the contrary,the expression of SLPI is positively correlated with canonical genes in the NE pathway(EN02,NCAM1,CXCR2,and SOX2).GSEA shows that the upregulation of SLPI could activate the NE pathway in PCa.5.With the extension of ADT treatment time,the expression of SLPI in the luminal cells increases gradually,and the expression of NE marker is induced.On the contrary,the expression of the luminal marker is inhibited,and the cells transdifferentiates from luminal to NE phenotype gradually.To verify that this process is reversible,we knocked down SLPI in PC3 cells and found that the transcription levels of canonical NE markers EN02 and SYP are also significantly downregulated.6.SOX2 is a crucial driver regulating the lineage plasticity of PCa.The expression of SLPI and SOX2 is positively correlated in PCa,and this correlation is even more significant in metastatic PCa and NEPC.Then we found the specific binding sites of SOX2 in the promoter region of SLPI using the MAST tool in the MEME(Multiple Em for Motif Elicitation,http://meme-suite.org/).Overexpression of SOX2 in LNCaP cells can upregulate the expression of SLPI and NE markers,and downregulate the expression of AR related markers.Therefore,we hypothesized that SLPI might be regulated by transcription factor SOX2 and play a critical role in lineage plasticity.Conclusion:1.The expression of SLPI is related to the AR pathway activity of PCa and is downregulated in primary adenocarcinoma,while upregulated in metastatic PCa and NEPC in which the AR pathway was suppressed.2.The expression of SLPI was negatively correlated with the AR pathway and positively correlated with the NE pathway.3.SLPI promotes cell lineage transformation and NE differentiation in PCa via lineage plasticity under the regulation of SOX2,and SLPI knockdown could reverse the NE characteristics.