Cenomic Hallmarks Of Prostate Cancer And The Mechanisms Of Huaier Inhibiting Prostate Cancer Progression

BANKGROUND:Prostate cancer(PCa)is the most common cancer followed by lung cancer in men in 2020,accounting for more than 20%of new male cancer diagnoses in the US,and there will be 33,330 PCa-related deaths in the US.With dramatic economic growth and westernized lifestyle changes,the incidence rate of PCa increased rapidly in China with an annual percentage change of 12.6%since 2000.Thirty percent of Chinese patients are diagnosed with metastatic PCa,and this phenomenon that an aggressive phenotype of PCa at diagnosis is more common in Asian than in Western population was confirmed in other Asian countries.PCa is a typical cancer harboring not only intratumor heterogeneity but also intertumor heterogeneity,especially in patients with different racial and ethnic backgrounds.At present,most of the data of prostate cancer genomics come from Caucasians,and lack of data of prostate cancer genomics in Chinese population.Therefore,genomic molecular alterations in Chinese should be explored in detail to find actionable alterations,thus guiding precision cancer therapy.PCa is a kind of tumor which depend on androgen,and androgen deprivation therapy(ADT)is the basic therapy for PCa.Unfortunately,after an initial response to ADT,majority of cases will progress to the more aggressive stages,or castration-resistant prostate cancer(CRPC),within 3 years,and the average overall survival for metastatic CRPC patients is about 1.5 years.In support of the importance of ligand-driven full-length-AR(AR-FL)signaling in CRPCs,a number of clinically effective endocrine therapies(e.g.,abiraterone,enzalutamide)targeting AR-LBD were recently developed to treat patients with CRPCs,named the second generation of hormonal drugs.Nevertheless,the major of patients progress shortly after abiraterone/enzalutamide treatment.Nowadays,docetaxel-based chemotherapy is one of the available treatments for these patients.Unfortunately,only 50%patients with CRPC could benefit from the docetaxel therapy,and what's more challenging is that even there is an initial response to docetaxel,all patients will eventually get resistance to docetaxel.Therefore,finding new treatments for PCa has become an important task for clinical and basic researchers.In recent years,a number of basic studies and high-quality clinical studies have revealed the anti-tumor effect of Huaier.As a treasure of traditional Chinese medicine with a history of more than 1600 years,more and more oncologists have paid attention to its antitumor properties.Although the recent study revealed its antitumor activity in hormone-dependent tumor(breast cancer and ovarian carcinoma),the studies of huaier extract on prostate cancer are rare.Therefore,we will explore the antitumor effect of huaier in prostate cancer in this study,and provide the theoretical basis for huaier in treatment of hormone sensitive prostate cancer and drug resistant prostate cancer.METHODS AND RESULTS:In this study,we explored genomic hallmarks of prostate cancer in Chinese population and the antitumor effects of huaier in prostate cancer.Part ? Genomic hallmarks of prostate cancerMETHODS:We included a cohort of 94 patients with PCa who had undergone radical prostatectomy or prostatic biopsy from January 2018 to March 2019 at Qi Lu Hospital of Shandong University.Prostate tumor and matched normal tissues or blood samples were collected to perform next-generation targeted sequencing,which contained all exons of 450 cancer-related genes and selected introns of 36 genes.Fisher's exact test or the x2 test was performed to examine the association of gene aberrations with rapid biochemical recurrence(BCR).The OncoKB knowledge database was used to identify and classify actionable alterations.RESULTS:The most frequently aberrant genes in our cohort included TP53(22.3%),ERG(18.1%),SPOP(17.0%),BRCA2(7.4%),APC(6.4%),CDH1(5.3%)and LRP1B(5.3%).The aberrations of PTEN,CDK12,and SPOP in our HNPC samples were similar to those in the Western samples.However,we demonstrated an association of a high frequency of TP53 alterations(21/94)with a relatively higher percentage of alterations in the Wnt signaling pathway(15/94)in our HNPC.Additionally,we highlighted alterations of LRP1B as accounting for a high proportion of PCa and found more frequent alterations in CDH1 in our PCa.Of these,only CDH1 alteration was associated with rapid biochemical recurrence(BCR).However,we verified that TP53 status was at the core of the genomic alteration landscape in our HNPC with putative driver mutations because of the strong connections with other signaling pathways.The mutually exclusive relationship between alterations in TP53 and Wnt/CTNNB1 further molecularly characterizes subsets of prostate cancers.Moreover,the alteration of KMT2C was more likely to cooccur with TP53 alteration,indicating a more aggressive phenotype of PCa,which was associated with sensitivity to treatment with poly ADT-ribose polymerase(PARP)inhibitors.Part ? Huaier inhibits prostate cancer growth via targeting AR/AR-V7 pathway and re-sensitize enzalutamide treatmentMETHODS:We performed CCK8 assay,transwell assay and flow cytometry to examine the effect of huaier on cell proliferation,migration,invasion,cell apoptosis and cell cycle of androgen receptor positive prostate cancer cell lines.WB and qPCR were used to explore the effect of huaier on the expression of AR/AR-V7.Luciferase reporter assay and immunofluorescence assay were used to examine the nuclear translocation of AR/AR-V7 as well as their transcriptional function.The AR and AR-V7 overexpression plasmids were used to confirm whether overexpression of AR/AR-V7 could rescue the inhibitory effect of huaier on the growth of prostate cancer cells.To further explore the mechanism that huaier regulates AR/AR-V7 pathway,we performed siRNA-mediated knockdown and cDNAs-mediated overexpression assays for both SMYD3 and USP14,the upstream regulators of AR/AR-V7.Finally,we explored the effect of huaier on reversing the resistance to enzalutamide by CCK8 assay in vitro and tumorigenesis in nude mice in vivo.RESULTS:Huaier significantly inhibited the growth,migration and invasion of prostate cancer cells,and led to cell apoptosis and cell cycle arrest.WB,qPCR and rescue experiments confirmed that huaier inhibited the growth of prostate cancer cells by regulating AR/AR-V7 pathway.Through knockdown and overexpression experiments,we confirmed that huaier can reduce AR/AR-V7 mRNA expression by downregulating SMYD3,and induce proteasome-mediated degradation of AR-FL and AR-V7 by downregulation USP14.The results of luciferase reporter assay and immunofluorescence assay suggested that huaier not only can reduce the expression of AR/AR-V7,but also inhibit their nuclear translocation and transcription function.Additionally,CCK8 assay in vitro and tumorigenesis in nude mice in vivo confirmed that huaier can reverse the enzalutamide-resistance in PCa cells.Altogether,these data demonstrated that huaier inhibit the growth of hormone sensitive prostate cancer as well as castration resistant prostate cancer cells.Part ? Huaier reverses docetaxel resistance in prostate cancer via targeting c-Fos/FASN-mediated de novo lipogenesisMETHODS:Docetaxel resistant prostate cancer cell line was established by continuously increasing the concentration of docetaxel in the culture medium.The sensitivity of the cell to docetaxel was examined by CCK8 and flow cytometry.To investigate genomic alterations and the changes of signaling pathways between docetaxel-resistant cells and the parental cells,we collected RNA of p-DU145 and DU145-DoR to perform next-generation sequencing(NGS).WB and qPCR were used to examine the expression of FASN,siRNA-mediated knockdown and cDNAs plasmid-mediated overexpression were performed to explore the effect of FASN on de novo lipogenesis in docetaxel-resistant cell lines.CCK8 assay was performed to examine whether FASN inhibitor could re-sensitize docetaxel treatment in DU 145-DoR and 22Rv1-DoR.The upstream regulatory factors of FASN were deeply mined through transcription factor databases,and further verified by luciferase reporter assay and CHIP assay.In order to verify whether huaier can inhibit the growth of docetaxel resistant cells and enhance the sensitivity of docetaxel treatment,CCK8 assay and tumorigenesis in nude mice were carried out.Mechanistically,we examined whether huaier inhibited the growth of docetaxel resistant cells by regulating c-fos/FASN-mediated de novo lipogenesis through WB experiments.RESULTS:It took 9 months for the establishment of docetaxel resistance in DU145 cells,while 10 months for 22Rv1,and we named these two docetaxel-resistant cell lines:DU145-DoR and 22Rv1-DoR.the IC50 for parental DU145(p-DU145)and DU145-DoR were 5.7±0.1nM and 735.2±83.3nM,while the IC50 for parental 22Rv1(p-22Rv1)and 22Rv1-DoR were 31.6nM±3.6nM and 11.6±3.0?M.Flow cytometry also confirmed that the constructed cell line was resistant to docetaxel.The results of WB and qPCR showed that the expression of FASN in docetaxel-resistant cells was significantly increased.Oil Red O(ORO)staining showed that increased lipid deposits accumulation in docetaxel-resistant cells than that in parental cells,and this process was mediated by FASN in drug-resistant cells.CCK8 assay showed that FASN inhibitor could enhance the sensitivity of drug-resistant cells to docetaxel treatment.We identified c-Fos as the upstream transcription factor(TF)of FASN by cross comparison of TF databases animalTFDB,PROMO,and iRegulon,and the results of WB and qPCR showed that c-Fos was significantly increased in docetaxel-resistant cells.Knockdown or overexpression of c-Fos could also reduce or increase FASN expression and lipid deposits accumulation in docetaxel-resistant cells.Results of luciferase reporter assay and CHIP further confirmed that c-Fos can directly regulate the expression of FASN gene by binding to the promotor of FASN.In addition,results of CCK8 assay in vitro and tumorigenesis in vivo confirmed that huaier can inhibit the growth of docetaxel-resistant cells and enhance docetaxel treatment.Mechanistically,our results showed that huaier inhibited the growth of docetaxel resistant prostate cancer cells by inhibiting c-Fos/FASN-mediated de novo lipogenesis.CONCLUSIONS1.There is heterogeneity in genomic hallmarks of prostate cancer between Chinese(all cases came from Shandong Province,China)and Western population,especially for the TP53 alterations.2.Huaier inhibits prostate cancer growth via targeting AR/AR-V7 pathway and re-sensitize enzalutamide treatment.3.Huaier reverses docetaxel resistance in prostate cancer via targeting c-Fos/FASN-mediated de novo lipogenesis.SIGNIFICANCESIn this study,we performed NGS of prostate cancer clinical samples to reveal the genomic hallmarks of prostate cancer in Chinese population(all cases came from Shandong Province,China),and tried to elucidate the molecular mechanisms of heterogeneity in prostate cancer between Chinese and Western populations.Our data of HNPC indicate that it is essential to perform further targeted NGS for precision treatment after finding significant TP53 alterations in PCa.We further revealed the anti-tumor effect of huaier in prostate cancer,which provided a theoretical basis for the treatment of prostate cancer with huaier alone or in combination.