Cross-resistance Between Enzalutamide And Apalutamide Through The AKR1C3/AR-V7 Axis In Advanced Prostate Cancer

Objective:Although localized prostate cancer could be cured by radical prostatectomy or radiotherapy,advanced metastatic prostate cancer,especially metastatic castrationresistant prostate cancer(m CRPC),is still an incurable lethal disease.In the past two decades,with the gradual clarification of the molecular mechanism of prostate cancer disease progression,it is unequivocal that the main driving factor of the m CRPC stage disease progression is still the androgen signaling pathway.The success of clinical trials with various new drugs for the treatment of m HSPC and CRPC patients,including new generation antiandrogens,cabazitaxel,Radium223,and even Sipuleucel-T,as well as the new concept of upfront use of novel agents,have comprehensively improved the survival outcomes of patients with advanced prostate cancer.Objectively speaking,due to the complicated molecular mechanisms of m CRPC,patients with m CRPC would unavoidably be resistant to any type of standard care,which ultimately leads to a limited survival benefit improvement.The similarities in the molecular structure and mechanism of action of different new antiandrogens for androgen signaling indicate the possible cross-resistance between these drugs,which greatly affects the therapeutic potential of sequential treatment in prolonging the survival outcomes of advanced prostate cancer patients.Several studies have explored the potential cross-resistance among enzalutamide,abiraterone and docetaxel.However,for the recent-approved antiandrogen apalutamide,there’s currently no research focusing on its possible cross-resistance with other agents.Therefore,this study aims to explore the possible cross-resistance of the new generation antiandrogenic drugs enzalutamide and apalutamide in the treatment of prostate tumors,and to try to clarify the molecular mechanisms leading to crossresistance between the two drugs,so as to provide laboratory evidence and foundation for maximizing the therapeutic efficacy of different antiandrogens.Materials and Methods:Our project can be generally divided into two major parts: basic experiment part and clinical research part.The basic experiment part mainly includes the following steps: 1)Long-term culturing and regular passaging of C4-2B cells in medium containing enzalutamide or apalutamide with gradually increasing drug concentration,to obtain enzalutamide-resistant cell(C4-2B MDVR)and apalutamide-resistant cell(C4-2B APALR);2)Performing cell proliferation experiments and clone formation experiments to explore whether the enzalutamide-(or apalutamide-)resistant cell line C4-2B MDVR(or C4-2B APALR)is also resistant to apalutamide(or enzalutamide),so as to verify the potential cross-resistance between these two drugs;3)Exploring the changes in the transcriptome level of C4-2B cells before and after resistance to enzalutamide or apalutamide by RNA-seq,and verifying the sequencing results by q RT-PCR and Western Plot;4)Performing the luciferase reporter gene experiment to explore the inhibitory effect of enzalutamide and apalutamide on the activation of the androgen receptor signaling pathway caused by AR,AR splice variants and different AR mutants;5)Using si RNA or sh RNA to down-regulate the protein levels of AKR1C3 and AR-V7 in C4-2B MDVR cells,C4-2B APALR cells and CWR22Rv1 cells,and then,detecting the changes of these cells in drug-resistance and protein levels.6)Upregulating the AKR1C3 level in C4-2B parental cell line,and then testing its cell proliferation and drug-resistance;7)Exploring the interaction between AKR1C3 protein and AR-V7 protein through co-immunoprecipitation experiment;7)Treating C4-2B MDVR or C4-2B APALR cells with small molecule inhibitors of AKR1C3 and AR-V7,with single drug treatment or combined administration,and then,observing the changes in cell proliferation and drug-resistance in these drugresistant cells.The clinical research part mainly includes the following aspects: 1)Collecting1003 prostate biopsy specimens from our hospital and performing immunohistochemical staining of AKR1C3 and AR-V7 on these specimens.Analyzing the positivity of AKR1C3 and AR-V7 in prostate cancer patients with different Gleason scores(GS)and the correlation between AKR1C3 and AR-V7expression;2)Collecting the clinicopathological data and prostate biopsy specimens of 114 m CRPC patients receiving abiraterone as first-line treatment in our hospital and performing immunohistochemical staining of AKR1C3 and AR-V7 on these specimens;3)Evaluating the significance of AKR1C3 and AR-V7 in the above biopsy specimens in predicting the PSA response rate,PSA progression-free survival,radiological progression-free survival and overall survival of the first-line abiraterone treatment in m CRPC patients.4)Further exploring the prognostic value of AKR1C3 in different subgroups of m CRPC patients;5)Mining TCGA-PRAD data from the TCGA public database to further analyze the correlation between the AKR1C3 m RNA expression and the prognosis of localized prostate cancer patients after radical prostatectomy.Results:In the basic research part of this project,through cell proliferation experiments and clone formation experiments,we proved that enzalutamide-resistant prostate cancer cell lines C4-2B MDVR and CWR22Rv1 are also resistant to apalutamide.Western Blot experiment revealed that the protein levels of AR-V7 and AR-Vs in the drug-resistant cells C4-2B MDVR and CWR22Rv1 increased significantly against the C4-2B parental cells.This could not be inhibited by either enzalutamide or apalutamide treatment.In addition,the luciferase assay experiment also confirmed that the second-generation antiandrogens can only inhibit the activation of the AR signaling pathway caused by the AR receptor but not by AR-V7 or AR mutants.After si RNA interference or small molecule antagonist treatment in C4-2B MDVR cells and CWR22Rv1 cells to inhibit their AR-V7 activity,the drug-resistant trait of these cells was reversed.In addition,the results of RNA-seq also revealed that the C4-2B MDVR cell had a higher expression of genes related to the steroid hormone biosynthesis signal pathway than the C4-2B parental cell line.Notably,the expression level of AKR1C3 increased the most.Similarly,after using si RNA interference or small molecule antagonist treatment in C4-2B MDVR and CWR22Rv1 cells in an attempt to inhibit their AKR1C3 activity,the drug-resistance of these cells was also reversed.Western Blot experiments showed that changes in the protein level of AKR1C3 affected the protein level of AR-V7 in C4-2B MDVR and CWR22Rv1 cells.On the other hand,AR-V7 could not affect the protein level of AKR1C3.After up-regulating the expression level of AKR1C3 in C4-2B parental cells,the cells were induced to develop resistance to enzalutamide and apalutamide treatment,and the level of ARV7 protein also increased.Furthermore,the results of the co-immunoprecipitation experiment also showed that AKR1C3 can interact with AR-V7 at protein level.Taking together,AKR1C3 can regulate the AR-V7 protein by binding it.On the other hand,through cell proliferation experiments and clone formation experiments,we also proved that the apalutamide-resistant cell line C4-2B APALR is also resistant to enzalutamide.The results of RNA-seq also showed that C4-2B APALR cells had an enrichment of steroid hormone biosynthesis related genes.Similarly,after using different methods to inhibit AKR1C3 in C4-2B APALR cells,drug-resistance to either enzalutamide or apalutamide of the cells was reversed,and the level of AR-V7 protein was also inhibited.Treating C4-2B MDVR or C4-2B APALR cells with small molecule antagonists of AKR1C3 and AR-V7 in combination can more significantly inhibit tumor proliferation than single-agent treatment.The followings are the findings of the clinical research part.Among the 1003 prostate biopsy specimens in our hospital,the overall positivity of AKR1C3 and ARV7 were 276/1003(27.5%)and 22/1003(2.2%),respectively.The positivity of both proteins increases in men harbouring a higher GS.The proportion of AR-V7 positivity in patients with positive AKR1C3 expression was much higher against that of cases with negative AKR1C3(14/276 [5.1%] vs.8/727 [1.1%],P < 0.001).Among the 114 patients who progressed to the m CRPC stage and used abiraterone as first-line therapy,AKR1C3 expression was not significantly related to patient’s PSA response rate(20/38 [52.6%] vs.49/76 [64.5%];P = 0.223).It is worth noting that AKR1C3-positive patients have a much shorter median PSA progression-free survival time(5.7months vs.11.2 months,P < 0.001)and median radiological progression-free survival time(12.4 months vs.23.3 months;P = 0.048)compared to their AKR1C3-negative counterparts.These results were well validated in different subgroups of prostate cancer patients with different clinical pathological baselines.On the other hand,the AR-V7 positivity in the biopsy specimens of the primary prostate could not effectively predict patient’s prognosis in the first-line abiraterone treatment for m CRPC(PSA progression-free survival: HR,95%CI: 1.47,0.66-3.28,P = 0.349;radiological progression-free survival: HR,95% CI: 1.78,0.61-5.23,P = 0.294).We also conducted in-depth mining on the data of 444 patients with localized prostate cancer after radical prostatectomy in the TCGA-PRAD data from the public TCGA database.We found that the m RNA level of AKR1C3 was obviously much higher in cases with high GS,high T stage,and lymph node(+)than those with low GS,low T stage,and lymph node(-).The m RNA FPKM value of AKR1C3 in radical prostatectomy specimens was related to patients’ disease progression.After dividing all patients into two groups according to the median FPKM value of AKR1C3,the median progression-free survival time was significantly more unfavorable in the high AKR1C3-expression group against the low AKR1C3-expression group(71.0 months vs.115.1 months,P = 0.006).Conclusion:1)Cross-resistance exists between enzalutamide and apalutamide in the treatment of prostate cancer.2)Both experimental and clinical study supports that AKR1C3 can positively regulate the protein level of AR-V7.The activation of the AKR1C3/AR-V7 signal axis is an important factor leading to the above-mentioned cross-resistance;3)Small molecule antagonists of AKR1C3 or AR-V7 can reverse the cross-resistance of enzalutamide and apalutamide;4)Data mining of the TCGA database revealed that the m RNA level of AKR1C3 is associated with several highly aggressive clinical and pathological parameters of prostate cancer patients(such as high GS,high T stage,lymph node metastasis).The m RNA expression of AKR1C3 is closely related to the recurrence and progression of patients with localized prostate cancer;5)For m CRPC patients receiving first-line abiraterone therapy,the positive immunohistochemistry of AKR1C3 in prostate biopsy specimens indicates rapid disease progression and poor clinical outcomes.