A Positive Roleofc-Myc In Regulating Androgen Receptor And Its Splice Variants In Prostate Cancer

Androgen receptor(AR)is the main therapeutic target in prostate cancer.Androgen deprivation therapy,the first-line treatment for disseminated prostate cancer,disrupts AR signaling through castration or AR antagonists.However,almost all patients experience progression to the presently incurable stage,termed castrationresistant prostate cancer(CRPC).AR signaling is sustained and remains critical in CRPC,and several new and more potent AR-directed drugs have been developed to target the sustained AR activity in CRPC.Among these,the potent AR antagonist enzalutamide and the CYP17A1 inhibitor abiraterone have been approved by the FDA as second-line treatments for metastatic CRPC.However,the development of therapyresistant disease is an inevitable outcome for many patients.Increased expression of AR splice variants(AR-Vs)has been ascribed as an important mechanism of resistance to AR-directed therapies,including enzalutamide and abiraterone.AR-Vs are truncated AR isoforms that lack the functional ligandbinding domain,but the N-terminal transactivation domain and the DNA-binding domain remain intact in the majority of AR-Vs.As a result,many AR-Vs have been shown to have constitutive transcriptional activities.High levels of AR-Vs,specifically,AR-V7,ARv567 es,and AR-V9,have been linked to poor prognosis and short survival of CRPC patients.The critical involvement of AR-Vs in the progression of CRPC underscores the need to understand how they are generated so that effective therapeutic strategies can be derived to curb AR-V production.Co-expression of AR-FL and different AR-Vs is commonly observed in the same cells from CRPC patients.On the other hand,several splicing factors,such as U2AF65,ASF/SF2,hn RNPA1,and hn RNPF,along with the RNA-binding protein Sam68,the transcription factor YB-1,and the molecular chaperone HSP90,have been shown to selectively regulate AR-V7 splicing without affecting AR-FL splicing.In this study,we sought to identify a mechanism that could mediate the co-expression of AR-FL and different AR-Vs.This is in view of the tight correlation between the levels of AR-FL and AR-V transcripts that is commonly observed in individual clinical specimens as well as in xenograft tumors.Androgen deprivation has been shown to enhance the rate of AR-gene transcription to produce more AR pre-m RNA.This indicates that,in addition to alternative splicing,a transcriptional mechanism may drive the expression of AR-FL and AR-Vs after AR-directed therapies.In the present study,we investigated the role of the c-Myc transcription factor in accounting for this transcriptional mechanism of regulation.This is because: 1)c-Myc is one of the top overexpressed genes during prostate cancer progression to metastatic and is critically involved in prostate cancer progression,2)c-Myc can bind to regulatory regions of AR to induce the transcription of the AR gene,and 3)a previous gene expression study of 140 metastatic CRPC samples showed a frequent upregulation of c-Myc in CRPC and a positive correlation between c-Myc and AR m RNA levels in these samples.Here,we confirmed this positive correlation in an additional 159 metastatic CRPC and 2142 primary prostate cancer samples.We further show that a striking positive correlation also exists between the activity of the c-Myc pathway and the levels of AR-FL and different AR-Vs,between the level of c-Myc and the activity of the AR pathway,and between the activities of two pathways.We then use cell models and a patient-derived xenograft model to demonstrate the importance of c-Myc in regulating the expression of AR-FL and AR-Vs.Mechanistically,this is mediated through a transcriptional regulation coupled with modulation of protein stability without impacting AR RNA splicing.Together,this work provides a rationale for targeting c-Myc to curb AR-FL and AR-V expression for more effective treatment of prostate cancer.TMPRSS2-ERG gene fusions,which cause overexpression of the ERG gene by the TMPRSS2 promoter,are present in ~50% of human prostate cancer.About half of the fusions are generated through an intra-chromosomal(interstitial)deletion between these two genes(referred to as “deletion”),and the others are generated via insertional chromosomal rearrangement(referred to as “insertion”).The deletion subtype is generally more aggressive than the insertion subtype,as it is associated with higher tumor stage,presence of pelvic lymph node metastases,and poorer disease-specific and overall survival.Moreover,patients with two copies of deletion-generated fusions showed substantially worse survival than those with one deletion-generated fusion.Fusions detected in metastatic castration-resistant PC(m CRPC)are more frequently caused by deletion than those in hormone-na?ve cases(75% vs.25%).Strikingly,a study of a cohort of patients who died of m CRPC showed that all the metastatic sites harboring TMPRSS2-ERG fusions were of the deletion subtype.In addition to these clinical evidences,the more aggressive nature of the deletion subtype is also reflected in transgenic mouse models.Prostatic hyperplasia was detected in 19% of mice with deletion-derived TMPRSS2-ERG fusion but not in any of the mice with insertion-derived fusion.Likewise,in the background of biallelic Pten loss,almost 60% of the mice with deletion-derived fusions,but no mice with insertion-derived fusions,developed poorly differentiated invasive adenocarcinomas by 12 months.Collectively,these findings suggest a critical role of the interstitial deletion(between TMPRSS2 and ERG)in promoting PC progression and subsequent lethality.Despite the greater aggressiveness of the deletion subtype,our understanding of how interstitial gene loss leads to poor patient outcome is unacceptably poor.The interstitial region between TMPRSS2 and ERG encompasses 28 genes,including 16 coding,9 long non-coding RNAs,and 3 mi RNAs.Interstitial deletion leads to copy number loss of these genes concomitant with ERG activation.Among these 28 genes,only ETS2 has been shown to have tumor suppressor function in fusion-positive PC cells and in PC mouse models.FAM3B(aka PANDER)is a member of the FAM3 family of cytokine molecules that were initially described in 2002.FAM3 B expression is primarily localized to the endocrine pancreas and is secreted from both pancreatic ? and ?-cells.Initial characterization of FAM3 B revealed a potential role in pancreatic islet apoptosis.While some more recent animal models have indicated FAM3 B functions as a hormone by regulating glucose levels via interaction with both the liver and the endocrine pancreas.Here,we first characterized FAM3 B as a tumor suppressor in prostate cancer.We investigated the interstitial genes between TMPRSS2 and ERG among primary and metastatic samples.Remarkably,FAM3 B is the only one whose low expression is significantly associated with m CRPC,high Gleason score and shorter time to biochemical recurrence.We demonstrated the cell growth inhibitory function of FAM3 B in the cell models and the patient-derived xenograft models.Mechanistically,our GSEA analysis result showed FAM3 B high expression is associated with OXPHOS and low glycolysis.Further,we confirmed the impact of FAM3 B on OXPHOS and glycolysis via seahorse analysis and FAM3 B overexpression cells are less sensitive to a glycolysis inhibitor,2-DG.Together,our preliminary data indicates FAM3 B functions as a tumor suppressor and we aim to reveal a new mechanism of prostate cancer progression,identify a biomarker to better define aggressive prostate cancer,and provide a foundation for future design of a biomarker-driven therapeutic trial to use glycolysis inhibitors to treat patients with FAM3B-low aggressive prostate cancer.