Search

Emerging evidence indicates that various cancers can gain resistance to targeted therapies by acquiring lineage plasticity. Although various genomic and transcriptomic aberrations correlate with lineage plasticity, the molecular mechanisms enabling the acquisition of lineage plasticity have not been fully elucidated. We reveal that Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling is a crucial executor in promoting lineage plasticity-driven androgen receptor (AR)-targeted therapy resistance in prostate cancer. Importantly, ectopic JAK–STAT activation is specifically required for the resistance of stem-like subclones expressing multilineage transcriptional programs but not subclones exclusively expressing the neuroendocrine-like lineage program. Both genetic and pharmaceutical inhibition of JAK–STAT signaling resensitizes resistant tumors to AR-targeted therapy. Together, these results suggest that JAK–STAT are compelling therapeutic targets for overcoming lineage plasticity-driven AR-targeted therapy resistance.

RNASeq quality control data

In vitro proliferation of fibroblasts from AA and EA patients. Proliferation of PCa cell lines exposed to CM from AA and EA prostate fibroblasts

Differential expression of selected secreted cytokines and densitometric quantitation

Progress in prostate cancer racial disparity research has been hampered by a lack of appropriate research tools and better understanding of the tumor biology. Recent gene expression studies suggest that the tumor microenvironment (TME) may contribute to racially disparate clinical outcomes in prostate cancer. Analysis of the prostate TME has shown increased reactive stroma associated with chronic inflammatory infiltrates in African-American (AA) compared with European-American (EA) patients with prostate cancer. To better understand stromal drivers of changes in TME, we isolated prostate fibroblasts (PrF) from AA (PrF−AA) and EA (PrF−EA) prostate cancer tissues and studied their functional characteristics. PrF−AA showed increased growth response to androgens FGF2 and platelet-derived growth factor. Compared with PrF−EA, conditioned media from PrF−AA significantly enhanced the proliferation and motility of prostate cancer cell lines. Expression of markers associated with myofibroblast activation (αSMA, vimentin, and tenascin-C) was elevated in PrF−AA. In vivo tumorigenicity of an AA patient–derived prostatic epithelial cell line E006AA was significantly increased in the presence of PrF−AA compared with PrF−EA, and RNA-seq data and cytokine array analysis identified a panel of potential proinflammatory paracrine mediators (BDNF, CHI3L1, DPPIV, FGF7, IL18BP, IL6, and VEGF) to be enriched in PrF−AA. E006AA cell lines showed increased responsiveness to BDNF ligand compared with EA-derived LNCaP and C4-2B cells. Addition of a TrkB-specific antagonist significantly reduced the protumorigenic effects induced by PrF−AA compared with PrF−EA. These findings suggest that fibroblasts in the TME of AA patients may contribute to the health disparity observed in the incidence and progression of prostate cancer tumors.Significance: These findings suggest that stromal cells in the tumor microenvironment of African-American men promote progression of prostate cancer by increasing levels of a specific set of pro-inflammatory molecules compared with European-American men.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6134/F1.large.jpg. Cancer Res; 78(21); 6134–45. ©2018 AACR.

Supplementary Methods

PDF file, 315KB, Supplemental Figure 1: Validation of differential Meis1 and Meis2 expression between Prostate and Seminal Vesicle Epithelial Cells. Supplemental Figure 2: Gene expression profiles from 181 prostatectomy specimens of patients (Taylor dataset) using the PAM (Prediction Analysis for Microarrays) algorithm to stratify patients into two groups based on changes in expression of Meis1, Meis2, Pbx1, HoxA9, HoxB7. Supplemental Figure 3: Meis biomodule network analysis. Supplemental Methods: qPCR Analyses of Meis1 and Meis2. Supplemental Table 1: Historical datasets used in the analyses. Supplemental Table 2: Prostate epithelial cell (PrEC) versus seminal epithelial cell (SVEC) culture differentially expressed genes. Supplemental Table 3: Functional Annotation of the Prostate versus Seminal Vesicle Epithelium Signature. Supplementary Table 4: Univariate and Multivariate Cox Regression.

Assessment of collagen deposition and neoangiogenesis in PCa samples from AA and EA patients. Quantitation of IHC staining of stromal markers

IHC staining and quantitation of FAP in AA andEA patient samples

Fig. S1. Plasmid map of A.TSTAPEG101KB in pGL3- Basic Fig. S2. PEG10 staining in positive control and healthy tissue Fig. S3. TSTA Schematic Fig. S4. Luciferase expression in empty pGL3 vector Fig. S5. PEG10 expression by IHC in HT-29 xenografts Fig. S6. HSV-TK and PEG10 expression in CWR-R1 tumors Table S1. Primers for PEG10 promoter cloning.

Purpose: The aberrant activity of developmental pathways in prostate cancer may provide significant insight into predicting tumor initiation and progression, as well as identifying novel therapeutic targets. To this end, despite shared androgen-dependence and functional similarities to the prostate gland, seminal vesicle cancer is exceptionally rare.Experimental Design: We conducted genomic pathway analyses comparing patient-matched normal prostate and seminal vesicle epithelial cells to identify novel pathways for tumor initiation and progression. Derived gene expression profiles were grouped into cancer biomodules using a protein–protein network algorithm to analyze their relationship to known oncogenes. Each resultant biomodule was assayed for its prognostic ability against publically available prostate cancer patient gene array datasets.Results: Analyses show that the embryonic developmental biomodule containing four homeobox gene family members (Meis1, Meis2, Pbx1, and HoxA9) detects a survival difference in a set of watchful-waiting patients (n = 172, P = 0.05), identify men who are more likely to recur biochemically postprostatectomy (n = 78, P = 0.02), correlate with Gleason score (r = 0.98, P = 0.02), and distinguish between normal prostate, primary tumor, and metastatic disease. In contrast to other cancer types, Meis1, Meis2, and Pbx1 expression is decreased in poor-prognosis tumors, implying that they function as tumor suppressor genes for prostate cancer. Immunohistochemical staining documents nuclear basal-epithelial and stromal Meis2 staining, with loss of Meis2 expression in prostate tumors.Conclusion: These data implicate deregulation of the Hox protein cofactors Meis1, Meis2, and Pbx1 as serving a critical function to suppress prostate cancer initiation and progression. Clin Cancer Res; 18(16); 4291–302. ©2012 AACR.

The retrotransposon-derived paternally expressed gene 10 (PEG10) protein is ordinarily expressed at high levels in the placenta. Recently, it was discovered that PEG10 isoforms promote the progression of prostate cancer to a highly lethal androgen receptor (AR)-negative phenotype. The presence of PEG10 in other subtypes of prostate cancer has not been explored and a utility for PEG10 overexpression has not been developed. Here, we found that in addition to AR-null disease, PEG10 was also expressed in prostate cancer with constitutively active AR-splice variants. A molecular genetic imaging strategy for noninvasive imaging of AR-splice variant prostate cancer was developed by utilizing the cancer specificity of the PEG10 promoter to drive the expression of reporter genes. Plasmid insertion of a PEG10 promoter sequence optimized for enhanced output upstream of a reporter gene allowed detection of prostate cancer by near-infrared and positron emission tomography imaging after systemic administration of the plasmid in vivo. PEG10 expressing subcutaneous xenograft and intratibial tumor models were imaged by both modalities using this molecular genetic imaging strategy. This study demonstrates a preclinical proof-of-concept that the PEG10 promoter is a powerful and specific tool that can be utilized for noninvasive detection of aggressive prostate cancer subtypes.Significance:PEG10 is expressed by prostate cancer with constitutively active AR-splice variants that can be exploited for noninvasive molecular imaging of this aggressive prostate cancer subytpe.

Supplementary Figure 1 - PDF file 202K, (A), expression of RBM3 in different human tissues. Data are mean � s.d. (n = 3). (B), expression of CIRBP in CD133+ and CD133- PrEC cells. mRNA levels of RBM3 and CIRBP was determined by RT-PCR. Data are mean � s.d. (n = 4). (C), mRNA levels of RBM3 were detected in the PC3-GFP and PC3-RBM3 clones by RT-PCR. Data are mean � s.d. (n = 3), *P < 0.05. (D), RBM3 protein was detected in both cytoplasmic (Cyto) and nuclear (Nu) components of PC3-GFP and PC3-RBM3 clones. Tubulin and Lammn A/C were used as loading controls for cytoplasmic and nuclear fractions, respectively. In all panels: G#1, PC3-GFP-clone #1; G#2, PC3-GFP-clone #2; R#1, PC3-RBM3-clone #1; R#6, PC3-RBM3-clone #6

Supplementary Figure 2 - PDF file 68K, (A), expression of cancer-related miRNAs was detected in the PC3-RBM3 and PC3-GFP clones using a cancer-specific miRNA PCR array. Left panel, selected miRNAs whose expression levels differed more than 1.5 fold in the clone R#1 compared to G#1. Red bars, differentiation-related miRNAs; black bars, other cancer-related miRNAs. Right panel, the up-regulation of miR-9 in PC3-RBM3 clones was validated by RT-PCR. Values are mean � s.d. (n = 3), *P < 0.05. (B), left panel, β3-tubulin expression was detected by Western blot in PC3-GFP and PC3-RBM3 clones. Right panel, quantitative evaluation of protein levels of β3-tubulin. Values are mean � s.d. (n = 3), *P < 0.05. (C), the immunohistochemical staining of RBM3 and β3-tubulin in the nerve fibers observed in human prostate tissue. Upper panels, low magnification; lower panels, high magnification. Scale bars, 100 �m. In all panels: G#1, PC3-GFP-clone #1; G#2, PC3-GFP-clone #2; R#1, PC3-RBM3-clone #1; R#6, PC3-RBM3-clone #6.expression of CDH2 in PC3-GFP and PC3-RBM3 clones

Supplementary Table 2 - PDF file 33K, Calculated size of PCR products for CD44 splicing variants

Supplementary Figure 5 - PDF file 104K, (A), mRNA levels of CD44v8-v10 (CD44v) were determined in human normal and prostate cancer tissues by RT-PCR. Normal, normal prostate tissue (n = 19); OC-PCa, organ-confined prostate cancer (n = 79); Met-PCa, metastatic prostate cancer (n = 23). (B), mRNA levels of CD44v were compared between patients with recurrent PCa (Rec, n = 44) and those without recurrent disease (Non-rec, n = 30). (C) and (D), mRNA levels of standard CD44 (CD44s) (C) or CD44v (D) were compared between patients with low grade PCa (Gleason score � 6, n = 21) and those with high grade disease (Gleason score > 6, n = 53). All experiments were repeated three times. Bars indicate median values

Supplementary Figure 2 from Low-Calcium Serum-Free Defined Medium Selects for Growth of Normal Prostatic Epithelial Stem Cells

Stage-specific differentiation markers were used to evaluate the cellular composition and the origin of nonimmortalized (PrEC) and immortalized (PZ-HPV7, CA-HPV10, RWPE-1, and 957E/hTERT) human prostate cell lines. These studies documented that immortalized and nonimmortalized prostate epithelial cells established and maintained in low (i.e., 2+ serum-free defined (SFD) medium were all derived from normal nonmalignant prostate tissues and contain CD133+/ABCG2+/α2β1Hi/p63−/PSCA−/AR−/PSA− prostate stem cells. In these cultures, prostate stem cells are able to self-renew and generate two distinct cell lineages: the minor proliferatively quiescent neuroendocrine lineage and the major transit-amplifying cell lineage. Subsequently, CD133−/ABCG2−/α2β1Hi/p63+/PSCA−/AR−/PSA− transit-amplifying cells proliferate frequently and eventually mature into proliferatively quiescent CD133−/ABCG2−/α2β1Lo/p63−/PSCA+/AR−/PSA− intermediate cells. Such proliferatively quiescent intermediate cells, however, do not complete their full maturation into CD133−/ABCG2−/α2β1Lo/p63−/PSCA−/AR+/PSA+ luminal-secretory cells in low Ca2+ SFD medium. Addition of universal type I IFN and synthetic androgen (R1881) to culture medium resulted in up-regulation of androgen receptor protein expression. However, it failed to induce full differentiation of intermediate cells into AR+/PSA+ luminal-secretory cells. Our results indicate that such inability of prostate epithelial cells to complete their differentiation is due to continuous expression of Notch-1 receptor and its downstream effector, Hey-1 protein, which actively suppresses differentiation via its ability to transcriptionally repress a series of genes, including the GATA family of transcription factors. (Cancer Res 2006; 66(17): 8598-607)

Supplementary Figure 4 - PDF file 146K, mRNA expression of Cyclin D1 was evaluated in PC3 cells (A) and DU145 cells (B) that formed colonies in soft agar or those grew in the regular 2D culture condition by qRT-PCR. Values are mean � s.d. (n = 4). (C), PC3 cells were cultured under 32˚C for 0 hr to 144 hr, and mRNA level of cyclin D1 was detected by qRT-PCR. Values are mean � s.d. (n = 4). (D) Over-expression of CD44 variant and standard construct. LNCaP cells were transfected with pLenti-III-CD44s (CD44s), pLenti-III-CD44v8-v10 (CD44v8-10) or empty vector. After 48 hr of transfection, cells were collected and the expression of mRNA and protein of CD44 were detected by RT-PCR and Western blot, respectively. Data are mean � s.d. (n = 3), *P < 0.05

Supplementary Figure 3 - PDF file 244K, (A), CD44 variant mRNAs were amplified by PCR using the general primers c13 and hs3'. The PCR products at size of 650 bp and 250 bp were cut and subjected to direct DNA sequencing. The numbers in the block are exons number of CD44. N, negative control; G#1, PC3-GFP-clone #1. (B), schematic diagram of designing siRNAs specific to CD 44 variant exons v8-v10 (siC1, siC2) and primers specific to CD44v8-v10 (CD44v) or standard CD44 (CD44s). Primers c13 and hs3' can amplify both CD44s and CD44v. Primers p1 and p2 can only amplify CD44v8-v10, while primers p3 and p4 are specific to standard CD44s. Primer hs5' is the outside primer of the nest PCR. (C), the specificity of siRNAs targeting for CD44v8-v10. Variant exon-specific PCR was performed using primer p1 and p2. CD44s was amplified using CD44 standard-specific primer p3 and p4. siN, non-target siRNA. Data are mean � s.d. (n = 3), *P < 0.05. (D), the efficiency of siRNA pool targeting RBM3. Cells were treated with siRBM3 or non-targeting siRNA (siN) for 48 hr. The expression of mRNA and protein of CD44 were detected by RT-PCR and Western blot, respectively. R#1, PC3-RBM3-clone #1; R#6, PC3-RBM3-clone #6. Data are mean � s.d. (n = 3), *P < 0.05

Supplementary Table 1 - PDF file 43K, mRNA expression of stem cell-related gene using a Stem Cell PCR array

Supplementary documents - PDF file 122K, Supplementary methods and supplementary figures legends

Supplementary Figure 3 from Low-Calcium Serum-Free Defined Medium Selects for Growth of Normal Prostatic Epithelial Stem Cells

Supplementary Figure 1 from Low-Calcium Serum-Free Defined Medium Selects for Growth of Normal Prostatic Epithelial Stem Cells

Purpose: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, heterogeneous disease with few therapeutic strategies that significantly prolong survival. Innovative therapies for mCRPC are needed; however, the development of new therapies relies on accurate imaging to assess metastasis and monitor response. Standard imaging modalities for prostate cancer require improvement and there remains a need for selective and sensitive imaging probes that can be widely used in patients with mCRPC. Experimental Design: We evaluated the transmembrane protease fibroblast activation protein alpha (FAP) as a targetable cell surface antigen for mCRPC. Genomic and IHC analyses were performed to investigate FAP expression in prostate cancer. Our FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was evaluated by PET/CT imaging in preclinical prostate cancer models. Results: Analysis of patient data documented FAP overexpression in metastatic disease across tumor subtypes. PET imaging with [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term retention of the probe in the preclinical models examined. FAP-positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher than the isotype control with mean %ID/cc of 34.13 ± 1.99 versus 6.12 ± 2.03 (n = 3/group; P = 0.0006) at 72 hours. Ex vivo biodistribution corroborated these results documenting rapid blood clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG by 72 hours. Conclusions: Our study reveals FAP as a target for imaging the tumor microenvironment of prostate cancer. Validation of [89Zr]Zr-B12 IgG as a selective imaging probe for FAP-expressing tumors presents a new approach for noninvasive PET/CT imaging of mCRPC.

Benign prostatic hyperplasia (BPH) is a benign enlargement of the prostate in which incidence increases linearly with age, beginning at about 50 years old. BPH is a significant source of morbidity in aging men by causing lower urinary tract symptoms and acute urinary retention. Unfortunately, the etiology of BPH incidence and progression is not clear. This review highlights the role of the androgen receptor (AR) in prostate development and the evidence for its involvement in BPH. The AR is essential for normal prostate development, and individuals with defective AR signaling, such as after castration, do not experience prostate enlargement with age. Furthermore, decreasing dihydrotestosterone availability through therapeutic targeting with 5α-reductase inhibitors diminishes AR activity and results in reduced prostate size and symptoms in some BPH patients. While there is some evidence that AR expression is elevated in certain cellular compartments, how exactly AR is involved in BPH progression has yet to be elucidated. It is possible that AR signaling within stromal cells alters intercellular signaling and a “reawakening” of the embryonic mesenchyme, loss of epithelial AR leads to changes in paracrine signaling interactions, and/or chronic inflammation aids in stromal or epithelial proliferation evident in BPH. Unfortunately, a subset of patients fails to respond to current medical approaches, forcing surgical treatment even though age or associated co-morbidities make surgery less attractive. Fundamentally, new therapeutic approaches to treat BPH are not currently forthcoming, so a more complete molecular understanding of BPH etiology is necessary to identify new treatment options.

Per- and polyfluorinated alkyl substances (PFAS) are a large family of widely used synthetic chemicals that are environmentally and biologically persistent and present in most individuals. Chronic PFAS exposure have been linked to increased prostate cancer risk in occupational settings, however, underlying mechanisms have not been interrogated. Herein we examined exposure of normal human prostate stem-progenitor cells (SPCs) to 10 nM PFOA or PFOS using serial passage of prostasphere cultures. Exposure to either PFAS for 3–4 weeks increased spheroid numbers and size indicative of elevated stem cell self-renewal and progenitor cell proliferation. Transcriptome analysis using single-cell RNA sequencing (scRNA-seq) showed 1) SPC expression of PPARs and RXRs able to mediate PFAS effects, 2) the emergence of a new cell cluster of aberrantly differentiated luminal progenitor cells upon PFOS/PFOA exposure, and 3) enrichment of cancer-associated signaling pathways. Metabolomic analysis of PFAS-exposed prostaspheres revealed increased glycolytic pathways including the Warburg effect as well as strongly enrichment of serine and glycine metabolism which may promote a pre-malignant SPC fate. Finally, growth of in vivo xenografts of tumorigenic RWPE-2 human prostate cells, shown to contain cancer stem-like cells, was markedly enhanced by daily PFOS feeding to nude mice hosts. Together, these findings are the first to identify human prostate SPCs as direct PFAS targets with resultant reprogrammed transcriptomes and metabolomes that augment a preneoplastic state and may contribute to an elevated prostate cancer risk with chronic exposures.

Vitamin D deficiency is associated with an increased risk of prostate cancer (PCa) mortality and is hypothesized to contribute to PCa aggressiveness and disparities in African American populations. The prostate epithelium was recently shown to express megalin, an endocytic receptor that internalizes globulin-bound hormones, which suggests regulation of prostate hormone levels, in contrast to the free hormone hypothesis. Here, we demonstrated that megalin imports testosterone bound to sex hormone-binding globulin into prostate cells. Prostatic loss of Lrp2 (megalin) in a mouse model resulted in reduced prostate testosterone and dihydrotestosterone (DHT) levels. Megalin expression was regulated and suppressed by 25-hydroxyvitamin D (25D) in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants, indicating a negative feedback loop. In patent samples, the relationships between hormones support this feedback mechanism, as prostatic DHT levels are higher in African American men and are inversely correlated with serum 25D status. Megalin levels are reduced in localized PCa by the Gleason grade and in patients with future disease recurrence. Our findings suggest that the free hormone hypothesis should be revisited for testosterone and highlight the impact of vitamin D deficiency on prostate androgen levels, which are known drivers of PCa. Thus, we revealed a mechanistic link between vitamin D and PCa disparities observed in African Americans.

Emerging evidence indicates that various cancers can gain resistance to targeted therapies by acquiring lineage plasticity. Although various genomic and transcriptomic aberrations correlate with lineage plasticity-driven resistance, the molecular mechanisms of acquiring lineage plasticity have not been fully elucidated. Through integrated transcriptomic and single cell RNA-Seq (scRNA-Seq) analysis of more than 80,000 cells, we reveal for the first time that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is a crucial executor in promoting lineage plasticity-driven AR targeted therapy resistance in prostate cancer. Ectopic activation of JAK-STAT signaling is specifically required for the AR targeted therapy resistance of subclones expressing multilineage, stem-like and epithelial-to-mesenchymal transition (EMT) lineage transcriptional programs and represents a potential therapeutic target for overcoming AR targeted therapy resistance.One-Sentence SummaryJAK-STAT signaling is a crucial executor in promoting lineage plasticity-driven AR therapy resistance in prostate cancer.

The retrotransposon-derived paternally expressed gene 10 (PEG10) protein is ordinarily expressed at high levels in the placenta. Recently, it was discovered that PEG10 isoforms promote the progression of prostate cancer to a highly lethal androgen receptor (AR)-negative phenotype. The presence of PEG10 in other subtypes of prostate cancer has not been explored and a utility for PEG10 overexpression has not been developed. Here, we found that in addition to AR-null disease, PEG10 was also expressed in prostate cancer with constitutively active AR-splice variants. A molecular genetic imaging strategy for noninvasive imaging of AR-splice variant prostate cancer was developed by utilizing the cancer specificity of the PEG10 promoter to drive the expression of reporter genes. Plasmid insertion of a PEG10 promoter sequence optimized for enhanced output upstream of a reporter gene allowed detection of prostate cancer by near-infrared and positron emission tomography imaging after systemic administration of the plasmid in vivo. PEG10 expressing subcutaneous xenograft and intratibial tumor models were imaged by both modalities using this molecular genetic imaging strategy. This study demonstrates a preclinical proof-of-concept that the PEG10 promoter is a powerful and specific tool that can be utilized for noninvasive detection of aggressive prostate cancer subtypes. Significance: PEG10 is expressed by prostate cancer with constitutively active AR-splice variants that can be exploited for noninvasive molecular imaging of this aggressive prostate cancer subytpe.

Background The progression of castration-resistant prostate cancer (CRPC) still relies on the function of androgen receptor (AR), achieved by evolving mechanisms to reactivate AR signaling under hormonal therapy. Histone deacetylase inhibitors (HDACis) disrupt cytoplasmic AR chaperone heat shock protein 90 (Hsp90) via HDAC6 inhibition, leading to AR degradation and growth suppression of prostate cancer (PCa) cells. However, current HDACis are not effective in clinical trials treating CRPC. Methods We designed hybrid molecules containing partial chemical scaffolds of AR antagonist enzalutamide (Enz) and HDACi suberoylanilide hydroxamic acid (SAHA) as new anti-PCa agents. We previously demonstrated that Enz-HDACi hybrid drug 2-75 targets both AR and Hsp90, which inhibits the growth of Enz-resistant C4-2 cells. In the current study, we further investigate the molecular and cellular actions of 2-75 and test its anti-PCa effects in vivo. Results Compared with Enz, 2-75 had greater AR antagonistic effects by decreasing the stability, transcriptional activity, and nuclear translocation of intracellular AR. In addition to inhibition of full-length AR (FL AR), 2-75 downregulated the AR-V7 variant in multiple PCa cell lines. Mechanistic studies indicated that the AR affinity of 2-75 retains the drug in the cytoplasm of AR + PCa cells and further directs 2-75 to the AR-associated protein complex, which permits localized effects on AR-associated Hsp90. Further, unlike pan-HDACi SAHA, the cytoplasm-retaining property allows 2-75 to significantly inhibit cytoplasmic HDAC6 with limited impact on nuclear HDACs. These selective cytoplasmic actions of 2-75 overcome the unfavorable resistance and toxicity properties associated with classical AR antagonists, HDACis, and Hsp90 inhibitors. Finally, 2-75 showed greater antitumor activities than Enz in vivo on SQ xenografts derived from LNCaP cells. Conclusions Novel therapeutic strategy using newly designed 2-75 and related AR antagonist-HDACi hybrid drugs has great potential for effective treatment of CRPC.

Background Ductal adenocarcinoma of the prostate is an aggressive subtype, with high rates of biochemical recurrence and overall poor prognosis. It is frequently found coincident with conventional acinar adenocarcinoma. The genomic features driving evolution to its ductal histology and the biology associated with its poor prognosis remain unknown. Objective To characterize genomic features distinguishing ductal adenocarcinoma from coincident acinar adenocarcinoma foci from the same patient. Design, setting, and participants Ten patients with coincident acinar and ductal prostate cancer underwent prostatectomy. Laser microdissection was used to separately isolate acinar and ductal foci. DNA and RNA were extracted, and used for integrative genomic and transcriptomic analyses. Outcome measurements and statistical analysis Single nucleotide mutations, small indels, copy number estimates, and expression profiles were identified. Phylogenetic relationships between coincident foci were determined, and characteristics distinguishing ductal from acinar foci were identified. Results and limitations Exome sequencing, copy number estimates, and fusion genes demonstrated coincident ductal and acinar adenocarcinoma diverged from a common progenitor, yet they harbored distinct alterations unique to each focus. AR expression and activity were similar in both histologies. Nine of 10 cases had mutually exclusive CTNNB1 hotspot mutations or phosphatase and tensin homolog ( PTEN ) alterations in the ductal component, and these were absent in the acinar foci. These alterations were associated with changes in expression in WNT- and PI3K-pathway genes. Conclusions Coincident ductal and acinar histologies typically are clonally related and thus arise from the same cell of origin. Ductal foci are enriched for cases with either a CTNNB1 hotspot mutation or a PTEN alteration, and are associated with WNT- or PI3K-pathway activation. These alterations are mutually exclusive and may represent distinct subtypes. Patient summary The aggressive subtype ductal adenocarcinoma is closely related to conventional acinar prostate cancer. Ductal foci contain additional alterations, however, leading to frequent activation of two targetable pathways.

Background: Common genetic variation in the arsenic methyltransferase (AS3MT) gene region is known to be associated with arsenic metabolism efficiency (AME), measured as the percentage of dimethylarsinic acid (DMA%) in the urine. Rare, protein-altering variants in AS3MT could have even larger effects on AME, but their contribution to AME has not been investigated. Objectives: We estimated the impact of rare, protein-coding variation in AS3MT on AME using a multi-population approach to facilitate the discovery of population-specific and shared causal rare variants. Methods: We generated targeted DNA sequencing data for the coding regions of AS3MT for three arsenic-exposed cohorts with existing data on arsenic species measured in urine: Health Effects of Arsenic Longitudinal Study (HEALS, n=2,434), Strong Heart Study (SHS, n=868), and New Hampshire Skin Cancer Study (NHSCS, n=666). We assessed the collective effects of rare (allele frequency

Background: Common genetic variation in the arsenic methyltransferase (AS3MT) gene region is known to be associated with arsenic metabolism efficiency (AME), measured as the percentage of dimethyla...

The molecular roles of HOX transcriptional activity in human prostate epithelial cells remain unclear, impeding the implementation of new treatment strategies for cancer prevention and therapy. MEIS proteins are transcription factors that bind and direct HOX protein activity. MEIS proteins are putative tumor suppressors that are frequently silenced in aggressive forms of prostate cancer. Here we show that MEIS1 expression is sufficient to decrease proliferation and metastasis of prostate cancer cells in vitro and in vivo murine xenograft models. HOXB13 deletion demonstrates that the tumor-suppressive activity of MEIS1 is dependent on HOXB13. Integration of ChIP-seq and RNA-seq data revealed direct and HOXB13-dependent regulation of proteoglycans including decorin (DCN) as a mechanism of MEIS1-driven tumor suppression. These results define and underscore the importance of MEIS1-HOXB13 transcriptional regulation in suppressing prostate cancer progression and provide a mechanistic framework for the investigation of HOXB13 mutants and oncogenic cofactors when MEIS1/2 are silenced., eLife digest Decisions regarding the treatment of patients with early-stage prostate cancer are often based on the risk that the cancer could grow and spread quickly. However, it is not always straightforward to predict how the cancer will behave. Studies from 2017 and 2018 found that samples of less aggressive prostate cancer have higher levels of a group of proteins called MEIS proteins. MEIS proteins help control the production of numerous other proteins, which could affect the behavior of prostate cancer cells in many ways. VanOpstall et al. – including some of the researchers that performed the 2017 and 2018 studies – have investigated how MEIS proteins affect prostate cancer. When prostate cancer cells are implanted into mice, they result in tumors. VanOpstall et al. found that tumors that produced MEIS proteins grew more slowly. Next, MEIS proteins were extracted from the prostate cancer cells and were found to interact with another protein called HOXB13, which regulates the activity of numerous genes. When the cells were genetically modified to prevent HOXB13 being produced, the protective effect of MEIS proteins was lost. MEIS proteins work with HOXB13 to regulate the production of several other proteins, in particular a protein called Decorin that can suppress tumors. When MEIS proteins and HOXB13 are present, the cell produces more Decorin and the tumors grow more slowly and are less likely to spread. VanOpstall et al. found that blocking Decorin production rendered MEIS proteins less able to slow the spread of prostate cancer. These results suggest that MEIS proteins and HOXB13 are needed to stop tumors from growing and spreading, and some of this ability is by prompting production of Decorin. This study explains how MEIS proteins can reduce prostate cancer growth, providing greater confidence in using them to determine whether aggressive treatment is needed. A greater understanding of this pathway for tumor suppression could also provide an opportunity for developing anti-cancer drugs.

Approximately 140 million people worldwide are exposed to inorganic arsenic through contaminated drinking water. Chronic exposure increases risk for cancers as well as cardiovascular, respiratory, and neurologic diseases. Arsenic metabolism involves the AS3MT (arsenic methyltransferase) gene, and arsenic metabolism efficiency (AME, measured as relative concentrations of arsenic metabolites in urine) varies among individuals. Inherited genetic variation in the 10q24.32 region, containing AS3MT, influences AME, but the mechanisms remain unclear. To better understand these mechanisms, we use tissue-specific expression data from GTEx (Genotype-tissue Expression project) to identify cis-eQTLs (expression quantitative trait loci) for AS3MT and other nearby genes. We combined these data with results from a genome-wide association study of AME using “colocalization analysis,” to determine if 10q24.32 SNPs (single nucleotide polymorphisms) that affect AME also affect expression of AS3MT or nearby genes. These analyses identified cis-eQTLs for AS3MT in 38 tissue types. Colocalization results suggest that the casual variant represented by AME lead SNP rs4919690 impacts expression of AS3MT in 13 tissue types (> 80% probability). Our results suggest this causal SNP also regulates/coregulates expression of nearby genes: BORCS7 (43 tissues), NT5C2 (2 tissues), CYP17A1-AS1 (1 tissue), and RP11-724N1.1 (1 tissue). The rs4919690 allele associated with decreased AME is associated with decreased expression of AS3MT (and other coregulated genes). Our study provides a potential biological mechanism for the association between 10q24.32 variation and AME and suggests that the causal variant, represented by rs4919690, may impact AME (as measured in urine) through its effects on arsenic metabolism occurring in multiple tissue types.

Background Homeobox B13 (HOXB13) expression regulates normal prostate development and mutations are associated with prostate cancer (PCa) formation. Objective To assess the role of HOXB13 mRNA expression in PCa progression following radical prostatectomy. Design, setting, and participants Genome-wide expression profiles were queried from two retrospective prostatectomy cohorts with follow-up data (Mayo Clinic, n=780; Johns Hopkins Medical Institute [JHMI], n=355), and a prospective genomic registry (n=5239). Outcome measurements and statistical analysis Multivariable Cox regressions were used to analyze metastasis-free survival (MFS). Results and limitations HOXB13 expression in primary PCa increased with increasing tumor grade and with high metastatic potential based on a genomic signature. The highest quartile of HOXB13 expression was associated with worse MFS compared with the lowest quartile (Mayo Clinic: adjusted hazard ratio [AHR] 1.46, 95% confidence interval [CI] 1.03–2.06, and JHMI: AHR 1.80, 95% CI 1.02–3.19). The combinations of high HOXB13 expression and low expression of its binding partner, MEIS1 (AHR 2.03, 95% CI 1.54–2.66) or MEIS2 (AHR 1.73, 95% CI 1.33–2.26), portended worse MFS. Additionally, high HOXB13 expression in combination with low MEIS1/2 expression correlated with high expression of androgen receptor-mediated genes. The retrospective nature of this study subjects the findings to a bias due to unmeasured variables. Conclusions Primary PCa tumors with increased HOXB13 expression have an increased propensity for metastases following prostatectomy, particularly in the setting of low MEIS1/2 expression. High androgen receptor output may account for worse outcomes for these tumors and suggests heightened sensitivity to androgen suppression. Patient summary Using genomic data from a large number of prostate cancer (PCa) tumors, we found that increased expression of homeobox B13 (HOXB13), a gene related to normal prostate development, was associated with worse outcomes following surgery for PCa. A biomarker signature suggests that these tumors would be more susceptible to androgen suppression, a common treatment for PCa. Take Home Messagece:: In multiple large cohorts, prostate cancer tumors with high homeobox B13 (HOXB13) expression and low expression of its binding partner MEIS1/2 were enriched with high androgen receptor output and had an increased propensity for metastases following surgery.

Purpose: Germline mutations within the MEIS-interaction domain of HOXB13 have implicated a critical function for MEIS–HOX interactions in prostate cancer etiology and progression. The functional and predictive role of changes in MEIS expression within prostate tumor progression, however, remain largely unexplored. Experimental Design: Here we utilize RNA expression datasets, annotated tissue microarrays, and cell-based functional assays to investigate the role of MEIS1 and MEIS2 in prostate cancer and metastatic progression. Results: These analyses demonstrate a stepwise decrease in the expression of both MEIS1 and MEIS2 from benign epithelia, to primary tumor, to metastatic tissues. Positive expression of MEIS proteins in primary tumors, however, is associated with a lower hazard of clinical metastasis (HR = 0.28) after multivariable analysis. Pathway and gene set enrichment analyses identified MEIS-associated networks involved in cMYC signaling, cellular proliferation, motility, and local tumor environment. Depletion of MEIS1 and MEIS2 resulted in increased tumor growth over time in vivo, and decreased MEIS expression in both patient-derived tumors and MEIS-depleted cell lines was associated with increased expression of the protumorigenic genes cMYC and CD142, and decreased expression of AXIN2, FN1, ROCK1, SERPINE2, SNAI2, and TGFβ2. Conclusions: These data implicate a functional role for MEIS proteins in regulating cancer progression, and support a hypothesis whereby tumor expression of MEIS1 and MEIS2 expression confers a more indolent prostate cancer phenotype, with a decreased propensity for metastatic progression. Clin Cancer Res; 24(15); 3668–80. ©2018 AACR.

The current paradigm in the development of new cancer therapies is the ability to target tumor cells while avoiding harm to noncancerous cells. Furthermore, there is a need to develop novel therapeutic options against drug-resistant cancer cells. Herein, we characterized the placental-derived stem cell (PLSC) exosomes (PLSCExo) and evaluated their anti-cancer efficacy in prostate cancer (PCa) cell lines. Nanoparticle tracking analyses revealed the size distribution (average size 131.4 ± 0.9 nm) and concentration of exosomes (5.23 × 1010±1.99 × 109 per ml) secreted by PLSC. PLSCExo treatment strongly inhibited the viability of enzalutamide-sensitive and -resistant PCa cell lines (C4-2B, CWR-R1, and LNCaP cells). Interestingly, PLSCExo treatment had no effect on the viability of a non-neoplastic human prostate cell line (PREC-1). Mass spectrometry (MS) analyses showed that PLSCExo are loaded with 241 proteins and mainly with saturated fatty acids. Further, Ingenuity Pathway Analysis analyses of proteins loaded in PLSCExo suggested the role of retinoic acid receptor/liver x receptor pathways in their biological effects. Together, these results suggest the novel selective anti-cancer effects of PLSCExo against aggressive PCa cells.

Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral androgen receptor inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand the exosome's role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4-2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2-4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride strongly decreased their cell viability. Mechanistic studies revealed upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific small interfering RNAs in Enz-resistant PCa cells (C4-2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4-2B and CWR-R1 cells. The Cancer Genome Atlas analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression-free survival in PCa patients. Importantly, IHC analysis showed higher syntaxin 6 expression in cancer tissues from Enz-treated patients compared to Enz naive patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.

Background WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. Methods Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. Results Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. Conclusions Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.