Development Of Castration-resistant Prostate Cancer Research Articles

Emerging Prognostic Groups Across the Spectrum of Metastatic Castration-Sensitive Prostate Cancer: Disease Outcomes and Genomics

Recent evidence has suggested metastatic cancer represents a disease spectrum rather than a binary state and metastasis-directed therapy (MDT) such as radiation may improve outcomes in some individuals with oligometastatic disease. Within metastatic castration-sensitive prostate cancer (mCSPC) several definitions categorize patients into low and high-volume disease. We assess these definitions, association with clinical outcome, and their mutational landscapes.We retrospectively reviewed patients with biochemically recurrent (BCR) or mCSPC who had undergone somatic next-generation sequencing (n = 294). Patients were classified by low and high-volume disease per CHAARTED criteria, STAMPEDE criteria, oligo- or polymetastatic ≤ 3 vs > 3 and ≤ 5 vs > 5 metastases (mets). Kaplan-Meier survival curves were generated for time to development of castrate-resistant prostate cancer (tdCRPC) for each definition and compared using log rank test. Pearson's χ2 of Fischer's exact test determined differences in driver mutations.Patients were classified with four different definitions as follows: CHAARTED low (53.1%) vs high volume (31.3%); STAMPEDE low (49.7%) vs high burden (35.4%); oligometastatic ≤ 3 (47.3%) vs > 3 (37.4%); and, ≤ 5 (54.1%) vs > 5 (30.6%). Median follow-up for the entire cohort was 52.3 months (mos). Median tdCRPC was not reached in patients with BCR; 85.4 vs 17.3 mos for CHAARTED low vs high, respectively; 99.3 vs 15.7 mos for STAMPEDE low vs high, respectively; 101.4 vs 15.7 mos for ≤ 3 vs > 3 mets respectively; and, 86.4 vs 15.4 mos for ≤ 5 vs > 5 mets respectively (P < 0.001 for all definitions). The frequency of mutations in WNT and cell cycle genes increased across the spectrum from BCR to highest volume of disease in all definitions. TP53 mutation frequency increased across the spectrum in the oligometastatic definitions (Table 1).The spectrum of mCSPC disease biology is generally confirmed across four different definitions of disease burden for tdCRPC and genomics. For purposes of implementing MDT, a practical approach including an enumeration-centric definition integrated with genomics may be best. Future work will involve examining these various definitions and genomics in de novo and metachronous subsets.

SPP1 Promotes Enzalutamide Resistance and Epithelial-Mesenchymal-Transition Activation in Castration-Resistant Prostate Cancer via PI3K/AKT and ERK1/2 Pathways.

The bottleneck arising from castration-resistant prostate cancer (CRPC) treatment is its high metastasis potential and antiandrogen drug resistance, which severely affects survival time of prostate cancer (PCa) patients. Secreted phosphoprotein 1 (SPP1) is a cardinal mediator of tumor-associated inflammation and facilitates metastasis. In our previous study, we firstly revealed SPP1 was a potential hub signature for predicting metastatic CRPC (mCRPC) development. Herein, we integrated multiple databases to explore the association of SPP1 expression with prognosis, survival, and metastatic levels in CRPC progression and investigated SPP1 expression in PCa tissues and cell lines. Next, PCa cell lines with overexpression or depletion of SPP1 were established to study the effect of SPP1 on enzalutamide sensitivity and adhesion and migration of prostate cancer cell lines and further explore the underlying regulatory mechanisms. Bioinformatics analysis, polymerase chain reaction (PCR), immunohistochemical staining, and western blot results suggested SPP1 upregulation had strong relationship with the malignant progression of CRPC and enzalutamide resistance. SPP1 knockdown enhanced enzalutamide sensitivity and repressed invasion and migration of prostate cancer cells. Importantly, upregulating SPP1 promoted, while silencing SPP1 attenuated epithelial-mesenchymal-transition (EMT). Our results further demonstrated that SPP1 overexpression maintains the activation of PI3K/AKT and ERK1/2 signaling pathways. Overall, our findings unraveled the functional role and clinical significance of SPP1 in PCa progression and help to discover new potential targets against mCRPC.

Intermittent Androgen Deprivation Therapy Plus Comprehensive Stereotactic Radiotherapy for Oligometastatic Prostate Cancer (CROP)

<h3>Purpose/Objective(s)</h3> The standard treatment for metastatic prostate cancer is systemic therapy. This prospective phase I/II study assessed the utility of SBRT to all tumor sites plus intermittent androgen deprivation therapy (ADT) in patients with hormone sensitive oligometastatic prostate cancer. Endpoints included incidence of SBRT induced late toxicities and clinical outcomes including cumulative incidences of developing biochemical progression, new metastases, restarting ADT, castration resistant prostate cancer (CRPC), and overall survival (OS). <h3>Materials/Methods</h3> Synchronous and metachronous metastatic disease presentations were eligible if there were ≤ 5 metastases, with ≤ 3 metastases in any one organ system. Conventional scans (CT/bonescan +/- MRI) were used to stage patients at baseline, although novel PET imaging was optional. SBRT was delivered to all sites of disease, including the prostate if not previously treated. SBRT dose was site dependent but was generally 30-35 Gy in 5 fractions for lymph nodes and non-spine bone lesions, 24-28 Gy in 2 fractions for spine, and 35 Gy in 5 fractions to the prostate. ADT was started prior to or immediately after SBRT and was continued for 1 year before moving to an intermittent approach. ADT was to be restarted when the PSA reached ≥ 10 ng/mL or earlier if clinically indicated (development of new metastases or rapidly rising PSA). Toxicity (CTCAE v4.0) and PSA measurements were collected every 4 months during follow-up. Conventional scans were performed at a minimum of once per year, but more frequent imaging was allowed at the discretion of the physician. Time zero was start of ADT or SBRT, whichever was earlier. <h3>Results</h3> 92 patients with 158 metastases were accrued with a median age of 74 years. Median follow-up time was 37 months. Median baseline PSA was 8.5 ng/mL (range 0.78 – 179.8). 39 (42%) patients had Gleason score of 8-10. 32 (35%) patients had synchronous disease presentation. 14 (15%) patients were staged with PET imaging. 5 (5%) patients developed late grade 3 GU toxicity and 6 (7%) patients developed a late SBRT induced bone fracture. There were no grade 4/5 toxicities. Median PSA nadir was 0.02 ng/mL. 50% of patients reached a PSA nadir of ≤ 0.02 ng/mL, while 92% of patients reached a PSA nadir of < 1 ng/mL. The cumulative incidence (CI) of biochemical progression (PSA nadir + 2 ng/mL) was 54% at 4 years. The 4-year CI of developing new metastases and local failure of irradiated sites was 42.8% and 7%, respectively. The 4-year CI of restarting ADT and developing CRPC was 62% and 25%, respectively. OS was 79% at 4 years. <h3>Conclusion</h3> The incidence of grade ≥ 3 toxicity was low when combining SBRT with intermittent ADT for hormone sensitive oligometastatic prostate cancer. With > 3 years of median follow-up, outcomes are promising compared to historical results of using ADT alone, particularly with regards to the development of CRPC. Further work will be done to identify potential predictors of clinical outcomes.

Androgen receptor (AR) heterogeneity in prostate cancer and therapy resistance.

Androgen receptor (AR), a ligand-dependent nuclear transcription factor and a member of steroid hormone receptor family, plays an important role in prostate organogenesis by regulating epithelial differentiation and restricting cell proliferation. Although rarely mutated or amplified in treatment-naïve prostate cancer (PCa), AR signaling drives tumor growth and as a result, therapies that aim to inhibit AR signaling, called ARSIs (AR signaling inhibitors), have been in clinical use for >70 years. Unfortunately, the clinical efficacy of ARSIs is short-lived and the majority of treated patients develop castration-resistant PCa (CRPC). Numerous molecular mechanisms have been proposed for castration resistance; however, the cellular basis for CRPC emergence has remained obscure. One under-appreciated cellular mechanism for CRPC development is the AR heterogeneity that pre-exists in treatment-naive primary tumors, i.e., although most PCa cells express AR (i.e., AR+), there is always a population of PCa cells that express no/low AR (i.e., AR-/lo). Importantly, this AR heterogeneity becomes accentuated during ARSI treatment and highly prominent in established CRPC. Here, we provide a succinct summary of AR heterogeneity across the PCa continuum and discuss its impact on PCa response to treatments. While AR+ PCa cells/clones exhibit exquisite sensitivities to ARSIs, AR-/lo PCa cells/clones, which are greatly enriched in stem cell signaling pathways, display de novo resistance to ARSIs. Finally, we offer several potential combinatorial strategies, e.g., ARSIs with stem cell targeting therapeutics, to co-target both AR+ and AR-/lo PCa cells and metastatic clones.

Blocking GRP/GRP-R signaling decreases expression of androgen receptor splice variants and inhibits tumor growth in castration-resistant prostate cancer

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.

Metformin mitigates PLCε gene expression and modulates the Notch1/Hes and androgen receptor signaling pathways in castration-resistant prostate cancer xenograft models.

The present study aimed to establish a mouse model of patient-derived castration-resistant prostate cancer (CRPC) xenograft tumors, and to evaluate the effects of various doses of metformin on phospholipase Cε (PLCε) expression and the neurogenic locus notch homolog protein 1 (Notch1)/hairy and enhancer of split 1 and androgen receptor (AR) signaling pathways via western blotting and reverse transcription-quantitative PCR. Additionally, phorbol 12-myristate 13-acetate was used to activate PLC, and Jagged1 was used as a Notch activator to verify whether metformin could suppress CRPC development via the PLCε/Notch1/AR pathways. The results confirmed that metformin may serve critical roles in CRPC by significantly inhibiting the occurrence, growth and proliferation of CRPC tumors by decreasing PLCε/Notch1 expression and AR nucleation.

The heterogeneity of prostate cancers lacking AR activity will require diverse treatment approaches.

The use of androgen deprivation therapy and second-line anti-androgens in prostate cancer has led to the emergence of tumors employing multiple androgen receptor (AR)-dependent and AR-independent mechanisms to resist AR-targeted therapies in castration-resistant prostate cancer (CRPC). While the AR signaling axis remains the cornerstone for therapeutic development in CRPC, a clearer understanding of the heterogeneous biology of CRPC tumors is needed for innovative treatment strategies. In this review, we discuss the characteristics of CRPC tumors that lack AR activity and the temporal and spatial considerations for the conversion of an AR-dependent to an AR-independent tumor type. We describe the more prevalent treatment-emergent phenotypes arising in the CRPC disease continuum, including amphicrine, AR-low, double-negative, neuroendocrine and small cell phenotypes. We discuss the association between the loss of AR activity and tumor plasticity with a focus on the roles of transcription factors like SOX2, DNA methylation, alternative splicing, and the activity of epigenetic modifiers like EZH2, BRD4, LSD1, and the nBAF complex in conversion to a neuroendocrine or small cell phenotype in CRPC. We hypothesize that only a subset of CRPC tumors have the propensity for tumor plasticity and conversion to the neuroendocrine phenotype and outline how we might target these plastic and emergent phenotypes in CRPC. In conclusion, we assess the current and future avenues for treatment and determine that the heterogeneity of CRPCs lacking AR activity will require diverse treatment approaches.

KAT2A-mediated AR translocation into nucleus promotes abiraterone-resistance in castration-resistant prostate cancer

Abiraterone, a novel androgen synthesis inhibitor, has been approved for castration-resistant prostate cancer (CRPC) treatment. However, most patients eventually acquire resistance to this agent, and the underlying mechanisms related to this resistance remain largely unelucidated. Lysine acetyltransferase 2 A (KAT2A) has been reported to enhance transcriptional activity for certain histone or non-histone proteins through the acetylation and post-translational modification of the androgen receptor (AR). Therefore, we hypothesised that KAT2A might play a critical role in the resistance of prostate tumours to hormonal treatment. In this study, we found that KAT2A expression was increased in abiraterone-resistant prostate cancer C4-2 cells (C4-2-AbiR). Consistently, elevated expression of KAT2A was observed in patients with prostate cancer exhibiting high-grade disease or biochemical recurrence following radical prostatectomy, as well as in those with poor clinical survival outcomes. Moreover, KAT2A knockdown partially re-sensitised C4-2-AbiR cells to abiraterone, whereas KAT2A overexpression promoted abiraterone resistance in parental C4-2 cells. Consistent with this finding, KAT2A knockdown rescued abiraterone sensitivity and inhibited the proliferation of C4-2-AbiR cells in a mouse model. Mechanistically, KAT2A directly acetylated the hinge region of the AR, and induced AR translocation from the cytoplasm to the nucleus, resulting in increased transcriptional activity of the AR-targeted gene prostate specific antigen (PSA) leading to resistance to the inhibitory effect of abiraterone on proliferation. Taken together, our findings demonstrate a substantial role for KAT2A in the regulation of post-translational modifications in AR affecting CRPC development, suggesting that targeting KAT2A might be a potential strategy for CRPC treatment.

IRE1α Expedites the Progression of Castration-Resistant Prostate Cancers via the Positive Feedback Loop of IRE1α/IL-6/AR.

Castration-resistant prostate cancer (CRPC) is the lethal form of prostate cancer (PCa), and the underlying molecular mechanism has not been fully elucidated. Inositol requiring enzyme 1 alpha (IRE1α), a key regulator of unfolded protein response (UPR), is intimately associated with PCa progression. However, whether IRE1α is implicated in CRPC development remains unknown. Here, we showed that IRE1α expression was significantly increased in CRPC tissues and high-grade PCa tissues. Overexpression of IRE1α promoted PCa cell proliferation under the androgen deficiency condition in vitro and in vivo. Mechanistically, increased IRE1α expression induced IL-6 secretion via the IRE1α/XBP-1s signal pathway. IRE1α-induced IL-6 activated androgen receptor (AR), and the activation of AR by IL-6, in turn, promoted IRE1α expression. IRE1α formed a positive feedback loop with IL-6 and AR to promote prostate cancer cell proliferation under the androgen-deficient condition. In clinical PCa samples, high IRE1α expression correlated with elevated IL-6 and increased PSA expression. Our findings demonstrated a novel mechanism of CRPC progression and suggest targeting IRE1α may be a potential target for the prevention and treatment of CRPC.

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer.

Simple SummaryProstate cancer is the principal cause of cancer-related mortality in men. While localized tumors can be successfully treated by orchiectomy or medical castration, most of the patients ultimately progress to the castration-resistant prostate cancer (CRPC) stage, which is incurable at present. Thus, uncovering the underlying mechanisms that cause CRPC could result in promising therapeutics. Our laboratory has identified LIMK2 kinase as an actionable target for CRPC. LIMK2 is vastly expressed in CRPC but minimally in normal prostates. LIMK2 knockout mice are healthy, indicating that LIMK2 inhibition should have minimal toxicity. LIMK2 is also expressed in other aggressive cancers; however, the molecular mechanisms leading to malignancy remain mostly unknown. This study identified that LIMK2 downregulates a prostate-specific tumor suppressor protein-NKX3.1 using two mechanisms. NKX3.1 loss is strongly associated with prostate cancer. Thus, LIMK2 inhibitor provides a powerful opportunity to rescue NKX3.1 loss, thereby preventing and/or delaying prostate cancer progression.NKX3.1’s downregulation is strongly associated with prostate cancer (PCa) initiation, progression, and CRPC development. Nevertheless, a clear disagreement exists between NKX3.1 protein and mRNA levels in PCa tissues, indicating that its regulation at a post-translational level plays a vital role. This study identified a strong negative relationship between NKX3.1 and LIMK2, which is critical in CRPC pathogenesis. We identified that NKX3.1 degradation by direct phosphorylation by LIMK2 is crucial for promoting oncogenicity in CRPC cells and in vivo. LIMK2 also downregulates NKX3.1 mRNA levels. In return, NKX3.1 promotes LIMK2’s ubiquitylation. Thus, the negative crosstalk between LIMK2-NKX3.1 regulates AR, ARv7, and AKT signaling, promoting aggressive phenotypes. We also provide a new link between NKX3.1 and PTEN, both of which are downregulated by LIMK2. PTEN loss is strongly linked with NKX3.1 downregulation. As NKX3.1 is a prostate-specific tumor suppressor, preserving its levels by LIMK2 inhibition provides a tremendous opportunity for developing targeted therapy in CRPC. Further, as NKX3.1 downregulates AR transcription and inhibits AKT signaling, restoring its levels by inhibiting LIMK2 is expected to be especially beneficial by co-targeting two driver pathways in tandem, a highly desirable requisite for developing effective PCa therapeutics.

β-elemonic acid inhibits growth and triggers apoptosis in human castration-resistant prostate cancer cells through the suppression of JAK2/STAT3/MCL-1 and NF-ĸB signal pathways

Castration-resistant prostate cancer (CRPC) has become a significant problem in the current treatment of prostate cancer (PCa) with the characteristics of high metastatic potential, resistance and easy recurrence. The abnormal activation of JAK2/STAT3/MCL-1 and NF-κB has been confirmed as the main reason for the development of CRPC. We previously found that β-elemonic acid (β-EA) as a natural triterpene has potential anti-inflammatory and anti-osteosarcoma effects with lower toxicity. But it remains unknown whether it had effects on CRPC. The present research in vitro and in vivo systematically investigates anti-cancer effects and mechanisms of β-EA on human CRPC. β-EA treatment resulted in apoptotic cell death in human PCa cells by mitochondrial apoptotic pathways (including up-regulation of cleaved caspase-3, cleaved PARP, and Bax or down-regulation of Bcl-2). Besides, β-EA at relatively lower levels inhibited colony-forming, the migration and invasion potential of PCa cells, indicating its anti-proliferation and anti-metastasis activities. After exploring the potential mechanism, our results suggested that it subsequently inhibited the activation of JAK2/STAT3/MCL-1 and NF-κB signaling pathway by the administration of β-EA. The silencing of NF-κB/p65, JAK2 and STAT3, respectively, increased the sensitivity of the PCa cells to β-EA induced apoptosis. Moreover, β-EA exhibited a strong affinity with its essential proteins JAK2, RELA/p65, NF-κBIα/IκBα by molecular docking analysis. Importantly, β-EA retards tumor growth in a murine xenograft model, consistent with our study in vitro. Taken together, findings from this study reveal for the first time the potential role and mechanisms of β-EA on CRPC.

Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is a terminal disease and the molecular underpinnings of CRPC development need to be better understood in order to improve its treatment. Here, we report that a transcription factor Yin Yang 1 (YY1) is significantly overexpressed during prostate cancer progression. Functional and cistrome studies of YY1 uncover its roles in promoting prostate oncogenesis in vitro and in vivo, as well as sustaining tumor metabolism including the Warburg effect and mitochondria respiration. Additionally, our integrated genomics and interactome profiling in prostate tumor show that YY1 and bromodomain-containing proteins (BRD2/4) co-occupy a majority of gene-regulatory elements, coactivating downstream targets. Via gene loss-of-function and rescue studies and mutagenesis of YY1-bound cis-elements, we unveil an oncogenic pathway in which YY1 directly binds and activates PFKP, a gene encoding the rate-limiting enzyme for glycolysis, significantly contributing to the YY1-enforced Warburg effect and malignant growth. Altogether, this study supports a master regulator role for YY1 in prostate tumorigenesis and reveals a YY1:BRD2/4-PFKP axis operating in advanced prostate cancer with implications for therapy.

Androgen receptor enhancer amplification in matched patient‐derived xenografts of primary and castrate‐resistant prostate cancer

Amplifications of the androgen receptor (AR) occur in up to 80% of men with castration-resistant prostate cancer (CRPC). Recent studies highlighted that these amplifications not only span the AR gene, but usually encompass a distal enhancer. This represents a newly recognised, non-coding mechanism of resistance to AR-directed therapies, including enzalutamide. To study disease progression before and after AR amplification, we used tumour samples from a castrate-sensitive primary tumour and castrate-resistant metastasis of the same patient. For subsequent functional and genomic studies, we established serially transplantable patient-derived xenografts (PDXs). Whole genome sequencing showed that alterations associated with poor prognosis, such as TP53 and PTEN loss, existed before androgen deprivation therapy, followed by co-amplification of the AR gene and enhancer after the development of metastatic CRPC. The PDX of the primary tumour, without the AR amplification, was sensitive to AR-directed treatments, including castration, enzalutamide, and apalutamide. The PDX of the metastasis, with the AR amplification, had higher AR and AR-V7 expression in castrate conditions, and was resistant to castration, apalutamide, and enzalutamide in vivo. Treatment with a BET inhibitor outperformed the AR-directed therapies for the metastasis, resulting in tumour regression for some, but not all, grafts. Therefore, this study provides novel matched PDXs to test potential treatments that target the over-abundance of AR in tumours with AR enhancer amplifications. This article is protected by copyright. All rights reserved.

Decoding the heterogeneous landscape in the development prostate cancer.

Prostate cancer (PCa) is characterized as being histologically and molecularly heterogeneous; however, this is not only incorrect among individuals, but also at the multiple foci level, which originates in the prostate gland itself. The reasons for such heterogeneity have not been fully elucidated; however, understanding these may be crucial in determining the course of the disease. PCa is characterized by a complex network of chromosomal rearrangements, which simultaneously deregulate multiple genes; this could explain the appearance of exclusive events associated with molecular subtypes, which have been extensively investigated to establish clinical management and the development of therapies targeted to this type of cancer. From a clinical aspect, the prognosis of the patient has focused on the characteristics of the index lesion (the largest focus in PCa); however, a significant percentage of patients (11%) also exhibit an aggressive secondary foci, which may determine the prognosis of the disease, and could be the determining factor of why, in different studies, the classification of the subtypes does not have an association with prognosis. Due to the aforementioned reasons, the analysis of molecular subtypes in several foci, from the same individual could assist in determining the association between clinical evolution and management of patients with PCa. Castration-resistant PCa (CRPC) has the worst prognosis and develops following androgen ablation therapy. Currently, there are two models to explain the development of CRPC: i) The selection model and ii) the adaptation model; both of which, have been found to include alterations described in the molecular subtypes, such as Enhancer of zeste 2 polycomb repressive complex 2 subunit overexpression, isocitrate dehydrogenase (NAPD+)1 and forkhead box A1 mutations, suggesting that the presence of specific molecular alterations could predict the development of CRPC. This type of analysis could lead to a biological understanding of PCa, to develop personalized medicine strategies, which could improve the response to treatment thus, avoiding the development of resistance. Therefore, the present review discusses the primary molecular factors, to which variable heterogeneity in PCa progress has been attributed.

ZRSR2 overexpression is a frequent and early event in castration-resistant prostate cancer development

BackgroundAndrogen deprivation therapy (ADT) remains the leading systemic therapy for locally advanced and metastatic prostate cancers (PCa). While a majority of PCa patients initially respond to ADT, the durability of response is variable and most patients will eventually develop incurable castration-resistant prostate cancer (CRPC). Our research objective is to identify potential early driver genes responsible for CRPC development.MethodsWe have developed a unique panel of hormone-naïve PCa (HNPC) patient-derived xenograft (PDX) models at the Living Tumor Laboratory. The PDXs provide a unique platform for driver gene discovery as they allow for the analysis of differentially expressed genes via transcriptomic profiling at various time points after mouse host castration. In the present study, we focused on genes with expression changes shortly after castration but before CRPC has fully developed. These are likely to be potential early drivers of CRPC development. Such genes were further validated for their clinical relevance using data from PCa patient databases. ZRSR2 was identified as a top gene candidate and selected for further functional studies.ResultsZRSR2 is significantly upregulated in our PDX models during the early phases of CRPC development after mouse host castration and remains consistently high in fully developed CRPC PDX models. Moreover, high ZRSR2 expression is also observed in clinical CRPC samples. Importantly, elevated ZRSR2 in PCa samples is correlated with poor patient treatment outcomes. ZRSR2 knockdown reduced PCa cell proliferation and delayed cell cycle progression at least partially through inhibition of the Cyclin D1 (CCND1) pathway.ConclusionUsing our unique HNPC PDX models that develop into CRPC after host castration, we identified ZRSR2 as a potential early driver of CRPC development.

Phage display screening identifies a prostate specific antigen (PSA)–/lo prostate cancer cell specific peptide to retard castration resistance of prostate cancer

Patients with prostate cancer (PCa) will eventually progress to castrate-resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT) treatment. Prostate-specific antigen (PSA)−/lo cells which harbor self-renewing long-term tumor-propagating cells that can be enriched using ALDH+CD44+α2β1+ and can initiate tumor development may represent a critical source of CRPC cells. Our purpose was to find a peptide that specifically targets PSA−/lo PCa cells to retard the development of CRPC. PSA+ and PSA−/lo cells were successfully separated from LNCaP xenograft tumors after prostate- PSAP-GFP vector infection and FACS. A variety of PSA−/lo cells specifically targeting peptide (named as “TAP1” targeted affinity peptide 1) was identified by using phage display library screening. The highest binding rate in TAP1 binding cell subpopulations are identified to be among ALDH+CD44+CXCR4+CD24+ cells. TAP1 significantly inhibited PCa growth both in vitro and in vivo. TAP1 significantly improved the anti-proliferation effect of the anti-androgens (Charcoal dextran-stripped serum (CDSS)+Bicalutamide, Enzalutamide) and chemotherapeutic agents (Abiraterone, Docetaxel, Etoposide) in vitro. TAP1 treatment shortens the length of telomeres in ALDH+CD44+CXCR4+CD24+ cells and significantly reduces the expression of Homeobox B9 (HOXB9) and TGF-β2. In conclusion, PSA−/lo PCa cell-specific targeting peptide (TAP1) that suppressed PCa cell growth both in vitro and in vivo and improved the drug sensitivities of anti-androgens and chemotherapeutic agents at least through shortening the length of telomere and reducing the expression of HOXB9 and TGF-β2. Therapeutic peptides that specifically target prostate cancer stem cell might be a very valuable and promising approach to overcome chemoresistance and prevent recurrence in patients with PCa.

Mesoporous silica nanoparticles combined with AKR1C3 siRNA inhibited the growth of castration-resistant prostate cancer by suppressing androgen synthesis in vitro and in vivo

Intracrine androgen synthesis plays a critical role in the development of castration-resistant prostate cancer (CRPC). Aldo-keto reductase family 1 member C3 (AKR1C3) is a vital enzyme in the intracrine androgen synthesis pathway. In this study, mesoporous silica nanoparticles (MSNs) were employed to deliver small interfering RNA targeting AKR1C3 (siAKR1C3) to downregulate AKR1C3 expression in CPRC cells. The optimal weight ratio of MSNs/siAKR1C3 was determined by a gel retardation assay. Prostate cancer cells such as VCaP cells, which intracrinally express AKR1C3, and LNCaP-AKR1C3 cells stably transfected with AKR1C3 were used to investigate the antitumour effect of MSNs-siAKR1C3. Fluorescence detection and Western blot analyses were applied to confirm the entrance of MSNs-siAKR1C3 into the cells. A SRB (Sulforhodamine B) assay was employed to assess the cell viability, and a radioimmunoassay was used to measure the androgen concentration. Moreover, real-time PCR (RT-PCR), Western blot analysis and ELISA were used to determine the transcription and expression of prostate-specific antigen (PSA), AKR1C3 and androgen receptor (AR). Meanwhile, a reporter gene assay was performed to determine the AR activity. Additionally, a castrated nude mouse xenograft tumour model was produced to verify the inhibitory effect of MSNs-siAKR1C3 in vivo. The results showed that the optimal weight ratio of MSNs/siAKR1C3 was 140:1, and the complex could effectively enter cells, downregulate AKR1C3 expression, reduce the androgen concentration, inhibit AR activation, and inhibit CRPC development both in vitro and in vivo. These results indicate that decreasing intracrine androgen synthesis and inactivating AR signals by MSNs-siAKR1C3 may be a potential effective method for CRPC treatment.

The Mutational Landscape of Metastatic Castration-sensitive Prostate Cancer: The Spectrum Theory Revisited

BackgroundEmerging data suggest that metastasis is a spectrum of disease burden rather than a binary state, and local therapies, such as radiation, might improve outcomes in oligometastasis. However, current definitions of oligometastasis are solely numerical. ObjectiveTo characterize the somatic mutational landscape across the disease spectrum of metastatic castration-sensitive prostate cancer (mCSPC) to elucidate a biological definition of oligometastatic CSPC. Design, setting, and participantsThis was a retrospective study of men with mCSPC who underwent clinical-grade sequencing of their tumors (269 primary tumor, 25 metastatic sites). Patients were classified as having biochemically recurrent (ie, micrometastatic), metachronous oligometastatic (≤5 lesions), metachronous polymetastatic (>5 lesions), or de novo metastatic (metastasis at diagnosis) disease. Outcome measurements and statistical analysisWe measured the frequency of driver mutations across metastatic classifications and the genomic associations with radiographic progression-free survival (rPFS) and time to castrate-resistant prostate cancer (CRPC). Results and limitationsThe frequency of driver mutations in TP53 (p = 0.01), WNT (p = 0.08), and cell cycle (p = 0.04) genes increased across the mCSPC spectrum. TP53 mutation was associated with shorter rPFS (26.7 vs 48.6 mo; p = 0.002), and time to CRPC (95.6 vs 155.8 mo; p = 0.02) in men with oligometastasis, and identified men with polymetastasis with better rPFS (TP53 wild-type, 42.7 mo; TP53 mutated, 18.5 mo; p = 0.01). Mutations in TP53 (incidence rate ratio [IRR] 1.45; p = 0.004) and DNA double-strand break repair (IRR 1.61; p < 0.001) were associated with a higher number of metastases. Mutations in TP53 were also independently associated with shorter rPFS (hazard ratio [HR] 1.59; p = 0.03) and the development of CRPC (HR 1.71; p = 0.01) on multivariable analysis. This study was limited by its retrospective nature, sample size, and the use of commercially available sequencing platforms, resulting in a limited predefined set of genes examined. ConclusionsSomatic mutational profiles reveal a spectrum of metastatic biology that helps in redefining oligometastasis beyond a simple binary state of lesion enumeration. Patient summaryOligometastatic prostate cancer is typically defined as less than three to five metastatic lesions and evidence suggests that using radiation or surgery to treat these sites improves clinical outcomes. As of now, treatment decisions for oligometastasis are solely defined according to the number of lesions. However, this study suggests that tumor mutational profiles can provide a biological definition of oligometastasis and complement currently used numerical definitions.

Targeting KDM4B that coactivates c-Myc-regulated metabolism to suppress tumor growth in castration-resistant prostate cancer.

Rationale: The progression of prostate cancer (PCa) to castration-resistant PCa (CRPC) despite continuous androgen deprivation therapy is a major clinical challenge. Over 90% of patients with CRPC exhibit sustained androgen receptor (AR) signaling. KDM4B that removes the repressive mark H3K9me3/2 is a transcriptional activator of AR and has been implicated in the development of CRPC. However, the mechanisms of KDM4B involvement in CRPC remain largely unknown. Here, we sought to demonstrate the molecular pathway mediated by KDM4B in CRPC and to provide proof-of-concept evidence that KDM4B is a potential CRPC target.Methods: CRPC cells (C4-2B or CWR22Rv1) depleted with KDM4B followed by cell proliferation (in vitro and xenograft), microarray, qRT-PCR, Seahorse Flux, and metabolomic analyses were employed to identify the expression and metabolic profiles mediated by KDM4B. Immunoprecipitation was used to determine the KDM4B-c-Myc interaction region. Reporter activity assay and ChIP analysis were used to characterize the KDM4B-c-Myc complex-mediated mechanistic actions. The clinical relevance between KDM4B and c-Myc was determined using UCSC Xena analysis and immunohistochemistry.Results: We showed that KDM4B knockdown impaired CRPC proliferation, switched Warburg to OXPHOS metabolism, and suppressed gene expressions including those targeted by c-Myc. We further demonstrated that KDM4B physically interacted with c-Myc and they were co-recruited to the c-Myc-binding sequence on the promoters of metabolic genes (LDHA, ENO1, and PFK). Importantly, KDM4B and c-Myc synergistically promoted the transactivation of the LDHA promoter in a demethylase-dependent manner. We also provided evidence that KDM4B and c-Myc are co-expressed in PCa tissue and that high expression of both is associated with worse clinical outcome.Conclusions: KDM4B partners with c-Myc and serves as a coactivator of c-Myc to directly enhance c-Myc-mediated metabolism, hence promoting CRPC progression. Targeting KDM4B is thus an alternative therapeutic strategy for advanced prostate cancers driven by c-Myc and AR.

Identification of marker genes and cell subtypes in castration-resistant prostate cancer cells

The diverse tumor cell populations may be the critical roles in relapse and resistance to treatment in prostate cancer patients. This study aimed to identify new marker genes and cell subtypes among castration-resistant prostate cancer (CRPC) cells. We downloaded single-cell RNA seq profiles (GSE67980) from the Gene Expression Omnibus (GEO) database. Principal component (PC) analysis and t-Distributed Stochastic Neighbor Embedding (TSNE) analysis were performed to identify marker genes. CRPC cells were clustered and annotated. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses among marker genes were performed. A total of 1500 genes with larger standardized variance were obtained. The top 20 genes were demonstrated in each identified 20 PCs. PC with P-value < 0.05 was selected, including PC1, PC7, PC8, and PC14. The TSNE analysis classified cells as two clusters. The top 6 genes in cluster 0 included HBB, CCL5, SLITRK4, GZMB, BBIP1, and PF4V1. Plus, the top 6 genes in cluster 1 included MLEC, CCT8, CCT3, EPCAM, TMPRSS2, EIF4G2. The GO analysis revealed that these marker genes were mainly enriched in RNA catabolic process, translational initiation, mitochondrial inner membrane, cytosolic part, ribosome, cell adhesion molecule binding, cadherin binding, and structural constituent of ribosome. The KEGG analysis showed that these marker genes mainly enriched in metabolism associated pathways, including carbon metabolism, cysteine and methionine metabolism, propanoate metabolism, pyruvate metabolism, and citrate cycle pathways. To conclude, our results provide essential insights into the spectrum of cellular heterogeneity within human CRPC cells. These marker genes, GO terms and pathways may be critical in the development and progression of human CRPC.