Pathogenesis Of Castration-resistant Prostate Cancer Research Articles

Role of gut microbiota in the pathogenesis of castration-resistant prostate cancer: a comprehensive study using sequencing and animal models.

CRPC remains a significant challenge in prostate cancer research. We aimed to elucidate the role of gut microbiota and its specific mechanisms in CRPC using a multidisciplinary approach. We analyzed 16S rRNA sequencing data from mouse fecal samples, revealing substantial differences in gut microbiota composition between CRPC and castration-sensitive prostate cancer mice, particularly in Firmicutes and Bacteroidetes. Functional analysis suggested different bacteria may influence CRPC via the α-linolenic acid metabolism pathway. In vivo, experiments utilizing mouse models and fecal microbiota transplantation (FMT) demonstrated that FMT from healthy control mice could decelerate tumor growth in CRPC mice, reduce TNF-α levels, and inhibit the activation of the TLR4/MyD88/NF-κB signaling pathway. Transcriptome sequencing identified crucial genes and pathways, with rescue experiments confirming the gut microbiota's role in modulating CRPC progression through the TLR4/MyD88/NF-κB pathway. The activation of this pathway by TNF-α has been corroborated by in vitro cell experiments, indicating its role in promoting prostate cancer cell proliferation, migration, and invasion while inhibiting apoptosis. Gut microbiota dysbiosis may promote CRPC development through TNF-α activation of the TLR4/MyD88/NF-κB signaling pathway, potentially linked to α-linolenic acid metabolism.

Identification of Molecular Markers Associated with Prostate Cancer Subtypes: An Integrative Bioinformatics Approach.

Prostate cancer (PCa) is characterised by androgen dependency. Unfortunately, under anti-androgen treatment pressure, castration-resistant prostate cancer (CRPC) emerges, characterised by heterogeneous cell populations that, over time, lead to the development of different androgen-dependent or -independent phenotypes. Despite important advances in therapeutic strategies, CRPC remains incurable. Context-specific essential genes represent valuable candidates for targeted anti-cancer therapies. Through the investigation of gene and protein annotations and the integration of published transcriptomic data, we identified two consensus lists to stratify PCa patients' risk and discriminate CRPC phenotypes based on androgen receptor activity. ROC and Kaplan-Meier survival analyses were used for gene set validation in independent datasets. We further evaluated these genes for their association with cancer dependency. The deregulated expression of the PCa-related genes was associated with overall and disease-specific survival, metastasis and/or high recurrence risk, while the CRPC-related genes clearly discriminated between adeno and neuroendocrine phenotypes. Some of the genes showed context-specific essentiality. We further identified candidate drugs through a computational repositioning approach for targeting these genes and treating lethal variants of PCa. This work provides a proof-of-concept for the use of an integrative approach to identify candidate biomarkers involved in PCa progression and CRPC pathogenesis within the goal of precision medicine.

Silencing of matrix metalloprotease-12 delays the progression of castration-resistant prostate cancer by regulating autophagy and lipolysis.

The complex pathogenesis of castration-resistant prostate cancer (CRPC) makes it challenging to identify effective treatment methods. Matrix metalloproteinase (MMP)-12 can degrade elastin as well as various extracellular matrix (ECM) components, which is associated with cancer progression. However, the relationship between MMP-12 and CRPC progression is poorly understood. In this study, we observed the effect of MMP-12 on the progression of CRPC and further explored its potential mechanism of action. High levels of MMP-12 were observed in patients with CRPC. We therefore developed cell co-culture and mouse models to study the function of MMP-12. Silencing MMP-12 in CRPC cells disrupted lipid utilization and autophagy marker expression via the CD36/CPT1 and P62/LC3 pathways, respectively, leading to reduced CRPC cell migration and invasion. Moreover, animal experiments confirmed that MMP-12-knockdown CRPC xenograft tumors exhibited reduced tumor growth, and the mechanisms involved the promotion of cancer cell autophagy and the inhibition of lipid catabolism. According to our results, MMP-12 played important roles in the progression of CRPC by disrupting adipocyte maturation and regulating cancer migration and invasion via the modulation of autophagy and lipid catabolism pathways.

Current and emerging approaches to noncompetitive AR inhibition.

The androgen receptor (AR) has been shown to be a key determinant in the pathogenesis of castration-resistant prostate cancer (CRPC). The current standard of care therapies targets the ligand-binding domain of the receptor and can afford improvements to life expectancy often only in the order of months before resistance occurs. Emerging preclinical and clinical compounds that inhibit receptor activity via differentiated mechanisms of action which are orthogonal to current antiandrogens show promise for overcoming treatment resistance. In this review, we present an authoritative summary of molecules that noncompetitively target the AR. Emerging small molecule strategies for targeting alternative domains of the AR represent a promising area of research that shows significant potential for future therapies. The overall quality of lead candidates in the area of noncompetitive AR inhibition is discussed, and it identifies the key chemotypes and associated properties which are likely to be, or are currently, positioned to be first in human applications.

Inorganic pyrophosphatase, a human prostate cancer activator

Prostate cancer (PCa) represents a common tumor in men. Inorganic pyrophosphatase (PPA1) can promote inorganic pyrophosphate (PPi) hydrolysis. However, whether PPA1 participates in castration-resistant prostate cancer (CRPC) is unclear. PPA1 expression was herein determined by immunohistochemistry along with analysis of cell behaviors by flow cytometry and CCK-8 assay. Cell invasion was measured by BD Matrigel matrix. PPA1 was found to be significantly upregulated and positively correlated with Gleason grade of PCa. PPA1 expression was significantly higher in the PC-3 and DU145 cells than LNCaP cells and Benign prostatic hyperplasia (BPH)-1 cells. Additionally, stable overexpression of AR reduced PPA1 expression, implying the important roles of PPA1 in CRPC pathogenesis. Moreover, knockdown of PPA1 inhibited cell activities and behaviors. Furthermore, silencing of PPA1 reduced the expression of cell proliferation markers Ki-67 and PCNA, cell cycle protein cyclin D1, cyclin E, c-Myc, and MMP-2/9. Moreover, it also inhibited epithelial to mesenchymal transition (EMT) by upregulating E-cadherin and ZO-1, but downregulating N-cadherin and ZEB-2. Mechanistically, silencing of PPA1 inhibited NF-κB activation through reduction of IκBα and p65 phosphorylation. Our findings provide a critical link between PPA1 and CRPC.

Advances in the Current Understanding of the Mechanisms Governing the Acquisition of Castration-Resistant Prostate Cancer.

Simple SummaryCastration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer with a low survival rate, as the CRPC patients only survive for 9–13 months on average. In this narrative review, we first outline the most common androgen receptor (AR) receptor-related mechanisms, highlighting the important role of ARs in the development of CRPC. We also discuss the key importance of non-coding RNAs (ncRNAs) in this setting, including long ncRNAs and microRNAs. Overall, studies of the molecular biological mechanisms governing the CRPC will facilitate the development of appropriate targeted therapeutics, improving treatment options for the CRPC patients.Despite aggressive treatment and androgen-deprivation therapy, most prostate cancer patients ultimately develop castration-resistant prostate cancer (CRPC), which is associated with high mortality rates. However, the mechanisms governing the development of CRPC are poorly understood, and androgen receptor (AR) signaling has been shown to be important in CRPC through AR gene mutations, gene overexpression, co-regulatory factors, AR shear variants, and androgen resynthesis. A growing number of non-AR pathways have also been shown to influence the CRPC progression, including the Wnt and Hh pathways. Moreover, non-coding RNAs have been identified as important regulators of the CRPC pathogenesis. The present review provides an overview of the relevant literature pertaining to the mechanisms governing the molecular acquisition of castration resistance in prostate cancer, providing a foundation for future, targeted therapeutic efforts.

Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models

BackgroundNKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1’s levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date.MethodsA chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells.ResultsWe uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it.ConclusionsNKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.

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.

Molecular Interplay between AURKA and SPOP Dictates CRPC Pathogenesis via Androgen Receptor.

Simple SummaryProstate cancer is the most common type of cancer in men. Early stage prostate cancer is treatable. However, remission eventually occurs in vast majority of patients, giving rise to castration-resistant prostate cancer (CRPC), which is incurable with current therapies. Aurora A (AURKA) is overexpressed in all stages of prostate cancer including CRPC. AURKA inhibition has shown efficacy in reducing cancer burden in clinical trials, however, no AURKA inhibitor has been approved by the FDA, primarily because AURKA inhibition is toxic to normal cells. Thus, identification of downstream targets of AURKA provides an alternative approach to regulate AURKA-mediated malignancy. We identified a tumor suppressor protein named SPOP as AURKA target. SPOP is rendered ineffective in prostate cancer by genomic mutations promoting cancer. We show that AURKA degrades SPOP, which promotes malignancy and drug-resistance. Thus, AURKA inhibition provides a powerful tool to retain SPOP, thereby treating the disease and inhibiting its progression. SPOP, an adaptor protein for E3 ubiquitin ligase can function as a tumor-suppressor or a tumor-enhancer. In castration-resistant prostate cancer (CRPC), it inhibits tumorigenesis by degrading many oncogenic targets, including androgen receptor (AR). Expectedly, SPOP is the most commonly mutated gene in CRPC (15%), which closely correlates with poor prognosis. Importantly, 85% of tumors that retain wild-type SPOP show reduced protein levels, indicating that SPOP downregulation is an essential step in CRPC progression. However, the underlying molecular mechanism remains unknown. This study uncovered the first mechanism of SPOP regulation in any type of cancer. We identified SPOP as a direct substrate of Aurora A (AURKA) using an innovative technique. AURKA directly phosphorylates SPOP at three sites, causing its ubiquitylation. SPOP degradation drives highly aggressive oncogenic phenotypes in cells and in vivo including stabilizing AR, ARv7 and c-Myc. Further, SPOP degrades AURKA via a feedback loop. SPOP upregulation is one of the mechanisms by which enzalutamide exerts its efficacy. Consequently, phospho-resistant SPOP fully abrogates tumorigenesis and EMT in vivo, and renders CRPC cells sensitive to enzalutamide. While genomic mutations of SPOP can be treated with gene therapy, identification of AURKA as an upstream regulator of SPOP provides a powerful opportunity for retaining WT-SPOP in a vast majority of CRPC patients using AURKA inhibitors ± enzalutamide, thereby treating the disease and inhibiting its progression.

Negative cross talk between LIMK2 and PTEN promotes castration resistant prostate cancer pathogenesis in cells and in vivo.

Androgen deprivation therapy (ADT) and androgen receptor (AR) signaling inhibitors are front-line treatments for highly aggressive prostate cancer. However, prolonged inhibition of AR triggers a compensatory activation of PI3K pathway, most often due to the genomic loss of tumor suppressor PTEN, driving progression to the castration-resistant prostate cancer (CRPC) stage, which has very poor prognosis. We uncovered a novel mechanism of PTEN downregulation triggered by LIMK2, which contributes significantly to CRPC pathogenesis. LIMK2 is a CRPC-specific target. Its depletion fully reverses tumorigenesis in vivo. LIMK2 phosphorylates PTEN at five sites, degrading and inhibiting its activity, thereby driving highly aggressive oncogenic phenotypes in cells and in vivo. PTEN also degrades LIMK2 in a feedback loop, which was confirmed in prostates from PTEN-/- and PTEN+/+ mice. LIMK2 is also the missing link between hypoxia and PTEN degradation in CRPC. This is the first study to show a feedback loop between PTEN and its regulator. Uncovering the LIMK2-PTEN loop provides a powerful therapeutic opportunity to retain the activity and stability of PTEN protein by inhibiting LIMK2, thereby halting the progression to CRPC, ADT-resistance and drug-resistance.

Annexin A1 promotes the nuclear localization of the epidermal growth factor receptor in castration-resistant prostate cancer.

Epidermal growth factor receptor is a cancer driver whose nuclear localization has been associated with the progression of prostate cancer to the castration-resistant phenotype. Previous reports indicated a functional interaction between this receptor and the protein Annexin A1, which has also been associated with aggressive tumors. The molecular pathogenesis of castration-resistant prostate cancer remains largely unresolved, and herein we have demonstrated the correlation between the expression levels and localization of the epidermal growth factor receptor and Annexin A1 in prostate cancer samples and cell lines. Interestingly, a higher expression of both proteins was detected in castration-resistant prostate cancer cell lines and the strongest correlation was seen at the nuclear level. We verified that Annexin A1 interacts with the epidermal growth factor receptor, and by using prostate cancer cell lines knocked down for Annexin A1, we succeeded in demonstrating that Annexin A1 promotes the nuclear localization of epidermal growth factor receptor. Finally, we showed that Annexin A1 activates an autocrine signaling in castration-resistant prostate cells through the formyl peptide receptor 1. The inhibition of such signaling by Cyclosporin H inhibits the nuclear localization of epidermal growth factor receptor and its downstream signaling. The present work sheds light on the functional interaction between nuclear epidermal growth factor receptor and nuclear Annexin A1 in castration-resistant prostate cancer. Therefore, strategies to inhibit the nuclear localization of epidermal growth factor receptor through the suppression of the Annexin A1 autocrine loop could represent an important intervention strategy for castration-resistant prostate cancer.

MYSM1-AR complex-mediated repression of Akt/c-Raf/GSK-3β signaling impedes castration-resistant prostate cancer growth.

Epigenetic alterations that lead to dysregulated gene expression in the progression of castration-resistant prostate cancer (CRPC) remain elusive. Here, we investigated the role of histone deubiquitinase MYSM1 in the pathogenesis of prostate cancer (PCa). Tissues and public datasets of PCa were evaluated for MYSM1 levels. We explored the effects of MYSM1 on cell proliferation, senescence and viability both in vitro and in vivo. Integrative database analyses and co-immunoprecipitation assays were performed to elucidate genomic association of MYSM1 and MYSM1-involved biological interaction network in PCa. We observed that MYSM1 were downregulated in CRPC compared to localized prostate tumors. Knockdown of MYSM1 promoted cell proliferation and suppressed senescence of CRPC cells under condition of androgen ablation. MYSM1 downregulation enhanced the tumorigenic ability in nude mice. Integrative bioinformatic analyses of the significantly associated genes with MYSM1 revealed MYSM1-correlated pathways, providing substantial clues as to the role of MYSM1 in PCa. MYSM1 was able to bind to androgen receptor instead of increasing its expression and knockdown of MYSM1 resulted in activation of Akt/c-Raf/GSK-3β signaling. Together, our findings indicate that MYSM1 is pivotal in CRPC pathogenesis and may be established as a potential target for future treatment.

Abstract 415A: Regulation of NCAPG by miR-99a-3p (passenger strand) inhibits cancer cell aggressiveness and is involved in CRPC

Abstract In developed countries, prostate cancer (PCa) is one of the most commonly diagnosed cancers, identified by prostate-specific antigen (PSA) screening; PCa is also the third leading cause of cancer-related death among men. Most naïve PCa initially responds well to androgen-deprivation therapy (ADT). However, during ADT treatment, PCa cells acquire ADT treatment resistance and progress to a lethal pathology known as castration-resistant prostate cancer (CRPC). Cancer cells that have reached CRPC can cause distant metastasis, and effective treatments for patients with CRPC have not yet been established. Identification of the molecular pathogenesis underlying acquisition of androgen-independent and metastatic signaling pathways based on advanced genomic approaches is essential for further understanding of this disease.MicroRNAs (miRNAs) are endogenous small RNA (molecules 18-23 bases in length) that act as central players regulating the expression control of protein-coding and protein-noncoding RNAs. Interestingly, a single miRNA can directly regulate a vast number of RNAs in human cells. Therefore, aberrant expression of miRNAs can disrupt normal control of RNA expression in cancer cells. Furthermore, dysregulation of miRNAs is contributed to cancer cell malignancies, such as progression, metastasis, and treatment resistance. Analyses of RNA-sequencing of microRNA (miRNA) expression revealed that miR-99a-3p (passenger strand) is significantly downregulated in several types of cancers. In this study, we aimed to identify novel miR-99a-3p regulatory networks and therapeutic targets for CRPC. The expression levels of miR-99a-3p was significantly lower in PCa and CRPC tissues than in normal tissues (P = 0.0047 and P = 0.0001). Ectopic expression of miR-99a-3p significantly inhibited cancer cell proliferation, migration, and invasion in PCa cells (P < 0.0001). Non-SMC condensin I complex subunit G (NCAPG) was a direct target of miR-99a-3p in PCa cells. Overexpression of NCAPG was detected in CRPC clinical specimens and TCGA data showed that high NCAPG expression was significantly associated with shorter disease-free survival (P = 0.0009) and advanced clinical stage in PCa. Knockdown of NCAPG markedly inhibited cancer cell proliferation, migration, and invasion (P < 0.0001). Moreover, the proliferation, migration, and invasion abilities were recovered by cotransfection with NCAPG and miR-99a-3p. These results indicated that NCAPG affected the aggressiveness in PCa. The passenger strand miR-99a-3p acted as an antitumor miRNA in naïve PCa and CRPC. NCAPG was regulated by miR-99a-3p, and its overexpression was involved in CRPC pathogenesis. Involvement of passenger strand of miRNA in cancer pathogenesis is novel concept and identification of antitumor miRNA regulatory networks in CRPC might be provided novel prognostic markers and therapeutic targets for this disease. Citation Format: Takayuki Arai, Atsushi Okato, Yasutaka Yamada, Sho Sugawara, Akira Kurozumi, Satoko Kojima, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Regulation of NCAPG by miR-99a-3p (passenger strand) inhibits cancer cell aggressiveness and is involved in CRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 415A.

Regulation of the Antioxidant Response by MyoD Transcriptional Coactivator in Castration-resistant Prostate Cancer Cells

To reveal the potential role of the basic helix-loop-helix myogenic transcription regulator MyoD in the regulation of castration-resistant prostate cancer. Expression level of MyoD was assessed in prostate cancer tissues using quantitative reverse transcription polymerase chain reaction and immunohistochemistry and in experimentally induced castration-resistant LNCaP/R cells using quantitative reverse transcription polymerase chain reaction and immunoblotting. Effect of MyoD knockdown on LNCaP/R cell progression was determined by assessing cell proliferation, apoptosis, and colony formation rate. The effect of MyoD knockdown on the oxidative stress state in PC3 cells was determined by assessing antioxidant response gene expression and glutathione synthetase-to-glutathione ratio. Finally, the functional link between the nuclear factor erythroid-derived 2-related factor 1 (NRF1) and the regulation of antioxidant response element-driven transcription by MyoD was studied at both molecular and functional levels. MyoD expression was significantly upregulated in hormone-refractory prostate cancer tissues and in experimentally induced castration-resistant LNCaP/R cells, and MyoD knockdown effectively impaired LNCaP/R cell proliferation and promoted apoptosis under androgen-depleted condition. Moreover, MyoD enhanced the glutathione production and protected against oxidative stress by positively regulating a cluster of antioxidant genes known to be the downstream targets of NRF1. Mechanistically, MyoD could augment the antioxidant response element-driven transcription in an NRF1-dependent manner, and the stimulatory effect of MyoD on the antioxidant response was substantially compromised in the presence of NRF1 small interfering RNA treatment. We have identified an unexpected collaboration between MyoD and NRF1 under androgen-depleted condition, which may serve as an important adaptive mechanism during the pathogenesis of castration-resistant prostate cancer.

Regulation of NCAPG by miR-99a-3p (passenger strand) inhibits cancer cell aggressiveness and is involved in CRPC.

Effective treatments for patients with castration‐resistant prostate cancer (CRPC) have not yet been established. Novel approaches for identification of putative therapeutic targets for CRPC are needed. Analyses of RNA sequencing of microRNA (miRNA) expression revealed that miR‐99a‐3p (passenger strand) is significantly downregulated in several types of cancers. Here, we aimed to identify novel miR‐99a‐3p regulatory networks and therapeutic targets for CRPC. Ectopic expression of miR‐99a‐3p significantly inhibited cancer cell proliferation, migration, and invasion in PCa cells. Non‐SMC condensin I complex subunit G (NCAPG) was a direct target of miR‐99a‐3p in PCa cells. Overexpression of NCAPG was detected in CRPC clinical specimens and was significantly associated with shorter disease‐free survival and advanced clinical stage. Knockdown of NCAPG inhibited cancer cell aggressiveness. The passenger strand miR‐99a‐3p acted as an antitumor miRNA in naïve PCa and CRPC. NCAPG was regulated by miR‐99a‐3p, and its overexpression was involved in CRPC pathogenesis. Involvement of passenger strand of miRNA in cancer pathogenesis is novel concept, and identification of antitumor miRNA regulatory networks in CRPC might be provided novel prognostic markers and therapeutic targets for this disease.

Abstract 1459: Dual-strand tumor-suppressor microRNA-145 (miR-145-5p and miR-145-3p) are involved in castration-resistant prostate cancer pathogenesis

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related death among men in developed countries. Androgen signaling through the androgen receptor (AR) is an important oncogenic pathway for PCa progression. Most patients initially respond to androgen-deprivation therapy (ADT), but eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). Although several clinical trials for CRPC have been carried out, resulting in the availability of novel chemotherapeutic agents, these treatments provide limited benefits and are not considered curative. Therefore, identification of effective biomarkers for detection of CRPC and understanding the molecular mechanisms of androgen-independent signaling and metastatic signaling pathways underlying PCa using current genomic approaches would help to improve therapies for and prevention of the disease. The discovery of microRNAs (miRNAs) has resulted in major advancements in cancer research. miRNAs are small noncoding RNAs that function to fine tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-depending manner. The unique characteristic function of miRNAs is to regulate RNA transcripts in human cells. Therefore, dysregulated expression of miRNAs can disrupt tightly regulated RNA networks in cancer cells. miRNAs play critical roles in various biological processes, and their dysregulation is shown in several human cancers. In this study, we constructed the miRNA expression signature of CRPC using clinical specimens because the development of therapeutic strategies is a central theme in the advancement of PCa treatments. Using CRPC expression signature data, we investigated the specific roles of miRNAs in PCa and CRPC oncogenesis by examining differentially expressed miRNAs. Based on the CRPC signature, we focused on the dual strand of pre-miR-145, miR-145-5p and miR-145-3p because these miRNAs were significantly reduced in cancer tissues, suggesting these miRNAs act as antitumor miRNAs in this disease. In miRNA biogenesis, it is the general consensus that processing of the pre-miRNA through Dicer1 generates a miRNA duplex (a passenger strand and a guide strand), and that the passenger strand has degradation and no regulatory activity and disintegrates in cells. Our present data showed that both miRNAs, miR-145-5p and miR-145-3p significantly suppressed cancer cell migration and invasion. Moreover, Kaplan-Meier survival curves showed that low expression of miR-145-3p predicted a short duration of progression to CRPC. Dual strand of pre-miR-145 functioned as tumor suppressors based on the miRNA expression signature of CRPC. Identification of miRNA-mediated cancer networks may provide novel molecular pathogenesis of the disease. Citation Format: Mayuko Kato, Akira Kurozumi, Yusuke Goto, Nijiro Nohata, Takayuki Arai, Atsushi Okato, Keiichi Koshizuka, Satoko Kojima, Tomohiko Ichikawa, Naohiko Seki. Dual-strand tumor-suppressor microRNA-145 (miR-145-5p and miR-145-3p) are involved in castration-resistant prostate cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1459. doi:10.1158/1538-7445.AM2017-1459

Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy.

A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin (IL)-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT1 receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.

Dual strands of pre-miR‑150 (miR‑150‑5p and miR‑150‑3p) act as antitumor miRNAs targeting SPOCK1 in naïve and castration-resistant prostate cancer.

Analysis of our microRNA (miRNA) expression signature in human cancers has shown that guide and passenger strands of pre-miR‑150, i.e., miR‑150‑5p and miR‑150‑3p, are significantly downregulated in cancer tissues. In miRNA biogenesis, the passenger strand of miRNA is degraded and is thought to have no functions. Thus, the aim of this study was to investigate the functional significance of miR‑150‑5p and miR‑150‑3p in naïve prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Ectopic expression assays showed that both strands of miRNAs significantly suppressed cancer cell migration and invasion. Our strategies of miRNA target searching demonstrated that SPOCK1 (SPARC/osteonectin, cwcv and kazal like domains proteoglycan 1) was directly regulated by miR‑150‑5p and miR‑150‑3p. Knockdown of SPOCK1 by siRNA inhibited cancer cell aggressiveness. Moreover, overexpression of SPOCK1 was observed in naïve PCa and CRPC tissues. Taken together, dual strands of pre-miR‑150 (miR‑150‑5p and miR‑150‑3p) acted as antitumor miRNAs in naïve PCa and CRPC cells. Expression of oncogenic SPOCK1 was involved in naïve PCa and CRPC pathogenesis. Novel approaches to analysis of antitumor miRNA-regulated RNA networks in cancer cells may provide new insights into the pathogenic mechanisms of naïve PCa and CRPC.

Androgen Receptor Splice Variants Dimerize to Transactivate Target Genes.

Constitutively active androgen receptor splice variants (AR-V) lacking the ligand-binding domain have been implicated in the pathogenesis of castration-resistant prostate cancer and in mediating resistance to newer drugs that target the androgen axis. AR-V regulates expression of both canonical AR targets and a unique set of cancer-specific targets that are enriched for cell-cycle functions. However, little is known about how AR-V controls gene expression. Here, we report that two major AR-Vs, termed AR-V7 and AR(v567es), not only homodimerize and heterodimerize with each other but also heterodimerize with full-length androgen receptor (AR-FL) in an androgen-independent manner. We found that heterodimerization of AR-V and AR-FL was mediated by N- and C-terminal interactions and by the DNA-binding domain of each molecule, whereas AR-V homodimerization was mediated only by DNA-binding domain interactions. Notably, AR-V dimerization was required to transactivate target genes and to confer castration-resistant cell growth. Our results clarify the mechanism by which AR-Vs mediate gene regulation and provide a pivotal pathway for rational drug design to disrupt AR-V signaling as a rational strategy for the effective treatment of advanced prostate cancer.

Clinical development of novel therapeutics for castration‐resistant prostate cancer

There have been more drugs approved by the US Food and Drug Administration for the treatment of castration-resistant prostate cancer in the past 3 years than in the prior 3 decades, with additional drugs on the verge of approval based on the results of recently reported randomized trials. While an improvement in the understanding of the pathogenesis of castration-resistant prostate cancer has undeniably accelerated the transition of novel approaches from "bench to bedside," the recent successes in the treatment of prostate cancer are also a result of the efforts of clinical investigators to redefine the framework in which drugs for castration-resistant disease are evaluated. This review will explore the shifting paradigm in drug development for castration-resistant prostate cancer over the past several decades, and highlight how new definitions, trial designs, and endpoints have facilitated the emergence of new therapies for this challenging disease.