Hybrid Androgen Receptor Inhibitors Outperform Enzalutamide and EPI-001 in in vitro Models of Prostate Cancer Drug Resistance

Androgen receptor targeted therapies for prostate cancer have serious limitations in advanced stages of the disease. While resistance to the FDA-approved enzalutamide is extensively documented, novel therapies based on epichlorohydrin scaffolds (EPI) are currently in clinical trials, but display suboptimal pharmacokinetics. Herein, we report the synthesis and biological characterisation of a novel class of compounds designed through covalently linking enzalutamide and EPI-001 through various triazole based linkers. The compounds display an 18 to 53 fold improvement in the cell killing potency towards C4-2b prostate cancer (PCa) cells compared to the gold standards of therapy, enzalutamide and EPI-001. The most promising compounds were proven to exhibit their toxicity exclusively through androgen receptor (AR) mediated pathways. This work sets the basis for the first class of hybrid AR inhibitors which successfully combine two drug moieties – EPI-001 and enzalutamide – into the same molecule.     

Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model

Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX) rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF) embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231). The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT), revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient’s medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.     

Deficiency of NEIL3 Enhances the Chemotherapy Resistance of Prostate Cancer

Acquired treatment resistance is an important cause of death in prostate cancer, and this study aimed to explore the mechanisms of chemotherapy resistance in prostate cancer. We employed castration-resistant prostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), and chemotherapy-resistant prostate cancer datasets to screen for potential target genes. The Cancer Genome Atlas (TCGA) was used to detect the correlation between the target genes and prognosis and clinical characteristics. Nei endonuclease VIII-like 3 (NEIL3) knockdown cell lines were constructed with RNA interference. Prostate cancer cells were treated with enzalutamide for the androgen deprivation therapy (ADT) model, and with docetaxel and cisplatin for the chemotherapy model. Apoptosis and the cell cycle were examined using flow cytometry. RNA sequencing and western blotting were performed in the knockdown Duke University 145 (DU145) cell line to explore the possible mechanisms. The TCGA dataset demonstrated that high NEIL3 was associated with a high T stage and Gleason score, and indicated a possibility of lymph node metastasis, but a good prognosis. The cell therapy models showed that the loss of NEIL3 could promote the chemotherapy resistance (but not ADT resistance) of prostate cancer (PCa). Flow cytometry revealed that the loss of NEIL3 in PCa could inhibit cell apoptosis and cell cycle arrest under cisplatin treatment. RNA sequencing showed that the knockdown of NEIL3 changes the expression of neuroendocrine-related genes. Further western blotting revealed that the loss of NEIL3 could significantly promote the phosphorylation of ATR serine/threonine kinase (ATR) and ATM serine/threonine kinase (ATM) under chemotherapy, thus initiating downstream pathways related to DNA repair. In summary, the loss of NEIL3 promotes chemotherapy resistance in prostate cancer, and NEIL3 may serve as a diagnostic marker for chemotherapy-resistant patients.     

Enzalutamide Enhances PSMA Expression of PSMA-Low Prostate Cancer

Prostate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) prolongs overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, men with low PSMA expression are excluded from RLT. We explored the effect of androgen receptor blockade with enzalutamide on PSMA expression. Assessment of PSMA and androgen receptor (AR) expression on the human PC cell lines 22Rv1, C4-2, and LNCaP by immunohistochemistry and flow cytometry revealed low (22Rv1) and high (C4-2 and LNCaP) PSMA expression, and high, comparable AR positivity. Treatment with enzalutamide increased PSMA levels in 22Rv1, C4-2, and LNCaP (2.2/2.3/2.6-fold, p = 0.0005/0.03/0.046) after one week compared to DMSO-treated controls as assessed by flow cytometry. NOD/Scid mice bearing 22Rv1 tumors were treated with enzalutamide for two weeks. Positron emission tomography/computed tomography (PET/CT) demonstrated higher tumor uptake of ⁶⁸Ga-PSMA after enzalutamide treatment (p = 0.004). Similarly, a clinical case with low baseline PSMA avidity demonstrated increased uptake of ⁶⁸Ga-PSMA after enzalutamide on PET/CT and post-therapeutic ¹⁷⁷Lu-PSMA scintigraphy in a patient with mCRPC. Enzalutamide induced PSMA expression in the 22Rv1 xenograft model and in an mCRPC patient, both with low baseline tumoral PSMA levels. Therefore, enzalutamide pre-treatment might render patients with low PSMA expression eligible for ¹⁷⁷Lu-PSMA RLT.     

Delineating the Molecular Events Underlying Development of Prostate Cancer Variants with Neuroendocrine/Small Cell Carcinoma Characteristics

The treatment landscape of prostate cancer has changed dramatically following the advent of novel systemic therapies, most of which target the androgen receptor (AR). Agents such as abiraterone, enzalutamide, apalutamide, darolutamide were designed to further suppress androgen receptor signaling following gonadal suppression achieved by first-line androgen deprivation therapies. These potent AR targeting agents are increasingly used in the earlier stages of the disease spectrum with the goal of delaying disease progression and extending survival. Although these therapies are effective in controlling prostate tumors dependent on or addicted to AR signaling, prostate tumors surviving the onslaught of potent treatments may evolve and develop drug resistance. A substantial proportion of treatment failures can be explained by the development of treatment-induced aggressive prostate cancer variants such as neuroendocrine/small cell carcinoma. These emerging disease entities demand detailed characterization and precise definitions. We postulate that these treatment-induced prostate cancer entities should be defined molecularly to overcome the drawbacks associated with the current clinical and pathological definitions. A precise molecular definition conforms with current knowledge on the molecular evolution of this disease entity and will enable early detection and early intervention.     

The Crosstalk of Long Non-Coding RNA and MicroRNA in Castration-Resistant and Neuroendocrine Prostate Cancer: Their Interaction and Clinical Importance

Prostate cancer is featured by its heterogeneous nature, which indicates a different prognosis. Castration-resistant prostate cancer (CRPC) is a hallmark of the treatment-refractory stage, and the median survival of patients is only within two years. Neuroendocrine prostate cancer (NEPC) is an aggressive variant that arises from de novo presentation of small cell carcinoma or treatment-related transformation with a median survival of 1–2 years from the time of diagnosis. The epigenetic regulators, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), have been proven involved in multiple pathologic mechanisms of CRPC and NEPC. LncRNAs can act as competing endogenous RNAs to sponge miRNAs that would inhibit the expression of their targets. After that, miRNAs interact with the 3’ untranslated region (UTR) of target mRNAs to repress the step of translation. These interactions may modulate gene expression and influence cancer development and progression. Otherwise, epigenetic regulators and genetic mutation also promote neuroendocrine differentiation and cancer stem-like cell formation. This step may induce neuroendocrine prostate cancer development. This review aims to provide an integrated, synthesized overview under current evidence to elucidate the crosstalk of lncRNAs with miRNAs and their influence on castration resistance or neuroendocrine differentiation of prostate cancer. Notably, we also discuss the mechanisms of lncRNA–miRNA interaction in androgen receptor-independent prostate cancer, such as growth factors, oncogenic signaling pathways, cell cycle dysregulation, and cytokines or other transmembrane proteins. Conclusively, we underscore the potential of these communications as potential therapeutic targets in the future.     

Multicellular Modelling of Difficult-to-Treat Gastrointestinal Cancers: Current Possibilities and Challenges

Cancers affecting the gastrointestinal system are highly prevalent and their incidence is still increasing. Among them, gastric and pancreatic cancers have a dismal prognosis (survival of 5–20%) and are defined as difficult-to-treat cancers. This reflects the urge for novel therapeutic targets and aims for personalised therapies. As a prerequisite for identifying targets and test therapeutic interventions, the development of well-established, translational and reliable preclinical research models is instrumental. This review discusses the development, advantages and limitations of both patient-derived organoids (PDO) and patient-derived xenografts (PDX) for gastric and pancreatic ductal adenocarcinoma (PDAC). First and next generation multicellular PDO/PDX models are believed to faithfully generate a patient-specific avatar in a preclinical setting, opening novel therapeutic directions for these difficult-to-treat cancers. Excitingly, future opportunities such as PDO co-cultures with immune or stromal cells, organoid-on-a-chip models and humanised PDXs are the basis of a completely new area, offering close-to-human models. These tools can be exploited to understand cancer heterogeneity, which is indispensable to pave the way towards more tumour-specific therapies and, with that, better survival for patients.     

A Review of Prostate Organogenesis and a Role for iPSC-Derived Prostate Organoids to Study Prostate Development and Disease

The prostate is vulnerable to two major age-associated diseases, cancer and benign enlargement, which account for significant morbidity and mortality for men across the globe. Prostate cancer is the most common cancer reported in men, with over 1.2 million new cases diagnosed and 350,000 deaths recorded annually worldwide. Benign prostatic hyperplasia (BPH), characterised by the continuous enlargement of the adult prostate, symptomatically afflicts around 50% of men worldwide. A better understanding of the biological processes underpinning these diseases is needed to generate new treatment approaches. Developmental studies of the prostate have shed some light on the processes essential for prostate organogenesis, with many of these up- or downregulated genes expressions also observed in prostate cancer and/or BPH progression. These insights into human disease have been inferred through comparative biological studies relying primarily on rodent models. However, directly observing mechanisms of human prostate development has been more challenging due to limitations in accessing human foetal material. Induced pluripotent stem cells (iPSCs) could provide a suitable alternative as they can mimic embryonic cells, and iPSC-derived prostate organoids present a significant opportunity to study early human prostate developmental processes. In this review, we discuss the current understanding of prostate development and its relevance to prostate-associated diseases. Additionally, we detail the potential of iPSC-derived prostate organoids for studying human prostate development and disease.     

Effects of Sorafenib on C-Terminally Truncated Androgen Receptor Variants in Human Prostate Cancer Cells

Recent evidence suggests that the development of castration resistant prostate cancer (CRPCa) is commonly associated with an aberrant, ligand-independent activation of the androgen receptor (AR). A putative mechanism allowing prostate cancer (PCa) cells to grow under low levels of androgens, is the expression of constitutively active, C-terminally truncated AR lacking the AR-ligand binding domain (LBD). Due to the absence of a LBD, these receptors, termed ARΔLBD, are unable to respond to any form of anti-hormonal therapies. In this study we demonstrate that the multikinase inhibitor sorafenib inhibits AR as well as ARΔLBD-signalling in CRPCa cells. This inhibition was paralleled by proteasomal degradation of the AR- and ARΔLBD-molecules. In line with these observations, maximal antiproliferative effects of sorafenib were achieved in AR and ARΔLBD-positive PCa cells. The present findings warrant further investigations on sorafenib as an option for the treatment of advanced AR-positive PCa.     

Inhibition of N-Acetyltransferase 10 Suppresses the Progression of Prostate Cancer through Regulation of DNA Replication

Cancer suppression through the inhibition of N-acetyltransferase 10 (NAT10) by its specific inhibitor Remodelin has been demonstrated in a variety of human cancers. Here, we report the inhibitory effects of Remodelin on prostate cancer (PCa) cells and the possible associated mechanisms. The prostate cancer cell lines VCaP, LNCaP, PC3, and DU145 were used. The in vitro proliferation, migration, and invasion of cells were measured by a cell proliferation assay, colony formation, wound healing, and Transwell assays, respectively. In vivo tumor growth was analyzed by transplantation into nude mice. The inhibition of NAT10 by Remodelin not only suppressed growth, migration, and invasion in vitro, but also the in vivo cancer growth of prostate cancer cells. The involvement of NAT10 in DNA replication was assessed by EdU labeling, DNA spreading, iPOND, and ChIP-PCR assays. The inhibition of NAT10 by Remodelin slowed DNA replication. NAT10 was detected in the prereplication complex, and it could also bind to DNA replication origins. Furthermore, the interaction between NAT10 and CDC6 was analyzed by Co-IP. The altered expression of NAT10 was measured by immunofluorescence staining and Western blotting. Remodelin markedly reduced the levels of CDC6 and AR. The expression of NAT10 could be altered under either castration or noncastration conditions, and Remodelin still suppressed the growth of in vitro-induced castration-resistant prostate cancers. The analysis of a TCGA database revealed that the overexpression of NAT10, CDC6, and MCM7 in prostate cancers were correlated with the Gleason score and node metastasis. Our data demonstrated that Remodelin, an inhibitor of NAT10, effectively inhibits the growth of prostate cancer cells under either no castration or castration conditions, likely by impairing DNA replication.     

Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture

Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.     

Inhibition of CCL2 Signaling in Combination with Docetaxel Treatment Has Profound Inhibitory Effects on Prostate Cancer Growth in Bone

The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.     

Effect of AQP9 Expression in Androgen-Independent Prostate Cancer Cell PC3

It is known that aquaporin 9 (AQP9) in the prostate was strictly upregulated by androgen and may represent a novel therapeutic target for several cancers, but whether AQP9 plays a role in the regulation of androgen-independent prostate cancer still remains unclear. In the present study, AQP9 was determined in prostate cancer and adjacent cancer tissues; AQP9-siRNA was applied to silencing AQP9 in androgen-independent prostate cancer cell PC3 cell line. Western blot and flow cytometry analysis were employed to detect changes in related-function of control and AQP9-siRNA groups. The results showed that AQP9 is significantly induced in cancer tissues than that in adjacent cancer tissues. Moreover, knockdown of AQP9 in PC3 androgen-independent prostate cancer cell prostate cancer cells increased inhibition rates of proliferation. In addition, knockdown of AQP9 resulted in a significant decrease in the expression of the Bcl-2 and with a notable increase in the expression of Bax and cleaved caspase 3, indicated that AQP9 knockdown promoted apoptosis in prostate cancer cells. From wound healing assay and matrigel invasion, we suggested that AQP9 expression affects the motility and invasiveness of prostate cancer cells. Moreover, In order to explore the pathway may be involved in AQP9-mediated motility and invasion of prostate cancer cells, the phosphorylation of ERK1/2 was significant suppressed in AQP9 siRNA-transfected cells compared with that in control cells, suggesting that AQP9 is involved in the activation of the ERK pathway in androgen-independent prostate cancer cells.     

Inhibition of Stromal PlGF Suppresses the Growth of Prostate Cancer Xenografts

The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer.     

D-pinitol Inhibits Prostate Cancer Metastasis through Inhibition of αVβ3 Integrin by Modulating FAK,c-Src and NF-κB Pathways

Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145) at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvβ3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK) phosphorylation, c-Src kinase activity and NF-κB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.     

Aminomethylphosphonic Acid and Methoxyacetic Acid Induce Apoptosis in Prostate Cancer Cells

Aminomethylphosphonic acid (AMPA) and its parent compound herbicide glyphosate are analogs to glycine, which have been reported to inhibit proliferation and promote apoptosis of cancer cells, but not normal cells. Methoxyacetic acid (MAA) is the active metabolite of ester phthalates widely used in industry as gelling, viscosity and stabilizer; its exposure is associated with developmental and reproductive toxicities in both rodents and humans. MAA has been reported to suppress prostate cancer cell growth by inducing growth arrest and apoptosis. However, it is unknown whether AMPA and MAA can inhibit cancer cell growth. In this study, we found that AMPA and MAA inhibited cell growth in prostate cancer cell lines (LNCaP, C4-2B, PC-3 and DU-145) through induction of apoptosis and cell cycle arrest at the G1 phase. Importantly, the AMPA-induced apoptosis was potentiated with the addition of MAA, which was due to downregulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2), leading to activation of caspases 7 and 3. These results demonstrate that the combination of AMPA and MAA can promote the apoptosis of prostate cancer cells, suggesting that they can be used as potential therapeutic drugs in the treatment of prostate cancer.