Role of PI3K-AKT-mTOR Pathway as a Pro-Survival Signaling and Resistance-Mediating Mechanism to Therapy of Prostate Cancer

Androgen deprivation therapy (ADT) and androgen receptor (AR)-targeted therapy are the gold standard options for treating prostate cancer (PCa). These are initially effective, as localized and the early stage of metastatic disease are androgen- and castration-sensitive. The tumor strongly relies on systemic/circulating androgens for activating AR signaling to stimulate growth and progression. However, after a certain point, the tumor will eventually develop a resistant stage, where ADT and AR antagonists are no longer effective. Mechanistically, it seems that the tumor becomes more aggressive through adaptive responses, relies more on alternative activated pathways, and is less dependent on AR signaling. This includes hyperactivation of PI3K-AKT-mTOR pathway, which is a central signal that regulates cell pro-survival/anti-apoptotic pathways, thus, compensating the blockade of AR signaling. The PI3K-AKT-mTOR pathway is well-documented for its crosstalk between genomic and non-genomic AR signaling, as well as other signaling cascades. Such a reciprocal feedback loop makes it more complicated to target individual factor/signaling for treating PCa. Here, we highlight the role of PI3K-AKT-mTOR signaling as a resistance mechanism for PCa therapy and illustrate the transition of prostate tumor from AR signaling-dependent to PI3K-AKT-mTOR pathway-dependent. Moreover, therapeutic strategies with inhibitors targeting the PI3K-AKT-mTOR signal used in clinic and ongoing clinical trials are discussed.     

Gonadotropin-Releasing Hormone Receptors in Prostate Cancer: Molecular Aspects and Biological Functions

Pituitary Gonadotropin-Releasing Hormone receptors (GnRH-R) mediate the activity of the hypothalamic decapeptide GnRH, thus playing a key role in the regulation of the reproductive axis. Early-stage prostate cancer (PCa) is dependent on serum androgen levels, and androgen-deprivation therapy (ADT), based on GnRH agonists and antagonists, represents the standard therapeutic approach for PCa patients. Unfortunately, the tumor often progresses towards the more aggressive castration-resistant prostate cancer (CRPC) stage. GnRH receptors are also expressed in CRPC tissues, where their binding to both GnRH agonists and antagonists is associated with significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic effects, mediated by the Gαi/cAMP signaling cascade. GnRH agonists and antagonists are now considered as an effective therapeutic strategy for CRPC patients with many clinical trials demonstrating that the combined use of these drugs with standard therapies (i.e., docetaxel, enzalutamide, abiraterone) significantly improves disease-free survival. In this context, GnRH-based bioconjugates (cytotoxic drugs covalently linked to a GnRH-based decapeptide) have been recently developed. The rationale of this treatment is that the GnRH peptide selectively binds to its receptors, delivering the cytotoxic drug to CRPC cells while sparing nontumor cells. Some of these compounds have already entered clinical trials.     

Improving Strategies in the Development of Protein-Downregulation-Based Antiandrogens

The androgen receptor (AR) plays a crucial role in the occurrence and development of prostate cancer (PCa), and its signaling pathway remains active in castration-resistant prostate cancer (CRPC) patients. The resistance against antiandrogen drugs in current clinical use is a major challenge for the treatment of PCa, and thus the development of new generations of antiandrogens is under high demand. Recently, strategies for downregulating the AR have attracted significant attention, given its potential in the discovery and development of new antiandrogens, including G-quadruplex stabilizers, ROR-γ inhibitors, AR-targeting proteolysis targeting chimeras (PROTACs), and other selective AR degraders (SARDs), which are able to overcome current resistance mechanisms such as acquired AR mutations, the expression of AR variable splices, or overexpression of AR. This review summarizes the various strategies for downregulating the AR protein, at either the mRNA or protein level, thus providing new ideas for the development of promising antiandrogen drugs.     

Novel Strategy of Proxalutamide for the Treatment of Prostate Cancer through Coordinated Blockade of Lipogenesis and Androgen Receptor Axis

Objective: Prostate cancer (PCa) is the most common malignant tumor diagnosed in men in developed countries. In developing countries, the PCa morbidity and mortality rates are also increasing rapidly. Since androgen receptor (AR) is a key driver and plays a critical role in the regulation of PCa development, AR-targeted agents provide a key component of current therapy regimens. However, even new-generation AR antagonists are prone to drug resistance, and there is currently no effective strategy for overcoming advanced PCa aggressiveness, including drug-resistance progression. The aim of this study was to evaluate the potential efficacy and novel therapy strategy of proxalutamide (a newly developed AR antagonist) in PCa. Methods: Four PCa cell lines with various biological heterogeneities were utilized in this study, namely, androgen-sensitive/-insensitive with/without AR expression. Proliferation, migration and apoptosis assays in PCa cells were used to evaluate the effective therapeutic activity of proxalutamide. The changes in lipid droplet accumulation and lipidomic profiles were analyzed to determine the influence of proxalutamide on lipogenesis in PCa cells. The molecular basis of the effects of proxalutamide on lipogenesis and the AR axis was then further investigated. Results: Proxalutamide significantly inhibited the proliferation and migration of PCa cells, and its inhibitory effect was superior to that of enzalutamide (Enz, second-generation AR antagonist). Proxalutamide induced the caspase-dependent apoptosis of PCa cells. Proxalutamide significantly diminished the level of lipid droplets in PCa cells, changed the lipid profile of PCa cells and reduced the content of most lipids (especially triglycerides) in PCa cells. Proxalutamide attenuated de novo lipogenesis by inhibiting the expression of ATP citrate lyase (ACL), acetyl CoA carboxylase (ACC), fatty acid synthase (FASN) and sterol regulatory element-binding protein-1 (SREBP-1). Moreover, proxalutamide also decreased AR expression in PCa cells, and its inhibitory effect on lipogenesis did not depend on its ability to down-regulate AR expression. However, Enz had no effect on AR expression, lipid accumulation or lipid de novo synthesis in PCa cells. Conclusions: By co-targeting the AR axis and endogenous adipogenesis, a novel and promising strategy was established for proxalutamide to combat the progress of PCa. The unique effect of proxalutamide on the metabolic reprogramming of PCa provides a potential solution to overcome the resistance of current AR-targeted therapy, which will help to effectively prolong its clinical service life.     

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.     

Androgen Receptor Phosphorylation at Serine 308 and Serine 791 Predicts Enhanced Survival in Castrate Resistant Prostate Cancer Patients

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR⁹⁴), 308 (pAR³⁰⁸), 650(pAR⁶⁵⁰) and 791 (pAR⁷⁹¹). No correlations with clinical parameters were observed for pAR⁹⁴ or pAR⁶⁵⁰ in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR³⁰⁸ is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR⁷⁹¹ expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer.     

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients’ specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.     

Isolation and Characterization of Urinary Extracellular Vesicles from Healthy Donors and Patients with Castration-Resistant Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed malignancy among men in developed countries. The five-year survival rate for men diagnosed with early-stage PCa is approximately 100%, while it is less than 30% for castration-resistant PCa (CRPC). Currently, the detection of prostate-specific antigens as biomarkers for the prognosis of CRPC is criticized because of its low accuracy, high invasiveness, and high false-positive rate. Therefore, it is important to identify new biomarkers for prediction of CRPC progression. Extracellular vesicles (EVs) derived from tumors have been highlighted as potential markers for cancer diagnosis and prognosis. Specifically, urinary EVs directly reflect changes in the pathophysiological conditions of the urogenital system because it is exposed to prostatic secretions. Thus, detecting biomarkers in urinary EVs provides a promising approach for performing an accurate and non-invasive liquid biopsy for CPRC. In this study, we effectively isolated urinary EVs with low protein impurities using size-exclusion chromatography combined with ultrafiltration. After EV isolation and characterization, we evaluated the miRNAs in urinary EVs from healthy donors and patients with CRPC. The results indicated that miRNAs (miR-21-5p, miR-574-3p, and miR-6880-5p) could be used as potential biomarkers for the prognosis of CRPC. This analysis of urinary EVs contributes to the fast and convenient prognosis of diseases, including CRPC, in the clinical setting.     

The Emerging Clinical Role of Spermine in Prostate Cancer

Spermine, a member of polyamines, exists in all organisms and is essential for normal cell growth and function. It is highly expressed in the prostate compared with other organs and is detectable in urine, tissue, expressed prostatic secretions, and erythrocyte. A significant reduction of spermine level was observed in prostate cancer (PCa) tissue compared with benign prostate tissue, and the level of urinary spermine was also significantly lower in men with PCa. Decreased spermine level may be used as an indicator of malignant phenotype transformation from normal to malignant tissue in prostate. Studies targeting polyamines and key rate-limiting enzymes associated with spermine metabolism as a tool for PCa therapy and chemoprevention have been conducted with various polyamine biosynthesis inhibitors and polyamine analogues. The mechanism between spermine and PCa development are possibly related to the regulation of polyamine metabolism, cancer-driving pathways, oxidative stress, anticancer immunosurveillance, and apoptosis regulation. Although the specific mechanism of spermine in PCa development is still unclear, ongoing research in spermine metabolism and its association with PCa pathophysiology opens up new opportunities in the diagnostic and therapeutic roles of spermine in PCa management.     

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.     

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.     

Chimeric Antigen Receptor-Engineered T Cell Therapy for the Management of Patients with Metastatic Prostate Cancer: A Comprehensive Review

Prostate cancer (PCa) has a vast clinical spectrum from the hormone-sensitive setting to castration-resistant metastatic disease. Thus, chemotherapy regimens and the administration of androgen receptor axis-targeted (ARAT) agents for advanced PCa have shown limited therapeutic efficacy. Scientific advances in the field of molecular medicine and technological developments over the last decade have paved the path for immunotherapy to become an essential clinical modality for the treatment of patients with metastatic PCa. However, several immunotherapeutic agents have shown poor outcomes in patients with advanced disease, possibly due to the low PCa mutational burden. Adoptive cellular approaches utilizing chimeric antigen receptor T cells (CAR-T) targeting cancer-specific antigens would be a solution for circumventing the immune tolerance mechanisms. The immunotherapeutic regimen of CAR-T cell therapy has shown potential in the eradication of hematologic malignancies, and current clinical objectives maintain the equivalent efficacy in the treatment of solid tumors, including PCa. This review will explore the current modalities of CAR-T therapy in the disease spectrum of PCa while describing key limitations of this immunotherapeutic approach and discuss future directions in the application of immunotherapy for the treatment of metastatic PCa and patients with advanced disease.     

Non-Coding RNAs Set a New Phenotypic Frontier in Prostate Cancer Metastasis and Resistance

Prostate cancer (PCa) mortality remains a significant public health problem, as advanced disease has poor survivability due to the development of resistance in response to both standard and novel therapeutic interventions. Therapeutic resistance is a multifaceted problem involving the interplay of a number of biological mechanisms including genetic, signaling, and phenotypic alterations, compounded by the contributions of a tumor microenvironment that supports tumor growth, invasiveness, and metastasis. The androgen receptor (AR) is a primary regulator of prostate cell growth, response and maintenance, and the target of most standard PCa therapies designed to inhibit AR from interacting with androgens, its native ligands. As such, AR remains the main driver of therapeutic response in patients with metastatic castration-resistant prostate cancer (mCRPC). While androgen deprivation therapy (ADT), in combination with microtubule-targeting taxane chemotherapy, offers survival benefits in patients with mCRPC, therapeutic resistance invariably develops, leading to lethal disease. Understanding the mechanisms underlying resistance is critical to improving therapeutic outcomes and also to the development of biomarker signatures of predictive value. The interconversions between epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) navigate the prostate tumor therapeutic response, and provide a novel targeting platform in overcoming therapeutic resistance. Both microRNA (miRNA)- and long non-coding RNA (lncRNA)-mediated mechanisms have been associated with epigenetic changes in prostate cancer. This review discusses the current evidence-based knowledge of the role of the phenotypic transitions and novel molecular determinants (non-coding RNAs) as contributors to the emergence of therapeutic resistance and metastasis and their integrated predictive value in prostate cancer progression to advanced disease.     

Extracellular Vesicle Proteome in Prostate Cancer: A Comparative Analysis of Mass Spectrometry Studies

Molecular diagnostics based on discovery research holds the promise of improving screening methods for prostate cancer (PCa). Furthermore, the congregated information prompts the question whether the urinary extracellular vesicles (uEV) proteome has been thoroughly explored, especially at the proteome level. In fact, most extracellular vesicles (EV) based biomarker studies have mainly targeted plasma or serum. Therefore, in this study, we aim to inquire about possible strategies for urinary biomarker discovery particularly focused on the proteome of urine EVs. Proteomics data deposited in the PRIDE archive were reanalyzed to target identifications of potential PCa markers. Network analysis of the markers proposed by different prostate cancer studies revealed moderate overlap. The recent throughput improvements in mass spectrometry together with the network analysis performed in this study, suggest that a larger standardized cohort may provide potential biomarkers that are able to fully characterize the heterogeneity of PCa. According to our analysis PCa studies based on urinary EV proteome presents higher protein coverage compared to plasma, plasma EV, and voided urine proteome. This together with a direct interaction of the prostate gland and urethra makes uEVs an attractive option for protein biomarker studies. In addition, urinary proteome based PCa studies must also evaluate samples from bladder and renal cancers to assess specificity for PCa.     

Androgen Receptor Gene Pathway Upregulation and Radiation Resistance in Oligometastatic Prostate Cancer

Stereotactic ablative body radiotherapy (SABR) is currently used as a salvage intervention for men with oligometastatic prostate cancer (PC), and increasingly so since the results of the Stereotactic Ablative Body Radiotherapy for the Comprehensive Treatment of Oligometastatic Cancers (SABR-COMET) trial reported a significant improvement in overall survival with SABR. The addition of androgen deprivation therapy (ADT) to localised prostate radiotherapy improves survival as it sensitises PC to radiotherapy-induced cell death. The importance of the androgen receptor (AR) gene pathway in the development of resistance to radiotherapy is well established. In this review paper, we will examine the data to determine how we can overcome the upregulation of the AR pathway and suggest a strategy for improving outcomes in men with oligometastatic hormone-sensitive PC.