Cell-by-cell quantification of the androgen receptor in benign and malignant prostate leads to a better understanding of changes linked to cancer initiation and progression.

The androgen receptor (AR) plays a crucial role in the development and homeostasis of the prostate and is a key therapeutic target in prostate cancer (PCa). The gold standard therapy for advanced PCa is androgen deprivation therapy (ADT), which targets androgen production and AR signaling. However, resistance to ADT develops via AR-dependent and AR-independent mechanisms. As reports on AR expression patterns in PCa have been conflicting, we performed cell-by-cell AR quantification by immunohistochemistry in the benign and malignant prostate to monitor changes with disease development, progression, and hormonal treatment. Prostates from radical prostatectomy (RP) cases, both hormone-naïve and hormone-treated, prostate tissues from patients on palliative ADT, and bone metastases were included. In the normal prostate, AR is expressed in >99% of luminal cells, 51% of basal cells, and 61% of fibroblasts. An increase in the percentage of AR negative (%AR-) cancer cells along with a gradual loss of fibroblastic AR were observed with increasing Gleason grade and hormonal treatment. This was accompanied by a parallel increase in staining intensity of AR positive (AR+) cells under ADT. Staining AR with N- and C-terminal antibodies yielded similar results. The combination of %AR- cancer cells, %AR- fibroblasts, and AR intensity score led to the definition of an AR index, which was predictive of biochemical recurrence in the RP cohort and further stratified patients of intermediate risk. Lastly, androgen receptor variant 7 (ARV7)+ cells and AR- cells expressing neuroendocrine and stem markers were interspersed among a majority of AR+ cells in ADT cases. Altogether, the comprehensive quantification of AR expression in the prostate reveals concomitant changes in tumor cell subtypes and fibroblasts, emphasizing the significance of AR- cells with disease progression and palliative ADT.

Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer.

Androgen receptor (AR) signaling reprograms cellular metabolism to support prostate cancer (PCa) growth and survival. Another key regulator of cellular metabolism is mTOR, a kinase found in diverse protein complexes and cellular localizations, including the nucleus. However, whether nuclear mTOR plays a role in PCa progression and participates in direct transcriptional cross-talk with the AR is unknown. Here, via the intersection of gene expression, genomic, and metabolic studies, we reveal the existence of a nuclear mTOR-AR transcriptional axis integral to the metabolic rewiring of PCa cells. Androgens reprogram mTOR-chromatin associations in an AR-dependent manner in which activation of mTOR-dependent metabolic gene networks is essential for androgen-induced aerobic glycolysis and mitochondrial respiration. In models of castration-resistant PCa cells, mTOR was capable of transcriptionally regulating metabolic gene programs in the absence of androgens, highlighting a potential novel castration resistance mechanism to sustain cell metabolism even without a functional AR. Remarkably, we demonstrate that increased mTOR nuclear localization is indicative of poor prognosis in patients, with the highest levels detected in castration-resistant PCa tumors and metastases. Identification of a functional mTOR targeted multigene signature robustly discriminates between normal prostate tissues, primary tumors, and hormone refractory metastatic samples but is also predictive of cancer recurrence. This study thus underscores a paradigm shift from AR to nuclear mTOR as being the master transcriptional regulator of metabolism in PCa.

The dog prostate cancer (DPC-1) model: a reliable tool for molecular imaging of prostate tumors and metastases.

BACKGROUND: Clinical applicability of newly discovered reagents for molecular imaging is hampered by the lack of translational models. As the dog prostate cancer (DPC-1) model recapitulates in dogs the natural history of prostate cancer in man, we tested the feasibility of single-photon emission computed tomography (SPECT)/CT imaging in this model using an anti-prostate-specific membrane antigen (PSMA)/17G1 antibody as the radiotracer. METHODS: Immunoblots and immunohistochemistry (IHC) with 17G1 were performed on canine and human prostate cancer cell lines and tissues. Five dogs with DPC-1 tumors were enrolled for pelvic and, in some instances, thoracic SPECT/CT procedures, also repeated over time. Controls included (111)indium (In)-17G1 prior to DPC-1 implantation and (111)In-immunoglobulins (IgGs) prior to imaging with (111)In-17G1 in dogs bearing prostatic DPC-1 tumors. RESULTS: 17G1 cross-reactivity with canine PSMA (and J591) was confirmed by protein analyses on DPC-1, LNCaP, and PC-3 cell lines and IHC of dog vs. human prostate tissue sections. 17G1 stained luminal cells and DPC-1 cancer cells in dog prostates similarly to human luminal and cancer cells of patients and LNCaP xenografts. SPECT/CT imaging revealed low uptake (≤2.1) of both (111)In-17G1 in normal dog prostates and (111)In-IgGs in growing DPC-1 prostate tumors comparatively to (111)In-17G1 uptake of 3.6 increasing up to 6.5 values in prostate with DPC-1 lesions. Images showed a diffused pattern and, occasionally, a peripheral doughnut-shape-like pattern. Numerous sacro-iliac lymph nodes and lung lesions were detected with contrast ratios of 5.2 and 3.0, respectively. The highest values were observed in pelvic bones (11 and 19) of two dogs, next confirmed as PSMA-positive metastases. CONCLUSIONS: This proof-of-concept PSMA-based SPECT/CT molecular imaging detecting primary prostate tumors and metastases in canines with high cancer burden speaks in favor of this large model's utility to facilitate technology transfer to the clinic and accelerate applications of new tools and modalities for tumor staging in patients.

The Fer tyrosine kinase acts as a downstream interleukin-6 effector of androgen receptor activation in prostate cancer.

Castrate-resistant prostate cancer (CRPC) is invariably lethal and still poorly understood. IL-6/pSTAT3 appears critical as elevated IL-6 and pSTAT3 correlate with CRPC and poor prognosis. We previously reported on the Fer tyrosine kinase being an integral component of the IL-6 pathway in PC by controlling STAT3. Since IL-6 also controls androgen receptor (AR) signaling via pSTAT3, we tested if Fer participates in this cross-talk. We report for the first time that in addition to STAT3, Fer is required for IL-6 mediated AR activation by phosphorylating AR tyrosine 223 and binding via its SH2 domain. Fer controls IL-6 induced growth response and PSA expression, while modestly contributing to EGF and IGF-1 effects. Finally, Fer, AR and pSTAT3 co-localize in the PC cell nucleus, including in prostate tissues from CRPC patients. Altogether these findings support a Fer contribution to aberrant AR signaling via pSTAT3 cross-talks during CRPC progression.

Endoscopic vascular targeted photodynamic therapy with the photosensitizer WST11 for benign prostatic hyperplasia in the preclinical dog model.

PURPOSE: Vascular targeted photodynamic therapy with WST11 (TOOKAD® Soluble) is in phase III clinical trials of an interstitial transperineal approach for focal therapy of prostate cancer. We investigated the safety and efficacy of the endourethral route in the context of benign prostatic hyperplasia in the dog model. MATERIALS AND METHODS: An optical laser fiber was positioned in the prostatic urethra of 34 dogs, including 4 controls. It was connected to a 753 nm diode laser at 200 mW/cm fluence, delivering 200 to 300 J. WST11 (5 to 15 mg/kg) was infused intravenously in 2 modes, including continuous, starting 5 to 15 minutes before and during illumination, or a bolus 5 to 10 minutes before illumination. Prostate ultrasound, cystourethrogram, urodynamics and histopathology were performed. Followup was 1 week to 1 year. RESULTS: Endourethral WST11 vascular targeted photodynamic therapy was uneventful in all except 1 dog, which experienced urinary retention but reached the 1-week end point. All prostates except those in controls showed hemorrhagic lesions. They consisted of 2 levels of concentric alterations, including periurethral necrosis with endothelial layer destruction and adjacent inflammation/atrophy with normal blood vessels. Prostatic urethral width increased as early as 6 weeks after treatment, while prostatic volume decreased, reaching 25% by 18 to 26 weeks. A parallel decrease in urethral pressure at 6 weeks lasted up to 1 year. CONCLUSIONS: We confirmed the vascular effect of endourethral WST11 vascular targeted photodynamic therapy. To our knowledge we report for the first time that the resulting periurethral necrosis led to significant, sustained widening of the prostatic urethra, accompanied by long-term improvement in urodynamic parameters. These findings support future clinical applications of this minimally invasive approach to benign prostatic hyperplasia.

Botulinum toxin type A inhibits the growth of LNCaP human prostate cancer cells in vitro and in vivo.

INTRODUCTION AND OBJECTIVE: Botulinum toxin type A (BTA) intraprostatic injection induces an improvement of urinary symptoms related to benign prostatic hypertrophy (BPH). Infra-clinical prostate cancer (PCa) foci and pre-neoplasic lesions occur concomitantly with BPH in a significant number of patients. The objective of this study was to address whether BTA influences the growth of prostate tumors. METHODS: Proliferation of PC-3 and LNCaP cell lines exposed or not to BTA (Botox) was assessed and compared. Presence of synaptic vesicle 2 (SV2) protein, the membrane receptor of BTA, was studied in both cell lines. After nude mice bearing LNCaP xenografts received intra-tumoral BTA or saline injection, tumor volume, serum PSA, histopathology and detection of apoptosis were comparatively assessed. RESULTS: BTA significantly reduced LNCaP cell proliferation and increased apoptosis in a dose-dependent manner but did not affect PC-3. The SV2 receptor was present in both cell lines at a ratio of 4:1 (LNCaP/PC-3). One unit of BTA resulted in a significantly lower growth rate and slower PSA progression over 28 days compare to controls. The tumors were morphologically similar. There were significantly more apoptotic cells compared to controls. CONCLUSION: BTA inhibits the growth of LNCaP human PCa cells in vitro and in vivo. These findings indicate that intra-prostatic BTA injections to treat BPH are unlikely to promote the growth of co-existing infra-clinical PCa foci in men. A potential inhibitory effect of BTA on the growth of human PCa should be further studied.

The Fer tyrosine kinase cooperates with interleukin-6 to activate signal transducer and activator of transcription 3 and promote human prostate cancer cell growth.

Androgen withdrawal is the most effective form of systemic therapy for men with advanced prostate cancer. Unfortunately, androgen-independent progression is inevitable, and the development of hormone-refractory disease and death occurs within 2 to 3 years in most men. The understanding of molecular mechanisms promoting the growth of androgen-independent prostate cancer cells is essential for the rational design of agents to treat advanced disease. We previously reported that Fer tyrosine kinase level correlates with the development of prostate cancer and aggressiveness of prostate cancer cell lines. Moreover, knocking down Fer expression interferes with prostate cancer cell growth in vitro. However, the mechanism by which Fer mediates prostate cancer progression remains elusive. We present here that Fer and phospho-Y705 signal transducer and activator of transcription 3 (STAT3) are barely detectable in human benign prostate tissues but constitutively expressed in the cytoplasm and nucleus of the same subsets of tumor cells in human prostate cancer. The interaction between STAT3 and Fer was observed in all prostate cancer cell lines tested, and this interaction is mediated via the Fer Src homology 2 domain and modulated by interleukin-6 (IL-6). Moreover, IL-6 triggered a rapid formation of Fer/gp130 and Fer/STAT3 complexes in a time-dependent manner and consistent with changes in Fer and STAT3 phosphorylation and cytoplasmic/nuclear distribution. The modulation of Fer expression/activation resulted in inhibitory or stimulatory effects on STAT3 phosphorylation, nuclear translocation, and transcriptional activation. These effects translated in IL-6-mediated PC-3 cell growth. Taken together, these results support an important function of Fer in prostate cancer.