Eleonora Scarlata

Inhibition of mammalian target of rapamycin as a therapeutic strategy in the management of bladder cancer

We examined whether mTOR inhibition by RAD001 (Everolimus) could be therapeutically efficacious in the treatment of bladder cancer. RAD001 markedly inhibited proliferation of nine human urothelial carcinoma cell lines in dose- and sensitivity-dependent manners in vitro. FACS analysis showed that treatment with RAD001 for 48h induced a cell cycle arrest in the G0/G1 phase in all cell lines, without eliciting apoptosis. Additionally, RAD001 significantly inhibited the phosphorylation of S6 downstream of mTOR and VEGF production in all cell lines. We also found tumor weights from nude mice bearing human KU-7 subcutaneous xenografts treated with RAD001 were significantly reduced as compared to placebo-treated mice. This tumor growth inhibition was associated with significant decrease in cell proliferation rate and angiogenesis without changes in cell death. In conclusion inhibition of mTOR signaling in bladder cancer models demonstrated remarkable antitumor activity both in vitro and in vivo. This is the first study showing that RAD001 could be exploited as a potential therapeutic strategy in bladder cancer.

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. (Mol Cancer Res 2009;7(1):142–55)

Preclinical Study of the Novel Vascular Occluding Agent, WST11, for Photodynamic Therapy of the Canine Prostate

PURPOSE Vascular targeted photodynamic therapy with WST09 shows promise for recurrent prostate cancer after radiation but hydrophobicity in aqueous solutions limited application. We tested the safety and efficacy of WST11, a novel water soluble vascular occluding agent, for vascular targeted photodynamic therapy of the dog prostate and compared it to WST09 vascular targeted photodynamic therapy. MATERIALS AND METHODS Optical fibers were inserted in the prostate and connected to diode lasers. WST11 (Steba Biotech, Cedex, France) at varying doses, including a drug control with no light in 34 dogs, and WST09 (Steba Biotech) (2 mg/kg) in 3 dogs were infused during 10 minutes. Illumination was initiated at 5 or 10 minutes, and lasted up to 33.2 minutes based on laser fluence and delivered energy. Blood was collected for analysis and pharmacokinetics. The end point was at 1 week. RESULTS No vascular targeted photodynamic therapy associated change was observed in blood pressure or blood test values. Circulating WST11 increased with drug infusion and decreased rapidly during 1 hour to reach undetectable levels by 24 hours. All except 1 dog with bowel intussusception did well after vascular targeted photodynamic therapy with only mild urinary symptoms that resolved within 24 to 48 hours. Lung and liver were normal. Hemorrhage was present in all prostates except controls. This translated into necrosis at a WST11 threshold and within a window of doses at fixed illumination. Necrosis was associated with loss of the vessel endothelial layer. Fluence highly impacted necrosis. WST11 vascular targeted photodynamic therapy was advantageously comparable to WST09 vascular targeted photodynamic therapy, and optimally ablated about 5.0 cm(3) of tissue per lobe and about 10 cm(3) of the whole prostate. CONCLUSIONS The safety and efficacy of WST11 vascular targeted photodynamic therapy in the dog prostate support clinical applications for prostate cancer and benign prostatic hyperplasia.

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.

Electrochemical red-ox therapy of prostate cancer in nude mice.

Minimally invasive therapies are increasingly in demand for organ-confined prostate tumors. Electrochemical therapy (EChT) is attractive, as it relies on locally-induced reduction-oxidation reactions to kill tumor cells. Its efficacy for prostate cancer was assessed in human PC-3 and LNCaP tumor xenografts growing subcutaneously in nude mice (n = 80) by applying 2 Stainless Steel vs. 4 Platinum-Iridium (Pt-Ir) electrodes to deliver current densities of 10 to 35 mA/cm(2) for 30 or 60 min. The procedure was uneventful in 90% of mice. No difference in tumor vs. body temperature was observed. Changes at electrode-tumor junctions were immediate, with dryness and acidity (pH2-3) at the anode and oedema and alkalinity (pH10-12) at the cathode. This was accompanied by cellular alterations, found more pronounced at the cathode. Such acidic and alkaline conditions were cytotoxic in vitro and dissolved cells at pH>10. In mice, tumor destruction was extensive by 24h with almost undetectable blood prostate specific antigen (LNCaP model) and covered the whole tumor surface by 4 days. EChT was most efficient at 25-30 mA/cm(2) for 60 min, yielding the longest recurrence-free survival and higher cure rates, especially with 4 Pt-Ir electrodes. EChT is a promising option to optimize for organ-confined prostate tumors.

Isolation of Human Prostatic Epithelial Plasma Membranes for Proteomics Using Mirror Image Tissue Banking of Radical Prostatectomy Specimens

Purpose: To isolate human prostatic epithelial plasma membranes for the identification of cell surface proteins in the therapeutic targeting of cancer cells while permitting the retrieval of banked samples for clinical purposes. Experimental Design: Radical prostatectomies from 84 patients (median, 61 years; prostate-specific antigen, 5.9; 66% nonpalpable) were processed with alternate, mirror image slices submitted for histology and tissue banking. Benign and malignant foci were macrodissected from the banked sections using the pathologically mapped, mirror image histology sections as a guide. Epithelial plasma membranes were isolated using novel immunomagnetic purification and their purity was assessed. Tissue homogenates were probed by Western blot for malignant (AMACR) and benign (p63) markers to test the accuracy of this protocol. Selected banked tissue slices were retrieved, thawed, and compared pathologically to their corresponding routinely processed alternate slices. Results: Plasma membrane preparations showed the enrichment of epithelial plasma membrane markers (prostate-specific membrane antigen and epithelial-specific antigen) with minimal marker expression from nonepithelial cells or intracellular organelles. Cancer homogenates showed up-regulated AMACR and down-regulated p63, whereas benign homogenates showed up-regulated p63 and down-regulated AMACR. There was 30% benign (p63+) contamination in cancer slices and <6% cancer (AMACR+) contamination in benign slices. Retrieved tissues showed the retention of immunoreactivity while their histology was always adequate for diagnosis. Conclusions: We have successfully isolated purified epithelial plasma membranes from benign and malignant human prostates and provided validation data for the accuracy of our protocol in a prostate-specific antigen–screened cohort. Our method also enabled the retrieval of banked tissues for clinical purposes with the retention of good histologic and immunohistochemical quality.

Deficiency of peroxiredoxin 6 or inhibition of its phospholipase A 2 activity impair the in vitro sperm fertilizing competence in mice

Prdx6−/− male mice are subfertile, and the deficiency or inactivation of Peroxiredoxins (PRDXs) is associated with human male infertility. We elucidate the impact of the lack of PRDX6 or inhibition of its calcium-independent phospholipase A2 (Ca2+-iPLA2) activity by MJ33 on fertilization competence of mouse spermatozoa. Sperm motility, viability, fertilization and blastocyst rates were lower in Prdx6−/− spermatozoa than in C57BL/6J wild-type (WT) controls (p ≤ 0.05). MJ33 inhibited the PRDX6 Ca2+-iPLA2 activity and reduced these parameters in WT spermatozoa compared with controls (p ≤ 0.05). Levels of lipid peroxidation and of superoxide anion (O2•─) were higher in Prdx6−/− than in WT spermatozoa (p ≤ 0.05). MJ33 increased the levels of lipid peroxidation and mitochondrial O2•─ production in treated versus non-treated WT spermatozoa. Acrosome reaction, binding to zona pellucida and fusion with the oolemma were lower in Prdx6−/− capacitated spermatozoa than WT capacitated controls and lower in WT spermatozoa treated with the PRDX6 inhibitor. In conclusion, the inhibition of the PRDX6 Ca2+-iPLA2 activity promotes an oxidative stress affecting viability, motility, and the ability of mouse spermatozoa to fertilize oocytes. Thus, PRDX6 has a critical role in the protection of the mouse spermatozoon against oxidative stress to assure fertilizing competence.

Asporin is a stromally expressed marker associated with prostate cancer progression.

Background:Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes.Methods:We have used Kaplan–Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells.Results:We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan–Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p53 inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines.Conclusions:Our results suggest that ASPN is a stromally expressed biomarker that correlates with disease progression, and is observed in reactive stroma. ASPN expression in stroma may be part of a stromal response to aggressive tumour subtypes.

Biopsy characteristics in men with a preoperative diagnosis of prostatic adenocarcinoma with high Gleason score (8-10) predict pathologic outcome in radical prostatectomy

Even if limited to one biopsy core, most urologists and radiation oncologists use the highest Gleason score (GS) to guide therapy. To evaluate the suitability of using biopsy characteristics to predict tumor characteristics at radical prostatectomy (RP) in men with high biopsy GS (BGS) cancer to better select men who will most benefit from various local therapies, we retrospectively reviewed the biopsy and RP findings of 144 men with a BGS 8-10. One hundred six and 38 patients with a BGS of 8 and 9-10, respectively, were included. Forty-eight percent of cases were downgraded to a final GS of 7 at RP, including 54% of BGS 8, and 32% of BGS 9-10 group. Overall, 31% had pT2 disease at RP. Multiple biopsy features, including the GS, the number of positive cores, the number of cores with high-GS cancer, and the maximum volume of high-grade cancer per core (MVPC) consistently predicted final GS and RP tumor stage. Multivariate analysis showed that biopsy GS and MVPC were independent predictors of final GS, while MVPC was also an independent predictor for final pT stage. Patients with high BGS are not a homogeneous group in terms of local tumor characteristics. In addition to BGS (9-10 being worse than 8), other biopsy findings, especially the number of involved cores, number of cores with high-BGS cancer, and MVPC are important predictors of findings at RP that should be incorporated in the decision treatment planning. Most patients with only one core BGS 8 cancer harbor GS 7 cancer.

PD65-12 THE ANDROGEN RECEPTOR BECOMES A NOVEL PREDICTIVE BIOMARKER OF PROSTATE CANCER PROGRESSION WHEN POST TRANSLATIONALLY ACTIVATED BY THE FER KINASE ON TYROSINE 223

INTRODUCTION AND OBJECTIVES: Prostate cancer is a heterogeneous disease both in terms of clinical presentation and pathology which can lead to very different clinical outcomes. Conventional prognostic factors, including serum PSA levels, Gleason score and pathological stage are often inaccurate and histological biomarkers could be useful in distinguishing indolent from aggressive prostate cancers. Tissue microarrays (TMAs) are useful for validating protein biomarker expression in large cohorts of patient samples using immunohistochemistry (IHC) but are often created from radical prostatectomy specimens which do not accurately represent diagnostic biopsies. The limited tumour availability in biopsy samples has led us to develop an improved method for constructing TMAs to study multiple biomarkers simultaneously on biopsy tissues. Objectives: Validate a new method of constructing TMA blocks from prostate needle biopsies and study the link between well known biomarkers (PSA, PSMA, p63,MSMB and AMACR) and mp-MRI data METHODS: Patients attending UCLH with suspected prostate cancer were recruited to the PICTURE study and underwent a diagnostic mp-MRI scan and subsequent image-guided biopsy. This was analysed by a pathologist to confirm tumour Grade. Clinical and MRI data were routinely collected. We extracted the regions of tumour within biopsy samples and re-embedded them so that they could easily be repositioned into a recipient TMA block. Blocks were sectioned and stained using automated IHC for established prostate cancer biomarkers including p63, AMACR, PSMA, MSMB and PSA. RESULTS: We have successfully produced TMA blocks containing representative regions of benign and tumour samples for 200 patients. 99.4% of the cores included were recovered in the TMAs slides. Biomarker expression correlates with Grade of cancer for PSA (p1⁄40.01), MSMB (p1⁄40.016) p63 (p<0.0001), AMACR (p<0.0001) and PSMA (p<0.0001). Expression also correlates with Likert score for PSMA (p1⁄40.009), p63 (p1⁄40.023) and AMACR (p<0.0001). CONCLUSIONS: This new method of constructing TMA blocks is effective at utilising interesting regions of biopsy tissue. It allows multiple biomarkers to be assessed quickly from large cohort studies that accurately represent the tissue routinely used for diagnosis.