Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

Quality of life and genetics in men with prostate cancer

As family history has been established as a risk factor for prostate cancer, attempts have been made to isolate predisposing genetic variants that are related to hereditary prostate cancer. With many genetic variants still to be identified and investigated, it is not yet possible to fully understand the impact of genetic variants on prostate cancer development. The high survival rates among men with prostate cancer have meant that other issues, such as quality of life (QoL), have also become important. Through their effect on a person’s health, a range of inherited genetic variants may potentially influence QoL in men with prostate cancer, even prior to treatment. Until now, limited research has been conducted on the relationship between genetics and QoL. Thus, this study contributes to an emerging field by aiming to identify certain genetic variants related to the QoL found in men with prostate cancer. It is hoped that this study may lead to future research that will identify men who have an increased risk of a poor QoL following prostate cancer treatment, which will aid in developing treatments that are individually tailored to support them. Previous studies have established that genetic variants of Vascular Endothelial Growth Factor (VEGF) and Insulin-like Growth Factor 1 (IGF-1) may play a role in prostate cancer development. VEGF and IGF-1 have also been reported to be associated with QoL in people with ovarian cancer and colorectal cancer, respectively. This study completed a series of secondary analyses using two major data-sets (from 850 men newly diagnosed with prostate cancer, and approximately 550 men from the general Queensland population), in which genetic variants of VEGF and IGF-1 were investigated for associations with prostate cancer susceptibility and QoL. The first aim of this research was to investigate genetic variants in the VEGF and IGF-I gene for an association with the risk of prostate cancer. It was found that one IGF-1 genetic variant (rs35765) had a statistically significant association with prostate cancer (p = 0.04), and one VEGF genetic variant (rs2146323) had a statistically significant association with advanced prostate cancer (p = 0.02). The estimates suggest that carriers of the CA and AA genotype for rs35765 may have a reduced risk of developing prostate cancer (Odds Ratio (OR) = 0.72, 95% Confidence Interval (CI) = 0.55, 0.95, OR = 0.60, 95% CI = 0.26, 1.39, respectively). Meanwhile, carriers of the CA and AA genotype for rs2146323 may be at increased risk of advanced prostate cancer, which was determined by a Gleason score of above 7 (OR = 1.72, 95% CI = 1.12, 2.63, OR = 1.90, 95% CI = 1.08, 3.34, respectively). Utilising the widely used short-form health survey, the SF-36v2, the second aim of this study was to investigate the relationship between prostate cancer and QoL prior to treatment. Assessing QoL at this time-point was important as little research has been conducted to evaluate if prostate cancer affects QoL regardless of treatment. The analyses found that mean SF-36v2 scale scores related to physical health were higher by at least 0.3 Standard Deviations (SD) among men with prostate cancer than the general population comparison group. This difference was considered clinically significant (defined by group differences in mean SF-36v2 scores by at least 0.3 SD). These differences were also statistically significant (p<0.05). Mean QoL scale scores related to mental health were similar between men with prostate cancer and those from the general population comparison group. The third aim of this study was to investigate genetic variants in the VEGF and IGF-1 gene for an association with QoL in prostate cancer patients prior to their treatment. It was essential to evaluate these relationships prior to treatment, before the involvement of these genes was potentially interrupted by treatment. The analyses found that some genetic variants had a small clinically significant association (0.3 SD) to some QoL domains experienced by these men. However, most relationships were not statistically significant (p>0.05). Most of the associations found identified that a small sub-group of men with prostate cancer (approximately 2%) reported, on average, a slightly better QoL than the majority of the prostate cancer patients. The fourth aim of this research was to investigate whether associations between genetic variants in VEGF and IGF-1 and QoL were specific to men with prostate cancer, or were also applicable to the general male population. It was found that twenty out of one-hundred relationships between the genetic variants of VEGF and IGF-1 and QoL health-measures and scales examined differed between these groups. In the majority of the relationships involving VEGF SNPs that differed, a clinically significant difference (0.3 or more SD) between mean scores among the genotype groups in prostate cancer patients was found, while mean scores among men from the general-population comparison group were similar. For example, prostate cancer participants who carried at least one T allele (CT or TT genotype) for rs3024994 had a clinically significant higher (0.3 SD) mean QoL score in terms of the role-physical scale, than participants who carried the CC genotype. This was not seen among men from the general population sample, as the mean score was similar between genotype groups. The opposite was seen in regards to the IGF-1 SNPs examined. Overall, these relationships were not considered to directly impact on the clinical options for men with prostate cancer. As this study utilised secondary data from two separate studies, there are a number of important limitations that should be acknowledged including issues of multiple comparisons, power, and missing or unavailable data. It is recommended that this study be replicated as a better-designed study that takes greater consideration of the many factors involved in prostate cancer and QoL. Investigation into other genetic variants of VEGF or IGF-1 is also warranted, as is consideration of other genes and their relationship with QoL. Through identifying certain genetic variants that have a modest association to prostate cancer, this project adds to the knowledge surrounding VEGF and IGF-1 and their role in prostate cancer susceptibility. Importantly, this project has also introduced the potential role genetics plays in QoL, through investigating the relationships between genetic variants of VEGF and IGF-1 and QoL.

The interactions between insulin and androgens in progression to castrate resistant prostate cancer

An association between the metabolic syndrome and reduced testosterone levels has been identified, and a specific inverse relationship between insulin and testosterone levels suggests that an important metabolic crosstalk exists between these two hormonal axes; however, the mechanisms by which insulin and androgens may be reciprocally regulated are not well described. Androgen-dependant gene pathways regulate the growth and maintenance of both normal and malignant prostate tissue, and androgen-deprivation therapy (ADT) in patients exploits this dependence when used to treat recurrent and metastatic prostate cancer resulting in tumour regression. A major systemic side effect of ADT includes induction of key features of the metabolic syndrome and the consistent feature of hyperinsulinaemia. Recent studies have specifically identified a correlation between elevated insulin and high-grade PCa and more rapid progression to castrate resistant disease. This paper examines the relationship between insulin and androgens in the context of prostate cancer progression. Prostate cancer patients present a promising cohort for the exploration of insulin stabilising agents as adjunct treatments for hormone deprivation or enhancers of chemosensitivity for treatment of advanced prostate cancer.

The role of the Eph and ephrin proteins in prostate cancer

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer related deaths in Australian men. Treatment in the early stages of the disease involves surgery, radiation and/or hormone therapy. However, in late stages of the disease these treatments are no longer effective and only palliative care is available. Therefore, there is a focus on exploration of novel therapies to increase survival and treatment efficacy. Advanced prostate cancer is characterised by bone or other distant metastasis. Spreading of the primary tumour to a secondary location is a complex process requiring an initial loss in cell-cell adhesion followed by increased cell migration and invasion. One gene family that has been known to affect cell-to-cell contact in other model systems are the Eph receptor tyrosine kinases. They are the largest family of receptor tyrosine kinases made up of 14 vertebrate Eph receptors that bind to nine cell membrane bound ephrin ligands. Eph-ephrin interaction is crucial in regulating cell behaviour in developmental processes and it is now thought that the underlying mechanisms involved in development may also be involved in cancer. Aberrant expression has been reported in many human malignancies including prostate cancer. Furthermore, expression has been linked with metastasis and poor prognosis in other tumour models. This study explores the potential role of the Eph receptor family in prostate cancer, in particular the roles of EphA2, EphA3 and ephrin-A5. Gene expression profiles were established for the Eph family in a series of prostate cancer cell lines using quantitative real time RT-PCR. A smaller subset of the most prominently expressed genes was chosen to screen a cohort of clinical samples. Elevated levels of EphA2, EphA3 and their ligands, ephrin-A1 and ephrin-A5 were observed in individual cell lines. Interestingly high EphA3 expression was observed in the androgen responsive cell lines while EphA2 was more prominent in the androgen independent cell lines. However, studies using 5-dihydrotestosterone suggest that EphA3 expression in not regulated by androgen. Cells expressing EphA2 showed a greater ability for migration and invasion while cells expressing EphA3 showed poor migration and invasion. Forced expression of EphA2 in the LNCaP cell line resulted in a more invasive phenotype while forced expression of EphA3 in the PC-3 cell line suggests a possible negative effect for EphA3 on cell migration and invasion. Cell signalling studies show activation of EphA2 decreases activity of proteins thought to be involved in pathways regulating cell movement including Akt, Src and FAK. Changes to the activation status of Rho family members, including RhoA and Rac1, associated with reorganisation of the actin cytoskeleton, an important part of cell migration was also observed. As a result, activation of EphA2 in PC-3 cells resulted in a less invasive phenotype. A novel finding in this study was the discovery of a combination of two EphA2 Mabs able to activate EphA2. Preliminary results show a potential for this antibody combination to reduce prostate cancer invasion in vitro. A unique aspect of Eph-ephrin interaction is the resulting bi-directional signalling that occurs through both the receptor and ligand. In this study a potential role for ephrin-A5 mediated signalling in prostate cancer was observed. LNCaP cells express high levels of EphA3 and its high affinity ligand ephrin-A5. In stripe assays, used to study guidance cues, LNCaP cells show strong attraction/migration to EphA3-Fc stripes but not ephrin-A5-Fc stripes suggesting ephrin-A5 mediated reverse cell signalling is involved. Knockdown of ephrin-A5 using shRNA resulted in a decrease in attraction/migration to EphA3-Fc stripes. Furthermore a reduction in proliferation was also observed in vitro. A subcutaneous xenograft model using ephrin-A5 shRNA cells versus controls showed a decrease in tumour formation. This study demonstrates a difference in EphA2 and EphA3 function in prostate cancer migration/invasion and a potential role for ephrin-A5 in prostate cancer cell adhesion and growth.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing,CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, preexisting SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SClike BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (ARlow) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The ARlow seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

Androgen-targeted therapy-induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer : an opportunity for intervention

Androgens regulate biological pathways to promote proliferation, differentiation, and survival of benign and malignant prostate tissue. Androgen receptor (AR) targeted therapies exploit this dependence and are used in advanced prostate cancer to control disease progression. Contemporary treatment regimens involve sequential use of inhibitors of androgen synthesis or AR function. Although targeting the androgen axis has clear therapeutic benefit, its effectiveness is temporary, as prostate tumor cells adapt to survive and grow. The removal of androgens (androgen deprivation) has been shown to activate both epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD) programs. EMT has established roles in promoting biological phenotypes associated with tumor progression (migration/invasion, tumor cell survival, cancer stem cell-like properties, resistance to radiation and chemotherapy) in multiple human cancer types. NEtD in prostate cancer is associated with resistance to therapy, visceral metastasis, and aggressive disease. Thus, activation of these programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression. Brachyury, Axl, MEK, and Aurora kinase A are molecular drivers of these programs, and inhibitors are currently in clinical trials to determine therapeutic applications. Understanding tumor cell plasticity will be important in further defining the rational use of androgen-targeted therapies clinically and provides an opportunity for intervention to prolong survival of men with metastatic prostate cancer.

Establishing prostate cancer patient derived xenografts: Lessons learned from older studies

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model

Prostate cancer organoids: A potential new tool for testing drug sensitivity

Recent technical advances have enabled for the first time, reliable in vitro culture of prostate cancer samples as prostate cancer organoids. This breakthrough provides the significant possibility of high throughput drug screening covering the spectrum of prostate cancer phenotypes seen clinically. These advances will enable precision medicine to become a reality, allowing patient samples to be screened for effective therapeutics ex vivo, with tailoring of treatments specific to that individual. This will hopefully lead to enhanced clinical outcomes, avoid morbidity due to ineffective therapies and improve the quality of life in men with advanced prostate cancer.

Extracellular vesicles mediate paracrine signalling in androgen deprived prostate cancer

The androgen receptor (AR) is the main therapeutic target for advanced prostate cancer (PCa). Current treatments have focused on inhibiting the transcriptional activity of the AR, however androgens can also induce non-genomic effects by facilitating the initiation of kinase signaling cascades in PCa. Cells, including PCa, secrete extracellular vesicles (EV), which are able to mediate communication between cells and can also contribute towards these processes.

MYC activity mitigates response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-binding protein 1-mediated inhibition of autophagy.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial

BACKGROUND: Long-term hormone therapy has been the standard of care for advanced prostate cancer since the 1940s. STAMPEDE is a randomised controlled trial using a multiarm, multistage platform design. It recruits men with high-risk, locally advanced, metastatic or recurrent prostate cancer who are starting first-line long-term hormone therapy. We report primary survival results for three research comparisons testing the addition of zoledronic acid, docetaxel, or their combination to standard of care versus standard of care alone.

METHODS: Standard of care was hormone therapy for at least 2 years; radiotherapy was encouraged for men with N0M0 disease to November, 2011, then mandated; radiotherapy was optional for men with node-positive non-metastatic (N+M0) disease. Stratified randomisation (via minimisation) allocated men 2:1:1:1 to standard of care only (SOC-only; control), standard of care plus zoledronic acid (SOC + ZA), standard of care plus docetaxel (SOC + Doc), or standard of care with both zoledronic acid and docetaxel (SOC + ZA + Doc). Zoledronic acid (4 mg) was given for six 3-weekly cycles, then 4-weekly until 2 years, and docetaxel (75 mg/m(2)) for six 3-weekly cycles with prednisolone 10 mg daily. There was no blinding to treatment allocation. The primary outcome measure was overall survival. Pairwise comparisons of research versus control had 90% power at 2·5% one-sided α for hazard ratio (HR) 0·75, requiring roughly 400 control arm deaths. Statistical analyses were undertaken with standard log-rank-type methods for time-to-event data, with hazard ratios (HRs) and 95% CIs derived from adjusted Cox models. This trial is registered at ClinicalTrials.gov (NCT00268476) and ControlledTrials.com (ISRCTN78818544).

FINDINGS: 2962 men were randomly assigned to four groups between Oct 5, 2005, and March 31, 2013. Median age was 65 years (IQR 60-71). 1817 (61%) men had M+ disease, 448 (15%) had N+/X M0, and 697 (24%) had N0M0. 165 (6%) men were previously treated with local therapy, and median prostate-specific antigen was 65 ng/mL (IQR 23-184). Median follow-up was 43 months (IQR 30-60). There were 415 deaths in the control group (347 [84%] prostate cancer). Median overall survival was 71 months (IQR 32 to not reached) for SOC-only, not reached (32 to not reached) for SOC + ZA (HR 0·94, 95% CI 0·79-1·11; p=0·450), 81 months (41 to not reached) for SOC + Doc (0·78, 0·66-0·93; p=0·006), and 76 months (39 to not reached) for SOC + ZA + Doc (0·82, 0·69-0·97; p=0·022). There was no evidence of heterogeneity in treatment effect (for any of the treatments) across prespecified subsets. Grade 3-5 adverse events were reported for 399 (32%) patients receiving SOC, 197 (32%) receiving SOC + ZA, 288 (52%) receiving SOC + Doc, and 269 (52%) receiving SOC + ZA + Doc.

INTERPRETATION: Zoledronic acid showed no evidence of survival improvement and should not be part of standard of care for this population. Docetaxel chemotherapy, given at the time of long-term hormone therapy initiation, showed evidence of improved survival accompanied by an increase in adverse events. Docetaxel treatment should become part of standard of care for adequately fit men commencing long-term hormone therapy.

FUNDING: Cancer Research UK, Medical Research Council, Novartis, Sanofi-Aventis, Pfizer, Janssen, Astellas, NIHR Clinical Research Network, Swiss Group for Clinical Cancer Research.

Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cells

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA re-expression was only attained GSTP1 and APC. Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.

Identifying the psychosexual implications of radical radiation treatment in prostate cancer

The purpose of this study was to determine whether there was any evidence of psychosexual morbidity among men who experienced radical radiation treatment for prostate cancer. With relatively little known or available retrospective data on the psychosexual implications of radical radiation treatment in men with prostate cancer, this study posited eight research questions which provided the basis for the research. Fifty men from Southern Ontario, between the ages of 52 to 78 years, were included in the study. They had been previously randomized to a clinical trial comparing radical radiation therapy by external beam radiation, or radical radiation using a combination of a temporary iridium implant plus external beam radiation, for localized or locally advanced prostate cancer. Assessment of sexual functioning, drive, attitudes, body image, and sexual satisfaction was drawn from a multidimensional approach, since psychosexuality was viewed as having an impact on biological, psychological, and sociological domains of functioning. Medical chart reviews, semi-structured interviews, demographical profiles of each participant, and the Derogatis Sexual Functioning Inventory (DSFI) were the methods used to collect data over a four-month period. Both quantitative and qualitative research methods were incorporated in the design and evaluation of the study. Frequencies, contingency analysis, Pearson's coefficient of correlation, t-tests, and ANOVA comprised the quantitative analysis. Data obtained from audio-taped interviews were analyzed qualitatively, and used for offering further insight and for facilitating the quantitative aspect of the analysis. Overall, there was sufficient evidence to suggest psychosexual morbidity among men who were treated with radiation therapy for prostate cancer. As well,there were a number of significant findings available to answer all of the posited research questions. The most significant findings were noted in post-treatment erectile ability and sexual activity. A post-treatment change in erectile ability was reported by eighty percent of men. Sixty percent of men noted a decrease in their ability to achieve an erection by reporting some morning stiffness only, penile rigidity insufficient for penetration, decreased control of erection, and loss of spontaneous erection. Other contributing factors associated with change in erectile status were: pain or altering sensation of orgasm, blood in ejaculate, pain and decreased amount of ejaculate, and penile numbness or pain. Eighty-two percent of men experienced a post-treatment change in sexual function, primarily due to the impact of decreasing erectile status. Only seven men reported that they experienced a decrease in desire mentally, whereas the vast majority did not experience any change in desire. Changes in foreplay, stress with optimal sexual positioning, and reduced spontaneity of sex, were other factors reported with the changes in sexual activity. The findings in this study broaden our understanding of what middle- to later-aged men feel and experience as they venture onward following treatment. This was the first study that evaluated available prospective data on pre-treatment erectile status and sexual activity. As well, this study was the first (with participant compliance rates of 100 percent) to have included an interview format to capture the views of such a large number of men. This study concluded with recommendations and implications for future research and practice as we move in the direction of understanding what is necessary for preserving psychosexual well being and enhancing quality of life in men treated with radiation therapy for prostate cancer.

Immuno-oncology of human prostate cancer : phenotypical characterization and study of the tumor-derived, androgen-regulated immunosuppressive microenvironment

Le cancer de la prostate est le cancer le plus fréquemment diagnostiqué chez les hommes canadiens et la troisième cause de décès relié au cancer. Lorsque diagnostiqué à un stade précoce de la maladie, le cancer de la prostate est traité de manière curative par chirurgie et radiothérapie. Par contre, les thérapies actuelles ne peuvent éradiquer la maladie lorsqu’elle progresse à des stades avancés. Ces thérapies, comme la chimiothérapie et l’hormonothérapie, demeurent donc palliatives. Il est primordial d’optimiser de nouvelles thérapies visant l’élimination des cellules cancéreuses chez les patients atteints des stades avancés de la maladie. Une de ces nouvelles options thérapeutiques est l’immunothérapie. L’immunothérapie du cancer a fait des progrès considérables durant les dernières années. Cependant, les avancements encourageants obtenus lors d’essais précliniques ne se sont pas encore traduits en des résultats cliniques significatifs. En ce qui concerne le cancer de la prostate, les résultats négligeables suivants des interventions immunothérapeutiques peuvent être causés par le fait que la plupart des études sur le microenvironnement immunologique furent effectuées chez des modèles animaux. De plus la majorité des études sur l’immunologie tumorale humaine furent effectuées chez des patients atteints d’autres cancers, tels que le mélanome, et non chez les patients atteints du cancer de la prostate. Donc, le but central de cette thèse de doctorat est d’étudier le microenvironnement immunologique chez les patients atteints du cancer de la prostate afin de mieux définir les impacts de la tumeur sur le développement de la réponse immunitaire antitumorale. Pour réaliser ce projet, nous avons établi deux principaux objectifs de travail : (i) la caractérisation précise des populations des cellules immunitaires infiltrant la tumeur primaire et les ganglions métastatiques chez les patients atteints du cancer de la prostate; (ii) l’identification et l’étude des mécanismes immunosuppressifs exprimés par les cellules cancéreuses de la prostate. Les résultats présentés dans cette thèse démontrent que la progression du cancer de la prostate est associée au développement d’un microenvironnement immunosuppressif qui, en partie, est régulé par la présence des androgènes. L’étude initiale avait comme but la caractérisation du microenvironnement immunologique des ganglions drainant la tumeur chez des patients du cancer de la prostate. Les résultats présentés dans le chapitre III nous a permis de démontrer que les ganglions métastatiques comportent des signes cellulaires et histopathologiques associés à une faible réactivité immunologique. Cette immunosuppression ganglionnaire semble dépendre de la présence des cellules métastatiques puisque des différences immunologiques notables existent entre les ganglions non-métastatiques et métastatiques chez un même patient. La progression du cancer de la prostate semble donc associée au développement d’une immunosuppression affectant les ganglions drainant la tumeur primaire. Par la suite, nous nous sommes intéressés à l’impact de la thérapie par déplétion des androgènes (TDA) sur le microenvironnement immunologique de la tumeur primaire. La TDA est associée à une augmentation marquée de l’inflammation prostatique. De plus, les protocoles d’immunothérapies pour le cancer de la prostate actuellement évalués en phase clinique sont dirigés aux patients hormonoréfractaires ayant subi et échoué la thérapie. Cependant, peu d’information existe sur la nature de l’infiltrat de cellules immunes chez les patients castrés. Il est donc essentiel de connaître la nature de cet infiltrat afin de savoir si celui-ci peut répondre de manière favorable à une intervention immunothérapeutique. Dans le chapitre IV, je présente les résultats sur l’abondance des cellules immunes infiltrant la tumeur primaire suivant la TDA. Chez les patients castrés, les densités de lymphocytes T CD3+ et CD8+ ainsi que des macrophages CD68+ sont plus importantes que chez les patients contrôles. Nous avons également observé une corrélation entre la densité de cellules NK et une diminution du risque de progression de la maladie (rechute biochimique). Inversement, une forte infiltration de macrophages est associée à un plus haut risque de progression. Conjointement, durant cette étude, nous avons développé une nouvelle approche informatisée permettant la standardisation de la quantification de l’infiltrat de cellules immunes dans les échantillons pathologiques. Cette approche facilitera la comparaison d’études indépendantes sur la densité de l’infiltrat immun. Ces résultats nous ont donc permis de confirmer que les effets pro-inflammatoires de la TDA chez les patients du cancer de la prostate ciblaient spécifiquement les lymphocytes T et les macrophages. L’hypothèse intéressante découlant de cette étude est que les androgènes pourraient réguler l’expression de mécanismes immunosuppressifs dans la tumeur primaire. Dans le chapitre V, nous avons donc étudié l’expression de mécanismes immunosuppressifs par les cellules cancéreuses du cancer de la prostate ainsi que leur régulation par les androgènes. Notre analyse démontre que les androgènes augmentent l’expression de molécules à propriétés immunosuppressives telles que l’arginase I et l’arginase II. Cette surexpression dépend de l’activité du récepteur aux androgènes. Chez les patients castrés, l’expression de l’arginase II était diminuée suggérant une régulation androgénique in vivo. Nous avons observé que l’arginase I et l’arginase II participent à la prolifération des cellules du cancer de la prostate ainsi qu’à leur potentiel immunosuppressif. Finalement, nous avons découvert que l’expression de l’interleukin-8 était aussi régulée par les androgènes. De plus, l’interleukin-8, indépendamment des androgènes, augmente l’expression de l’arginase II. Ces résultats confirment que les androgènes participent au développement d’une microenvironnement immunosuppressif dans le cancer de la prostate en régulant l’expression de l’arginase I, l’arginase II et l’interleukin-8. En conclusion, les résultats présentés dans cette thèse témoignent du caractère unique du microenvironnement immunologique chez les patients atteints du cancer de la prostate. Nos travaux ont également permis d’établir de nouvelles techniques basées sur des logiciels d’analyse d’image afin de mieux comprendre le dialogue entre la tumeur et le système immunitaire chez les patients. Approfondir les connaissances sur les mécanismes de régulation du microenvironnement immunologique chez les patients atteint du cancer de la prostate permettra d’optimiser des immunothérapies mieux adaptées à éradiquer cette maladie.