Marcus V. Cronauer

Enzalutamide in castration-resistant prostate cancer patients progressing after docetaxel and abiraterone.

BACKGROUND Abiraterone, an androgen synthesis inhibitor, has been successfully used in the treatment of castration-resistant prostate cancer (CRPC) for 2 yr. Enzalutamide is a second-generation nonsteroidal antiandrogen that has recently been approved for the same indication. OBJECTIVE This is the first study to evaluate the effectiveness of enzalutamide after failure of abiraterone. DESIGN, SETTING, AND PARTICIPANTS Thirty-five patients were identified as having received sequential therapy with abiraterone followed by enzalutamide. All patients had undergone prior docetaxel chemotherapy, and no patient had received ketoconazole. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Posttreatment changes in prostate-specific antigen (PSA) were used to determine the activity of enzalutamide in patients who had received prior abiraterone. RESULTS AND LIMITATIONS The median duration of abiraterone treatment was 9.0 mo (range: 2.0-19.0 mo). Of the 35 patients, 16 (45.7%) achieved a >50% decline in PSA, and 14 (40%) had a rising PSA as the best response. The median duration of subsequent enzalutamide treatment was 4.9 mo (Kaplan-Meier estimate; 95% confidence interval [CI], 2.4-7.4). Seven of 16 CRPC patients who were initially abiraterone-sensitive (43.8%) and 3 of 19 CRPC patients who were initially abiraterone-insensitive (15.8%) showed a >50% PSA decline while taking enzalutamide. Of the 35 patients, 17 (48.6%) were primarily enzalutamide-resistant and showed a rising PSA as the best response. Median time to progression was 4.0 mo (95% CI, 2.0-6.0) for 18 of 35 patients with at least one declining PSA value while taking enzalutamide (51.4%). Of the 17 patients who were assessable radiologically, only 1 (2.9%) attained a confirmed partial response. Small sample size was the major limitation. CONCLUSIONS Enzalutamide treatment achieved only a modest response rate in patients progressing after abiraterone. Although cross-resistance between abiraterone and enzalutamide was a common phenomenon, it was not inevitable, and a small but significant number of patients showed significant benefit from sequential treatment.

Detecting predictive androgen receptor modifications in circulating prostate cancer cells

Molecular modifications of the androgen receptor (AR) can cause resistance to androgen deprivation therapy (ADT) in prostate cancer patients. Since lack of representative tumor samples hinders therapy adjustments according to emerging AR-modifications, we evaluated simultaneous detection of the two most common AR modifications (AR-V7 splice variant and AR point mutations) in circulating tumor cells (CTCs). We devised a single-tube assay to detect AR-V7 splice variants and AR point mutations in CTCs using immunomagnetic cell isolation, followed by quantitative real-time PCR and DNA pyrosequencing. We prospectively investigated 47 patients with PSA progression awaiting therapy switch. Comparison of response to newly administered therapy and CTC-AR-status allowed effect size estimation. Nineteen (51%) of 37 patients with detectable CTCs carried AR-modifications. Seventeen patients carried the AR-V7 splice variant, one harbored a p.T878A point mutation and one harbored both AR-V7 and a p.H875Y mutation. We estimated a positive predictive value for response and non-response to therapy by AR status in CTCs of ~94%. Based on a conservative calculation, we estimated the effect size for molecularly-informed therapy switches for prospective clinical trial planning to ~27%. In summary, the ability to determine key resistance-mediating AR modifications in CTCs has the potential to considerably improve prostate cancer treatment.

NF-κB signaling in prostate cancer: A promising therapeutic target?

Prostate carcinoma (PCa) displays a wide variety of genetic alterations, versatile expression profiles as well as cell surface markers. Despite this heterogeneity, a common treatment for advanced PCa is androgen deprivation therapy (ADT). ADT targets the androgen receptor—a member of the nuclear receptor superfamily—which is required for development and function of the prostate and critical for PCa growth and survival. After an initial regression of the tumor during ADT, a large fraction of tumors progress to so-called castration-resistant prostate carcinoma (CRPca) which is highly resistant toward chemotherapy. The ensuing high mortality rates illustrate the importance of novel therapeutic targets for CRPCa. The transcription factor NF-κB was recently proposed as such a potential target for therapeutic intervention in CRPCa. Although NF-κB is essential for the regulation of innate and adaptive immunity recent data suggest a role of NF-κB in cancer initiation and progression. However, the exact function of NF-κB signaling in PCa is still a matter of debate. Here, we review known roles of NF-κB signaling in PCa and emphasize the crosstalk of NF-κB and androgen receptor signaling. Finally, we discuss potential therapeutic relevance of blocking NF-κB in PCa.

Inhibition of glycogen synthase kinase-3β promotes nuclear export of the androgen receptor through a CRM1-dependent mechanism in prostate cancer cell lines

The androgen receptor (AR) is a ligand‐dependent transcription factor belonging to the steroid hormone receptor superfamily. Under normal conditions, in the absence of a ligand, the AR is localized to the cytoplasm and is actively transported into the nucleus upon binding of androgens. In advanced prostate cancer (PCa) cell lines, an increased sensitivity to dihydrotestosterone (DHT), enabling the cells to proliferate under sub‐physiological levels of androgens, has been associated with increased stability and nuclear localization of the AR. There is experimental evidence that the glycogen synthase kinase‐3β (GSK‐3β), a multifunctional serine/threonine kinase is involved in estrogen and AR stability. As demonstrated in the following study by immunoprecipitation analysis, GSK‐3β binds to the AR forming complexes in the cytoplasm and in the nucleus. Furthermore, inhibition of GSK‐3β activity by pharmacological inhibitors like the maleimide SB216761, the chloromethyl‐thienyl‐ketone GSK‐3 inhibitor VI or the aminopyrazol GSK‐3 inhibitor XIII in cells grown in the presence of DHT triggered a rapid nuclear export of endogenous AR as well as of green fluorescent AR‐EosFP. The nuclear export of AR following GSK‐3β inhibition could be blocked by leptomycin B suggesting a CRM1‐dependent export mechanism. This assumption is supported by the localization of a putative CRM1 binding site at the C‐terminus of the AR protein. The results suggest that GSK‐3β is an important element not only in AR stability but also significantly alters nuclear translocation of the AR, thereby modulating the androgenic response of human PCa cells. J. Cell. Biochem. 109: 1192–1200, 2010.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells

BackgroundNitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC).MethodsProstate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay.ResultsThe NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway.ConclusionsOur results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.

Overexpression of p16INK4a in Urothelial Carcinoma In Situ Is a Marker for MAPK-Mediated Epithelial-Mesenchymal Transition but Is Not Related to Human Papillomavirus Infection

Background The role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16INK4a, a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16INK4a is associated with HPV infection and to identify mechanisms of p16INK4a upregulation in UCIS. Materials and Methods In 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16INK4a immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16INK4a applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16INK4a as well as markers of EMT. Results We found overexpression of p16INK4a in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16INK4a. High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16INK4a and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib. Conclusions Our results show that overexpression of p16INK4a in UCIS is neither associated with HPV infection nor p16INK4a gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.

Inhibition of Glycogen Synthase Kinase-3β Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.

High CRP values predict poor survival in patients with penile cancer

BackgroundHigh levels of circulating C-reactive protein (CRP) have recently been linked to poor clinical outcome in various malignancies. The aim of this study was to evaluate the prognostic significance of the preoperative serum CRP level in patients with squamous cell carcinoma (SCC) of the penis.MethodsThis retrospective analysis included 79 penile cancer patients with information about their serum CRP value prior to surgery who underwent either radical or partial penectomy at two German high-volume centers (Ulm University Medical Center and Hannover Medical School) between 1990 and 2010. They had a median (mean) follow-up of 23 (32) months.ResultsA significantly elevated CRP level (>15 vs. ≤ 15 mg/l) was found more often in patients with an advanced tumor stage (≥pT2) (38.9 vs. 11.6%, p=0.007) and in those with nodal disease at diagnosis (50.0 vs. 14.6%, p=0.007). However, high CRP levels were not associated with tumor differentiation (p=0.53). The Kaplan-Meier 5-year cancer-specific survival (CSS) rate was 38.9% for patients with preoperative CRP levels above 15 mg/l and 84.3% for those with lower levels (p=0.001). Applying multivariate analysis and focusing on the subgroup of patients without metastasis at the time of penile surgery, both advanced local tumor stage (≥pT2; HR 8.8, p=0.041) and an elevated CRP value (>15 mg/l; HR 3.3, p=0.043) were identified as independent predictors of poor clinical outcome in patients with penile cancer.ConclusionsA high preoperative serum CRP level was associated with poor survival in patients with penile cancer. If larger patient populations confirm its prognostic value, its routine use could enable better risk stratification and risk-adjusted follow-up of patients with SCC of the penis.

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.

AR-Q640X, a model to study the effects of constitutively active C-terminally truncated AR variants in prostate cancer cells

PurposeA recently identified mechanism allowing prostate cancer (PCa) cells to grow in the absence of androgens is the expression of constitutively active, C-terminally truncated androgen receptor (AR) variants lacking vast parts of the ligand-binding domain. These AR variants termed ARΔLBD are either products of alternative splicing, point mutations leading to premature stop codons or proteolytic cleavage of the AR. Some controversies exist about the requirement of additional full-length AR for the full transcriptional activity of the ARΔLBD. On basis of a mutated, C-terminally truncated AR termed Q640X, we developed an experimental model for the study of ARΔLBD in PCa cells.MethodsActivation of AR-dependent promoters was analyzed by reporter gene assays. Dimerization studies were conducted using a mammalian two-hybrid system.ResultsAlthough Q640X/Q640X homodimers were able to induce the expression of certain AR target genes, Q640X/AR heterodimers were necessary to activate the full panel of androgen-dependent genes under androgen-deprived conditions.ConclusionsThe following study supports the hypothesis that castration-resistant prostate cancer (CRPC) cells are able to activate specific androgen-dependent genes by selective modulation of the ratio between ARΔLBD and their putative dimerization partners like the full-length AR or other ARΔLBD in the absence of androgens. The present data suggest that AR-mutant Q640X is a powerful experimental tool for the functional analysis of ARΔLBD in CRPC.