Jonathan Melamed

Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence

Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.

A Novel Androgen Receptor Splice Variant Is Up-regulated during Prostate Cancer Progression and Promotes Androgen Depletion–Resistant Growth

The androgen receptor (AR) plays a key role in progression to incurable androgen ablation-resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand-binding domain (designated as AR3, AR4, and AR5) in hormone-insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active, and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly up-regulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells, whereas specific knockdown of AR3 expression (without altering AR level) in hormone-resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct, yet essential, role in ablation-independent growth through the regulation of a unique set of genes, including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation independence during PCA progression, and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available antiandrogen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA.

TWO CONSECUTIVE SETS OF TRANSRECTAL ULTRASOUND GUIDED SEXTANT BIOPSIES OF THE PROSTATE FOR THE DETECTION OF PROSTATE CANCER

PURPOSE We investigated the role of performing 2 consecutive sets of transrectal ultrasound guided sextant biopsies of the prostate in a single office visit as the protocol for detecting prostate cancer in men presenting for the first time with an abnormal digital rectal examination and/or elevated serum prostate specific antigen (PSA). MATERIALS AND METHODS A total of 137 consecutive men presenting for the first time with a clinically localized prostate nodule on digital rectal examination and/or elevated serum PSA based upon age specific reference ranges underwent 2 consecutive sets of sextant prostate biopsies under transrectal ultrasound guidance in a single office visit. The 2 sets of biopsies were processed and analyzed separately by pathologists. RESULTS Adenocarcinoma of the prostate was diagnosed in 43 of the patients (31%) undergoing biopsy. Adenocarcinoma of the prostate was diagnosed in only the second set of biopsies in 13 cases (10%). High grade prostatic intraepithelial neoplasia without adenocarcinoma of the prostate was observed in 18 of the first set of biopsies (15%). High grade intraepithelial neoplasia without adenocarcinoma of the prostate was the only pathological diagnosis in the second set of biopsies in 3 cases. The second set of biopsies provided important new clinical information related to prostate cancer in 20 cases (28%) and increased the number of cancers detected by 30%. In addition, 14 patients with high grade intraepithelial neoplasia who would have required a second set of biopsies were found not to have adenocarcinoma of the prostate. Prostate cancer was detected in 43, 27 and 24% of men with prostate volumes less than 30, 30 to 50 and greater than 50 cc, respectively. The percentage of prostate cancers detected only in the second set of biopsies was not significantly related to prostate size. CONCLUSIONS Two consecutive sets of transrectal ultrasound guided sextant biopsies of the prostate performed in a single office visit represent a cost-effective biopsy strategy for men presenting with an abnormal digital rectal examination and/or elevated serum PSA. The benefits include increasing the detection of adenocarcinoma of the prostate and providing the recommended second set of biopsies for high grade intraepithelial neoplasia without increased morbidity or cost.

Standards of Reporting for MRI-targeted Biopsy Studies (START) of the Prostate: Recommendations from an International Working Group.

BACKGROUND A systematic literature review of magnetic resonance imaging (MRI)-targeted prostate biopsy demonstrates poor adherence to the Standards for the Reporting of Diagnostic Accuracy (STARD) recommendations for the full and transparent reporting of diagnostic studies. OBJECTIVE To define and recommend Standards of Reporting for MRI-targeted Biopsy Studies (START). DESIGN, SETTING, AND PARTICIPANTS Each member of a panel of 23 experts in urology, radiology, histopathology, and methodology used the RAND/UCLA appropriateness methodology to score a 258-statement premeeting questionnaire. The collated responses were presented at a face-to-face meeting, and each statement was rescored after group discussion. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Measures of agreement and consensus were calculated for each statement. The most important statements, based on group median score, the degree of group consensus, and the content of the group discussion, were used to create a checklist of reporting criteria (the START checklist). RESULTS AND LIMITATIONS The strongest recommendations were to report histologic results of standard and targeted cores separately using Gleason score and maximum cancer core length. A table comparing detection rates of clinically significant and clinically insignificant disease by targeted and standard approaches should also be used. It was recommended to report the recruitment criteria for MRI-targeted biopsy, prior biopsy status of the population, a brief description of the MRI sequences, MRI reporting method, radiologist experience, and image registration technique. There was uncertainty about which histologic criteria constitute clinically significant cancer when the prostate is sampled using MRI-targeted biopsy, and it was agreed that a new definition of clinical significance in this setting needed to be derived in future studies. CONCLUSIONS Use of the START checklist would improve the quality of reporting in MRI-targeted biopsy studies and facilitate a comparison between standard and MRI-targeted approaches.

Stromal Cell–Derived Factor-1α and CXCR4 Expression in Hemangioblastoma and Clear Cell-Renal Cell Carcinoma: von Hippel-Lindau Loss-of-Function Induces Expression of a Ligand and Its Receptor

The genetic hallmark of hemangioblastomas and clear cell-renal cell carcinomas (CC-RCCs) is loss-of-function of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL is required for oxygen-dependent degradation of hypoxia-inducible factor-1alpha (HIF-1alpha). In hemangioblastomas and CC-RCCs, HIF-1alpha is constitutively overexpressed leading to increased transcription of HIF-1-regulated genes, including vascular endothelial growth factor (VEGF). Because loss of VHL function is associated with increased expression of the chemokine receptor CXCR4 in CC-RCCs, we investigated the expression of HIF-1alpha, CXCR4, and its ligand stromal cell-derived factor-1alpha (SDF-1alpha) in hemangioblastomas and CC-RCCs. Immunohistochemistry revealed overexpression of both CXCR4 and SDF-1alpha within tumor cells and endothelial cells of hemangioblastomas and CC-RCCs. HIF-1alpha was detected in tumor cell nuclei of both hemangioblastomas and CC-RCCs. A specific ELISA showed that hemangioblastomas and CC-RCCs expressed SDF-1alpha protein at levels that were significantly higher than those found in normal tissue. Analysis of the VHL-null RCC line 786-0 revealed that SDF-1alpha mRNA levels were 100-fold higher than in a subclone transfected with the wild-type VHL gene. Expression of CXCR4 and SDF-1alpha mRNA was significantly decreased in HIF-1alpha-null compared with wild-type mouse embryo fibroblasts (MEFs). ELISA and Western blot studies for SDF-1alpha and CXCR4 protein expression confirmed the RNA findings in RCC lines and MEFs. These results suggest that loss-of-function of a single tumor suppressor gene can up-regulate the expression of both a ligand and its receptor, which may establish an autocrine signaling pathway with important roles in the pathogenesis of hemangioblastoma and CC-RCC.

A Prospective, Blinded Comparison of Magnetic Resonance (MR) Imaging–Ultrasound Fusion and Visual Estimation in the Performance of MR-targeted Prostate Biopsy: The PROFUS Trial

BACKGROUND Increasing evidence supports the use of magnetic resonance (MR)-targeted prostate biopsy. The optimal method for such biopsy remains undefined, however. OBJECTIVE To prospectively compare targeted biopsy outcomes between MR imaging (MRI)-ultrasound fusion and visual targeting. DESIGN, SETTING, AND PARTICIPANTS From June 2012 to March 2013, prospective targeted biopsy was performed in 125 consecutive men with suspicious regions identified on prebiopsy 3-T MRI consisting of T2-weighted, diffusion-weighted, and dynamic-contrast enhanced sequences. INTERVENTION Two MRI-ultrasound fusion targeted cores per target were performed by one operator using the ei-Nav|Artemis system. Targets were then blinded, and a second operator took two visually targeted cores and a 12-core biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Biopsy information yield was compared between targeting techniques and to 12-core biopsy. Results were analyzed using the McNemar test. Multivariate analysis was performed using binomial logistic regression. RESULTS AND LIMITATIONS Among 172 targets, fusion biopsy detected 55 (32.0%) cancers and 35 (20.3%) Gleason sum ≥7 cancers compared with 46 (26.7%) and 26 (15.1%), respectively, using visual targeting (p=0.1374, p=0.0523). Fusion biopsy provided informative nonbenign histology in 77 targets compared with 60 by visual (p=0.0104). Targeted biopsy detected 75.0% of all clinically significant cancers and 86.4% of Gleason sum ≥7 cancers detected on standard biopsy. On multivariate analysis, fusion performed best among smaller targets. The study is limited by lack of comparison with whole-gland specimens and sample size. Furthermore, cancer detection on visual targeting is likely higher than in community settings, where experience with this technique may be limited. CONCLUSIONS Fusion biopsy was more often histologically informative than visual targeting but did not increase cancer detection. A trend toward increased detection with fusion biopsy was observed across all study subsets, suggesting a need for a larger study size. Fusion targeting improved accuracy for smaller lesions. Its use may reduce the learning curve necessary for visual targeting and improve community adoption of MR-targeted biopsy.

Association of P53 Nuclear Overexpression and Tumor Progression in Carcinoma in situ of the Bladder

We investigated the prevalence and clinical relevance of p53 nuclear overexpression, as detected by antibody PAb1801 and immunohistochemistry, in 33 patients with carcinoma in situ of the bladder. Median followup was 124 months. Disease progressed in 16 patients (48%) during followup. The association between p53 nuclear overexpression and tumor progression was assessed by multivariate analysis, controlling for possible confounding variables, such as patient age and sex, presence of associated stage Ta bladder tumor and adjuvant bacillus Calmette-Guerin therapy. Patients were stratified into 2 groups according to the per cent of tumor cells displaying p53 nuclear overexpression: group 1-18 with less than 20% tumor cells positive and group 2-15 with 20% or more tumor cells positive. Disease progressed in 3 patients (16.7%) in group 1 and in 13 (86.7%) in group 2 (p < 0.0001). Detection of p53 nuclear overexpression in 20% or more tumor cells was the only independent marker of tumor progression in univariate and multivariate analyses (p = 0.004, adjusted relative risk 8.6, 95% confidence interval 2 to 40). Death specifically from bladder cancer was also associated with this altered pattern of p53 expression (p = 0.01, Fishers exact test). We conclude that p53 nuclear overexpression is an early event in bladder cancer, occurring in 48% of cases of carcinoma in situ of the bladder. Our results also suggest that p53 nuclear overexpression offers significant clinical information and may be a useful tool in the selection of therapy for patients with carcinoma in situ of the bladder.

Prostate Cancer: Feasibility and Preliminary Experience of a Diffusional Kurtosis Model for Detection and Assessment of Aggressiveness of Peripheral Zone Cancer

PURPOSE To assess the feasibility of diffusional kurtosis (DK) imaging for distinguishing benign from malignant regions, as well as low- from high-grade malignant regions, within the peripheral zone (PZ) of the prostate in comparison with standard diffusion-weighted (DW) imaging. MATERIALS AND METHODS The institutional review board approved this retrospective HIPAA-compliant study and waived informed consent. Forty-seven patients with prostate cancer underwent 3-T magnetic resonance imaging by using a pelvic phased-array coil and DW imaging (maximum b value, 2000 sec/mm2). Parametric maps were obtained for apparent diffusion coefficient (ADC); the metric DK (K), which represents non-Gaussian diffusion behavior; and corrected diffusion (D) that accounts for this non-Gaussianity. Two radiologists reviewed these maps and measured ADC, D, and K in sextants positive for cancer at biopsy. Data were analyzed by using mixed-model analysis of variance and receiver operating characteristic curves. RESULTS Seventy sextants exhibited a Gleason score of 6; 51 exhibited a Gleason score of 7 or 8. K was significantly greater in cancerous sextants than in benign PZ (0.96±0.24 vs 0.57±0.07, P<.001), as well as in cancerous sextants with higher rather than lower Gleason score (1.05±0.26 vs 0.89±0.20, P<.001). K showed significantly greater sensitivity for differentiating cancerous sextants from benign PZ than ADC or D (93.3% vs 78.5% and 83.5%, respectively; P<.001), with equal specificity (95.7%, P>.99). K exhibited significantly greater sensitivity for differentiating sextants with low- and high-grade cancer than ADC or D (68.6% vs 51.0% and 49.0%, respectively; P≤.004) but with decreased specificity (70.0% vs 81.4% and 82.9%, respectively; P≤.023). K had significantly greater area under the curve for differentiating sextants with low- and high-grade cancer than ADC (0.70 vs 0.62, P=.010). Relative contrast between cancerous sextants and benign PZ was significantly greater for D or K than ADC (0.25±0.14 and 0.24±0.13, respectively, vs 0.18±0.10; P<.001). CONCLUSION Preliminary findings suggest increased value for DK imaging compared with standard DW imaging in prostate cancer assessment.

Regulation of androgen receptor transcriptional activity and specificity by RNF6-induced ubiquitination.

The androgen receptor (AR) plays a critical role in prostate cancer. We have identified a ubiquitin E3 ligase, RNF6, as an AR-associated protein in a proteomic screen. RNF6 induces AR ubiquitination and promotes AR transcriptional activity. Specific knockdown of RNF6 or mutation of RNF6-induced ubiquitination acceptor sites on AR selectively alters expression of a subset of AR target genes and diminishes recruitment of AR and its coactivators to androgen-responsive elements present in the regulatory region of these genes. Furthermore, RNF6 is overexpressed in hormone-refractory human prostate cancer tissues and required for prostate cancer cell growth under androgen-depleted conditions. Our data suggest that RNF6-induced ubiquitination may regulate AR transcriptional activity and specificity through modulating cofactor recruitment.

Heterogeneous Expression and Functions of Androgen Receptor Co-Factors in Primary Prostate Cancer

The androgen receptor (AR), a ligand-activated transcription factor of the steroid receptor superfamily, plays an important role in normal prostate growth and in prostate cancer. The recent identification of various AR co-factors prompted us to evaluate their possible roles in prostate tumorigenesis. To this end, we analyzed the expression of AR and eight of its co-factors by quantitative in situ RNA hybridization in 43 primary prostate cancers with different degrees of differentiation. Our results revealed nearly constant expression of AR and heterogeneous expression of AR co-factors, with increased expression of PIAS1 and Ran/ARA24, decreased expression of ELE1/ARA70, and no change in TMF1/ARA160, ARA54, SRC1, or TRAP220. Interestingly, whereas TMF1/ARA160, ELE1/ARA70, ARA54, RAN/ARA24, and PIAS1 were preferentially expressed in epithelial cells, another co-factor, ARA55, was preferentially expressed in stromal cells. Although the changes in levels of these co-activators did not correlate with Gleason score, their occurrence in high-grade prostatic intraepithelial neoplasia, suggests their involvement in initiation (or an early stage) of cancer. In addition, human prostate tumor cell proliferation and colony formation were markedly reduced by ELE1/ATRA70. Together, these findings indicate that changes in levels of expression of AR co-factors may play important, yet different, roles in prostate tumorigenesis.