Tobias Zellweger

Stat3 promotes metastatic progression of prostate cancer.

There are currently no effective therapies for metastatic prostate cancer because the molecular mechanisms that underlie the metastatic spread of primary prostate cancer are unclear. Transcription factor Stat3 is constitutively active in malignant prostate epithelium, and its activation is associated with high histological grade and advanced cancer stage. In this work, we hypothesized that Stat3 stimulates metastatic progression of prostate cancer. We show that Stat3 is active in 77% of lymph node and 67% of bone metastases of clinical human prostate cancers. Importantly, adenoviral gene delivery of wild-type Stat3 (AdWTStat3) to DU145 human prostate cancer cells increased the number of lung metastases by 33-fold in an experimental metastasis assay compared with controls. Using various methods to inhibit Stat3, we demonstrated that Stat3 promotes human prostate cancer cell migration. Stat3 induced the formation of lamellipodia in both DU145 and PC-3 cells, further supporting the concept that Stat3 promotes a migratory phenotype of human prostate cancer cells. Moreover, Stat3 caused the rearrangement of cytoplasmic actin stress fibers and microtubules in both DU145 and PC-3 cells. Finally, inhibition of the Jak2 tyrosine kinase decreased both activation of Stat3 and prostate cancer cell motility. Collectively, these data indicate that transcription factor Stat3 is involved in metastatic behavior of human prostate cancer cells and may provide a therapeutic target to prevent metastatic spread of primary prostate cancer.

Expression patterns of potential therapeutic targets in prostate cancer.

Androgen withdrawal is the only effective therapy for patients with advanced prostate cancer, but progression to androgen independence ultimately occurs in almost all patients. Novel therapeutic strategies targeting molecular mechanisms that mediate resistance to hormonal and chemotherapeutic treatment are highly warranted. Here, we aimed to evaluate the expression of potential therapeutic targets in advanced prostate cancer. A tissue microarray (TMA) containing samples from 535 tissue blocks was constructed, including benign prostatic hyperplasia as controls (n = 65), prostatic intraepithelial neoplasia (PIN; n = 78), clinically localized prostate cancers (n = 181), as well as hormone‐refractory local recurrences (n = 120) and distant metastases (n = 91). The expression of 13 different proteins was analyzed using immunohistochemistry (Bcl‐2, p53, ILK, Syndecan‐1, MUC‐1, EGFR, HER2/neu, HSP‐90, Ep‐CAM, MMP‐2, CD‐10, CD‐117 and Ki67). Significant overexpression in hormone‐refractory prostate cancer and metastatic tissue compared to localized prostate cancer was found for Ki67 (64% vs. 9%), Bcl‐2 (11% vs. 1%), p53 (35% vs. 4%), Syndecan‐1 (38% vs. 3%), EGFR (16% vs. 1%) and HER2/neu (16% vs. 0%). Overexpression of CD‐117 was restricted to 1 single metastasis. All other markers did not show relevant differences in expression between subgroups. Taken together, p53, Bcl‐2, Syndecan‐1, EGFR and HER2/neu are preferentially expressed in hormone‐refractory and metastatic prostate cancer. Selected inhibition of these targets might offer a strategy to treat advanced tumors and prevent further progression. Treatment decisions should not be based on findings in primary tumors but rather on tissues from recurrent or metastatic lesions.

Multi-target fluorescence in situ hybridization in bladder washings for prediction of recurrent bladder cancer

The objective of this study was to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for predicting recurrence of urothelial carcinoma (UC) after transurethral resection. One hundred and thirty‐eight patients (median age 68.5 years) with a history of UC were eligible for this prospective study. FISH was applied to cytospin specimens prepared from bladder washings taken during a negative control cystoscopy. The multi‐target FISH test UroVysion® (Abbott/Vysis) containing probes to the centromeres of chromosomes 3, 7, 17 and the 9p21 locus was used. UC recurrence was defined as a positive biopsy during follow‐up. The median follow‐up time was 19.2 (4–52) months. FISH was positive in 50 (36%) patients and negative in 88 (64%) patients. A recurrence occurred in 39% of the patients with a positive FISH test and in 21% of patients with a negative FISH test. FISH positivity according to manufacturers criteria, at the time of a negative cystoscopy, was not significantly associated with the risk of recurrence (p = 0.12). However, the sensitivity of the FISH test to predict recurrence was significantly improved by considering specimens with rare (≤10) tetraploid cells as negative (p < 0.006). In addition, presence of 9p21 deletion was significantly associated with recurrence (p < 0.01). Notably, positive standard cytology was an independent factor for subsequent recurrence in this study (p < 0.001). Taken together, multi‐target FISH may help to stratify the risk of recurrence of UC at the time of a negative follow‐up cystoscopy. Defining the optimal threshold for FISH positivity requires consideration of tetraploid pattern and 9p21 deletion. Our results also emphasize the paramount importance of conventional cytology for UC surveillance.

Activation of Signal Transducer and Activator of Transcription-5 in Prostate Cancer Predicts Early Recurrence

Purpose: We have shown previously that the signal transducer and activator of transcription-5 (Stat5) is a critical survival factor in human prostate cancer cells. In addition, we recently showed that Stat5 is activated at a high level, particularly in high-grade human prostate cancers. Here, we investigated whether activation of Stat5 in prostate cancer was linked to clinical outcome with disease recurrence as end point. Experimental Design: Immunohistochemistry was used to detect active, nuclear Stat5 in 357 paraffin-embedded prostate cancer specimens on a tissue microarray with clinical follow-up data. Stat5 activation status in prostate cancer specimens was analyzed by univariate and multivariate survival analysis to determine whether activation of Stat5 predicts earlier prostate cancer recurrence. Separate sets of statistical analysis were done for all patients regardless of Gleason grade and for patients with prostate cancer of intermediate Gleason grades (3 and 4). Results and Conclusions: Stat5 activation in prostate cancer was associated with early disease recurrence (P = 0.0399). Importantly, active Stat5 also predicted shorter progression-free survival in intermediate Gleason grade prostate cancers (P = 0.0409). Stat5 activation remained an independent prognostic marker after adjusting for Gleason grade, pT stage, perineural invasion, or seminal vesicle infiltration in all patients (P = 0.0565) and in Gleason grade 3 or 4 patients (P = 0.0582). The results of this work also confirmed our previous finding of association of Stat5 activation with a high histologic grade of prostate cancer (R = 0.11, P = 0.033). In summary, our study shows that active Stat5 distinguished prostate cancer patients whose disease is likely to progress earlier; therefore, active Stat5 may be a useful marker for selection of more individualized treatment. The results of this study need to be validated in a large prospective cohort.

Advancing a clinically relevant perspective of the clonal nature of cancer

Cancers frequently arise as a result of an acquired genomic instability and the subsequent clonal evolution of neoplastic cells with variable patterns of genetic aberrations. Thus, the presence and behaviors of distinct clonal populations in each patients tumor may underlie multiple clinical phenotypes in cancers. We applied DNA content-based flow sorting to identify and isolate the nuclei of clonal populations from tumor biopsies, which was coupled with array CGH and targeted resequencing. The results produced high-definition genomic profiles of clonal populations from 40 pancreatic adenocarcinomas and a set of prostate adenocarcinomas, including serial biopsies from a patient who progressed to androgen-independent metastatic disease. The genomes of clonal populations were found to have patient-specific aberrations of clinical relevance. Furthermore, we identified genomic aberrations specific to therapeutically responsive and resistant clones arising during the evolution of androgen-independent metastatic prostate adenocarcinoma. We also distinguished divergent clonal populations within single biopsies and mapped aberrations in multiple aneuploid populations arising in primary and metastatic pancreatic adenocarcinoma. We propose that our high-definition analyses of the genomes of distinct clonal populations of cancer cells in patients in vivo can help guide diagnoses and tailor approaches to personalized treatment.

Transcription Factor Stat5 Synergizes with Androgen Receptor in Prostate Cancer Cells

The molecular mechanisms underlying progression of prostate cancer to the hormone-independent state are poorly understood. Signal transducer and activator of transcription 5a and 5b (Stat5a/b) is critical for the viability of human prostate cancer cells. We have previously shown that Stat5a/b is constitutively active in high-grade human prostate cancer, but not in normal prostate epithelium. Furthermore, activation of Stat5a/b in primary human prostate cancer predicted early disease recurrence. We show here that transcription factor Stat5a/b is active in 95% of clinical hormone-refractory human prostate cancers. We show for the first time that Stat5a/b synergizes with androgen receptor (AR) in prostate cancer cells. Specifically, active Stat5a/b increases transcriptional activity of AR, and AR, in turn, increases transcriptional activity of Stat5a/b. Liganded AR and active Stat5a/b physically interact in prostate cancer cells and, importantly, enhance nuclear localization of each other. The work presented here provides the first evidence of synergy between AR and the prolactin signaling protein Stat5a/b in human prostate cancer cells.

The prognostic value of cytology and fluorescence in situ hybridization in the follow‐up of nonmuscle‐invasive bladder cancer after intravesical Bacillus Calmette‐Guérin therapy

Molecular markers reliably predicting failure or success of Bacillus Calmette‐Guérin (BCG) in the treatment of nonmuscle‐invasive urothelial bladder cancer (NMIBC) are lacking. The aim of our study was to evaluate the value of cytology and chromosomal aberrations detected by fluorescence in situ hybridization (FISH) in predicting failure to BCG therapy. Sixty‐eight patients with NMIBC were prospectively recruited. Bladder washings collected before and after BCG instillation were analyzed by conventional cytology and by multitarget FISH assay (UroVysion®, Abbott/Vysis, Des Plaines, IL) for aberrations of chromosomes 3, 7, 17 and 9p21. Persistent and recurrent bladder cancers were defined as positive events during follow‐up. Twenty‐six of 68 (38%) NMIBC failed to BCG. Both positive post‐BCG cytology and positive post‐BCG FISH were significantly associated with failure of BCG (hazard ratio (HR)= 5.1 and HR= 5.6, respectively; p < 0.001 each) when compared to those with negative results. In the subgroup of nondefinitive cytology (all except those with unequivocally positive cytology), FISH was superior to cytology as a marker of relapse (HR= 6.2 and 1.4, respectively). Cytology and FISH in post‐BCG bladder washings are highly interrelated and a positive result predicts failure to BCG therapy in patients with NMIBC equally well. FISH is most useful in the diagnostically less certain cytology categories but does not provide additional information in clearly malignant cytology.

Tumour growth fraction measured by immunohistochemical staining of Ki67 is an independent prognostic factor in preoperative prostate biopsies with small-volume or low-grade prostate cancer

Accurate prognostic parameters in prostate biopsies are needed to better counsel individual patients with prostate cancer. We evaluated the prognostic impact of morphologic and immunohistochemical parameters in preoperative prostate cancer biopsies. A consecutive series of prostate biopsies of 279 men (72% with clinical stage T1c and 23% with T2) who subsequently underwent radical prostatectomy was prospectively analysed for Gleason score, number and percentage of positive cores (NPC, PPC), total percentage of biopsy tissue with tumour (TPT), maximum tumour percentage per core (MTP), and expression of Ki67, Bcl‐2 and p53. All biopsy features were significantly associated with at least one feature of the radical prostatectomy specimen. pT stage was independently predicted by PSA, seminal vesicle invasion by Ki67 LI, positive margins by PSA and MTP, large tumour diameter by PSA and PPC, and Gleason score by biopsy Gleason score, MTP, and Ki67 LI, respectively. Biopsy Gleason score, NPC (1 vs. >1), TPT (<7 vs. ≥7%), and Ki67 LI (<10 vs. ≥10%) were significant predictors of biochemical recurrence after radical prostatectomy (p < 0.01, each). KI67 LI was the only independent prognostic factor in case of a low TPT (<7%) or low Gleason score (<7), the hazard ratio being 6.76 and 6.44, respectively. In summary, preoperative Gleason score, NPC, TPT and Ki67 LI significantly predict the risk of recurrence after radical prostatectomy, and Ki67 is an independent prognosticator in biopsies with low‐volume or low‐grade prostate cancer. Analysis of Ki67 LI in these biopsies may help to better identify patients with clinically insignificant prostate cancer.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration‐resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high‐resolution array‐CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC‐3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC‐3, but not in the non‐amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration‐resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation. Copyright

Estrogen receptor β expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naïve prostate cancer and are elevated in castration-resistant disease

Patients with advanced prostate cancer (PC) are usually treated with androgen withdrawal. While this therapy is initially effective, nearly all PCs become refractory to it. As hormone receptors play a crucial role in this process, we constructed a tissue microarray consisting of PC samples from 107 hormone-naïve (HN) and 101 castration-resistant (CR) PC patients and analyzed the androgen receptor (AR) gene copy number and the protein expression profiles of AR, Serin210-phosphorylated AR (pAR(210)), estrogen receptor (ER)β, ERα and the proliferation marker Ki67. The amplification of the AR gene was virtually restricted to CR PC and was significantly associated with increased AR protein expression (P<0.0001) and higher tumor cell proliferation (P=0.001). Strong AR expression was observed in a subgroup of HN PC patients with an adverse prognosis. In contrast, the absence of AR expression in CR PC was significantly associated with a poor overall survival. While pAR(210) was predominantly found in CR PC patients (P<0.0001), pAR(210) positivity was observed in a subgroup of HN PC patients with a poor survival (P<0.05). Epithelial ERα expression was restricted to CR PC cells (9%). ERβ protein expression was found in 38% of both HN and CR PCs, but was elevated in matched CR PC specimens. Similar to pAR(210), the presence of ERβ in HN patients was significantly associated with an adverse prognosis (P<0.005). Our results strongly suggest a major role for pAR(210) and ERβ in HN PC. The expression of these markers might be directly involved in CR tumor growth.