Validation of GEMCaP as a DNA Based Biomarker to Predict Prostate Cancer Recurrence after Radical Prostatectomy.

PURPOSE: We aimed to validate GEMCaP (Genomic Evaluators of Metastatic Cancer of the Prostate) as a novel copy number signature predictive of prostate cancer recurrence. MATERIALS AND METHODS: We randomly selected patients who underwent radical prostatectomy at Cleveland Clinic or University of Rochester from 2000 to 2005. DNA isolated from the cancer region was extracted and subjected to high resolution array comparative genomic hybridization. A high GEMCaP score was defined as 20% or greater of genomic loci showing copy number gain or loss in a given tumor. Cox regression was used to evaluate associations between the GEMCaP score and the risk of biochemical recurrence. RESULTS: We report results in 140 patients. Overall 38% of patients experienced recurrence with a median time to recurrence of 45 months. Based on the CAPRA-S (Cancer of the Prostate Risk Assessment Post-Surgical) score 39% of the patients were at low risk, 42% were at intermediate risk and 19% were at high risk. The GEMCaP score was high (20% or greater) in 31% of the cohort. A high GEMCaP score was associated with a higher risk of biochemical recurrence (HR 2.69, 95% CI 1.51-4.77) and it remained associated after adjusting for CAPRA-S score and age (HR 1.94, 95% CI 1.06-3.56). The C-index of GEMCaP alone was 0.64, which improved when combined with the CAPRA-S score and patient age (C-index = 0.75). CONCLUSIONS: A high GEMCaP score was associated with biochemical recurrence in 2 external cohorts. This remained true after adjusting for clinical and pathological factors. The GEMCaP biomarker could be an efficient and effective clinical risk assessment tool to identify patients with prostate cancer for early adjuvant therapy.

Increased androgen receptor gene copy number is associated with TMPRSS2-ERG rearrangement in prostatic small cell carcinoma.

Small cell carcinoma of the prostate (PSCC) is a highly aggressive malignancy that often develops in patients previously treated with hormonal therapy for metastatic prostatic acinar adenocarcinoma. The TMPRSS2-ERG gene rearrangement is highly specific for prostate cancer and shared by PSCC; however, the role of androgen receptor (AR) gene alterations and interaction with TMPRSS2-ERG rearrangement are incompletely understood in PSCC. Sixty-one cases of PSCC were examined for AR gene copy number and TMPRSS2-ERG rearrangement by fluorescence in situ hybridization (FISH) and AR protein expression by immunohistochemistry. Of 61 cases of PSCC, 51% (31/61) demonstrated increased AR gene copy number (FISH+), 54% (33/61) were positive for TMPRSS2-ERG gene fusion, and 38% (23/61) showed AR protein expression. Of the 31 AR FISH+ cases, 23 also showed TMPRSS2-ERG gene fusion, and 16 expressed AR protein. Of the 33 cases with TMPRSS2-ERG fusion, 28 were AR FISH+ or expressed AR protein. Statistically significant correlations were observed between AR gene copy number or AR protein expression and TMPRSS2-ERG gene fusion (P = 0.001 and P = 0.03, respectively). In summary, high AR gene copy number emerges during the development of PSCC, often in association with TMPRSS2-ERG rearrangement. This potential mechanism warrants further study. Improvement will come from understanding the biology of the disease and integrating new therapies into the treatment of this rare and aggressive tumor.

Prostate specific G protein coupled receptor is associated with prostate cancer prognosis and affects cancer cell proliferation and invasion.

BACKGROUND: There is limited information about the clinical and biological significance of prostate specific G protein coupled receptor (PSGR) in prostate cancer (PCa) initiation and progression. Here, we evaluated the expression of PSGR protein, studied its diagnostic and prognostic value in PCa, and also explored its role in cancer cell growth and invasion. METHODS: The expression of PSGR in paired adjacent normal prostate, high grade prostatic intraepithelial neoplasia (PIN), and PCa were determined by immunohistochemistry on tissue microarrays constructed from 150 radical prostatectomy specimens. The effects of PSGR on PCa cell growth and invasion were investigated using human PCa cell lines. RESULTS: Membranous and cytoplasmic PSGR staining was observed at luminal epithelial cells of prostate. PSGR protein expression was significantly higher in PIN compared to normal prostate. Interestingly, the expression of PSGR decreased as PIN progressed to PCa. Low PSGR expression in PCa was associated with high Gleason score, and poor overall survival. Activated PSGR increased cancer cell invasive ability, but retarded cell growth. PSGR did not affect mTOR activity, but suppressed P70 S6 kinase activity. CONCLUSIONS: PSGR may participate in PCa progression through affecting cell proliferation and invasion. High expression of PSGR in PIN may implicate its role in early neoplastic transformation of PCa. Low expression of PSGR in PCa may serve as a potential indicator for poor prognosis.

Expression of UDP-glucuronosyltransferase 1A in bladder cancer: association with prognosis and regulation by estrogen.

Although UDP-glucuronosyltransferase 1A (UGT1A) plays an important role in preventing bladder cancer initiation by detoxifying carcinogenic compounds, its contribution to bladder cancer progression is poorly understood. We immunohistochemically stained for UGT1A in bladder specimens. UGT1A was positive in 130/145 (90%; 28 [19%] weak, 53 [37%] moderate, and 49 [34%] strong) urothelial neoplasms, which was significantly weaker than in matched non-neoplastic urothelial tissues (100/101 [99%]; 2 [2%] weak, 17 [17%] moderate, and 81 [80%] strong). Fifty (98%) of 51 low-grade/79 (99%) of 80 non-muscle-invasive tumors were immunoreactive to UGT1A, whereas 80 (85%) of 94 high-grade/51 (78%) of 65 muscle-invasive tumors were UGT1A-positive. Kaplan-Meier analysis showed strong associations between lower UGT1A expression versus the risk of recurrence in high-grade non-muscle-invasive tumors (P = 0.038) or disease-specific mortality in muscle-invasive tumors (P = 0.016). Multivariate analysis further revealed UGT1A loss as an independent prognosticator for disease-specific mortality in patients with muscle-invasive tumor (P = 0.010). Additionally, the expression of UGT1A was positively and negatively correlated with those of estrogen receptor-α and estrogen receptor-ß, respectively. We then assessed UGT1A/Ugt1a levels in human cell lines/mouse tissues. 17ß-Estradiol increased and decreased UGT1A expression in normal urothelium and bladder cancer lines, respectively, and an anti-estrogen abolished these effects. Ovariectomy in mice resulted in down-regulation of Ugt1a subtypes. These results suggest the involvement of UGT1A in not only bladder carcinogenesis but tumor progression. Moreover, UGT1A is likely regulated by estrogens in non-neoplastic urothelium versus bladder tumor in opposite manners, which could be underlying mechanisms of gender-specific differences in bladder cancer incidence and progression.

Identifying additional lymph nodes in radical cystectomy lymphadenectomy specimens.

Lymph node count has prognostic implications in bladder cancer patients who are treated with radical cystectomy. Lymph nodes that are too small to identify grossly can easily be missed, potentially leading to missed nodal metastases and inaccurate nodal counts, resulting in inaccurate prognoses. We investigated whether there is a benefit to submitting the entire lymph node packet for histological examination to identify additional lymph nodes. We prospectively assessed 61 pelvic lymphadenectomy specimens in 14 consecutive patients undergoing radical cystectomy. The specimens were placed in Carnoy's solution overnight, then analyzed for lymph nodes. The residual tissue was entirely submitted to assess for additional lymph nodes. In 61 specimens, we identified 391 lymph nodes, ranging from 4-44 nodes per patient. We identified 238 (61%) lymph nodes with standard techniques and 153 (39%) lymph nodes in submitted residual tissue. The number of additional lymph nodes found in the residual tissue ranged from 0 to 26 (0-75%) per patient. These lymph nodes ranged in size from 0.05 to 1 cm. All additional lymph nodes were negative for metastatic disease. Submitting the entire specimen for histological examination allowed for identification of more lymph nodes in radical cystectomy pelvic lymphadenectomy specimens. However, as none of the additional lymph nodes contained metastatic disease, it is unclear if there is a clinical benefit in evaluating lymph nodes that are neither visible nor palpable in lymphadenectomy specimens.

Skp2 overexpression is associated with loss of BRCA2 protein in human prostate cancer.

BRCA2 (breast cancer 2, early onset) is a tumor suppressor gene that confers increased susceptibility for prostate cancer (PCa). Previous in vitro experiments demonstrated that Skp2, an E3 ubiquitin ligase aberrantly overexpressed in PCa, is involved in the proteolytic degradation of BRCA2 in PCa cells, suggesting that the BRCA2-Skp2 interaction may play a role in prostate tumorigenesis. Herein, we investigated BRCA2 and Skp2 expression during PCa development using a prostate TMA. Although luminal and basal benign prostate epithelium exhibited moderate to strong nuclear BRCA2 immunostaining, the intensity and number of positive nuclei decreased significantly in high-grade prostatic intraepithelial neoplasia and PCa. Decreased frequency and intensity of nuclear BRCA2 labeling were inversely correlated with Skp2 expression in high-grade prostatic intraepithelial neoplasia and PCa. To functionally assess the effects of BRCA2 and Skp2 expression on prostate malignant transformation, we overexpressed Skp2 in normal immortalized prostate cells. Skp2 overexpression reduced BRCA2 protein and promoted cell growth and migration. A similar phenotype was observed after reduction of BRCA2 protein levels using specific BRCA2 small-interfering RNA. Forced BRCA2 expression in Skp2-overexpressing stable transfectants inhibited the migratory and growth properties by >60%. These results show that loss of BRCA2 expression during prostate tumor development is strongly correlated with both migratory behavior and cancer growth and include Skp2 as a BRCA2 proteolytic partner in vivo.

Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder.

UNLABELLED: What's known on the subject? and What does the study add? Steroid hormone receptor signals have been implicated in bladder tumourigenesis and tumour progression. The expression of androgen and/or oestrogen receptors has been assessed in bladder cancer, leading to conflicting data of expression levels and their relationship to histopathological characteristics of the tumours. We simultaneously analyze three receptors in non-neoplastic bladder tissues as well as in primary and metastatic bladder tumour specimens. Our data demonstrate that the expression status correlates with tumour grades/stages and patients' outcomes. OBJECTIVE: To assess the expression of the androgen receptor (AR) and oestrogen receptors (ERs) in bladder tumours because recent studies have shown conflicting results and the prognostic significance of their expression remains unclear. PATIENTS AND METHODS: We investigated the expression of AR, ERα and ERß in 188 bladder tumour specimens, as well as matched 141 non-neoplastic bladder and 14 lymph node metastasis tissues, by immunohistochemistry. We then evaluated the relationships between their expression and the clinicopathological features available for the present patient cohort. RESULTS: AR/ERα/ERß was positive in 80%/50%/89% of benign urothelium, 50%/67%/41% of benign stroma, 42%/27%/49% of primary tumours and 71%/64%/71% of metastatic tumours. Significantly lower expression of AR/ERα was found in high-grade tumours (36%/23%) and tumours invading muscularis propria (33%/19%) compared to low-grade tumours (55%; P= 0.0232/38%; P= 0.0483) and tumours not invading muscularis propria (51%; P= 0.0181/35%; P= 0.0139), respectively. Significantly higher expression of ERß was found in high-grade tumours (58%) and tumours invading muscularis propria (67%) compared to low-grade tumours (29%; P= 0.0002) and tumours not invading muscularis propria (34%; P < 0.0001), respectively. Kaplan-Meier and log-rank tests further showed that positivity of ERß (but not AR or ERα) was associated with the recurrence of low-grade tumours (P= 0.0072); the progression of low-grade tumours (P= 0.0005), high-grade tumours not invading muscularis propria (P= 0.0020) and tumours invading muscularis propria (P= 0.0010); or disease-specific mortality in patients with tumours invading muscularis propria (P= 0.0073). CONCLUSIONS: Compared to benign bladders, a significant decrease in the expression of AR, ERα or ERß in bladder cancer was seen. Loss of AR or ERα was strongly associated with higher grade/more invasive tumours, whereas ERß expression was increased in high-grade/invasive tumours and predicted a worse prognosis.

Expression of semenogelins I and II and its prognostic significance in human prostate cancer.

BACKGROUND: Little is known about the role of semenogelins, seminal plasma proteins that play critical roles in semen clotting and subsequent liquefaction in the presence of zinc and prostate-specific antigen, in human malignancies. METHODS: We investigated the expression of semenogelins in four human prostate cancer lines by RT-PCR and Western blotting as well as in 70 radical prostatectomy specimens by immunohistochemistry. Effects of semenogelin overexpression on prostate cancer cell proliferation were also assessed. RESULTS: mRNA/protein signals for semenogelins I (SgI) and II (SgII) were detected only in androgen-sensitive LNCaP cells cultured with zinc. Transfection of SgI/SgII increased/decreased cell growth of androgen receptor (AR)-positive/semenogelin-negative CWR22Rv1 in the presence of zinc, whereas it showed marginal effects in AR-negative/semenogelin-negative PC-3 and DU145. Immunohistochemical studies showed that SgI and SgII stain positively in 55 (79%) and 31 (44%) cancer tissues, respectively, which was significantly higher than in corresponding benign tissues [SgI-positive in 13 (19%) cases (P < 0.0001) and SgII-positive in 15 (21%) cases (P = 0.0066)]. Among the histopathological parameters available for our patient cohort, there was an inverse association only between Gleason score (GS) and SgII expression (GS ≤ 7 vs. GS ≥ 8: P = 0.0150; GS7 vs. GS ≥ 8: P = 0.0111). Kaplan-Meier and log-rank tests further revealed that patients with SgI-positive/SgII-negative tumor have the highest risk for biochemical recurrence (P = 0.0242). CONCLUSIONS: These results suggest the involvement of semenogelins in prostate cancer and their prognostic values in predicting cancer progression after radical prostatectomy. Additional functional analyses of semenogelins are necessary to determine their biological significance in prostate cancer.

ERG-TMPRSS2 rearrangement is shared by concurrent prostatic adenocarcinoma and prostatic small cell carcinoma and absent in small cell carcinoma of the urinary bladder: evidence supporting monoclonal origin.

Prostatic carcinoma is a heterogeneous disease with frequent multifocality and variability in morphology. Particularly, prostatic small cell carcinoma is a rare variant with aggressive behavior. Distinction between small cell carcinoma of the prostate and urinary bladder may be challenging, especially in small biopsy specimens without associated prostatic adenocarcinoma or urothelial carcinoma. Recently, gene fusions between ETS genes, particularly ETS-related gene (ERG), and transmembrane protease, serine 2 (TMPRSS2) have been identified as a frequent event in prostate cancer. Thus, molecular methods may be helpful in determining the primary site of small cell carcinoma. Thirty cases of prostatic small cell carcinoma from the authors' archives were studied, among which 13 had concurrent prostatic adenocarcinoma. Tricolor fluorescence in situ hybridization (FISH) was performed on formalin-fixed paraffin-embedded tissue sections with a probe cocktail for 3'/5' ERG and TMPRSS2. Cases of small cell carcinoma of the bladder and conventional prostatic adenocarcinoma (25 each) were also tested as controls. ERG gene alterations were found only in prostate malignancies and not in benign prostatic tissue or bladder small cell carcinoma. TMPRSS2-ERG gene fusion was found in 47% (14/30) of prostatic small cell carcinoma. Of cases with concurrent prostatic adenocarcinoma, 85% (11/13) had identical findings in both components. In 20% of rearranged cases, the ERG abnormality was associated with 5' ERG deletion. In 17% (5/30) of cases, gain of the 21q22 locus was present. Two cases showed discordant aberrations in the small cell carcinoma and adenocarcinoma, one with deletion of 5' ERG and one with gain of chromosome 21q, both in only the adenocarcinoma component. Small cell carcinoma of the prostate demonstrates TMPRSS2-ERG rearrangement with comparable frequency to prostatic adenocarcinoma. In cases with concurrent adenocarcinoma and small cell carcinoma, the majority showed identical abnormalities in both components, indicating a likely common clonal origin. Discordant alterations were present in rare cases, suggesting that acquisition of additional genetic changes in multifocal tumors may be responsible for disease progression to a more aggressive phenotype. TMPRSS2-ERG fusion is absent in bladder small cell carcinoma, supporting the utility of FISH in distinguishing prostate from bladder primary tumors and identifying metastatic small cell carcinoma of unknown origin.

Near infrared fluorescence imaging with robotic assisted laparoscopic partial nephrectomy: initial clinical experience for renal cortical tumors.

PURPOSE: We evaluated the utility of near infrared fluorescence of intravenously injected indocyanine green in performing robotic assisted laparoscopic partial nephrectomy. In addition, we evaluated the initial performance of a novel near infrared fluorescence imaging system integrated into the da Vinci® Si Surgical System during robotic assisted laparoscopic nephrectomy. MATERIALS AND METHODS: Fluorescence imaging for the da Vinci Si Surgical System was used for all cases. Indocyanine green was injected before near infrared imaging. Immediate imaging assessed the renal vasculature while delayed imaging differentiated renal cortical tumors from normal parenchyma. The intraoperative performance of near infrared fluorescence of intravenous indocyanine green was evaluated for tumor appearance relative to surrounding renal parenchyma as well as identification of the renal vasculature. RESULTS: A total of 11 patients underwent robotic assisted laparoscopic nephrectomy with 2 converted to robotic assisted laparoscopic radical nephrectomy. Indocyanine green injections were repeated up to a total of 5 times depending on the goal of visualization. Of the 11 patients 10 demonstrated malignancy on final pathology. Of the malignant tumors 7 were hypofluorescent and 3 were isofluorescent compared to the surrounding renal parenchyma. Near infrared fluorescence imaging delineated the vascular anatomy in all cases. All surgical margins were negative on final pathology. CONCLUSIONS: Intraoperative imaging of indocyanine green with near infrared fluorescence is a safe and effective method to accurately identify the renal vasculature and to differentiate renal tumors from surrounding normal parenchyma. The capacity for multimodal imaging within the surgical console further facilitates this imaging. Further study is needed to determine if this technique will help improve outcomes of robotic assisted laparoscopic nephrectomy.

Thrombin expression in prostate: a novel finding.

INTRODUCTION: A variable repertoire of coagulation protein expression is observed in different cancers. We evaluated expression of thrombin in prostate tissue. METHODS: Detection of thrombin was performed using quantitative real-time PCR in fresh tissue and in situ hybridization (ISH) in archival prostate tissue and by immunohistochemistry of prostate tissue microarrays. RESULTS: (Pro)thrombin mRNA expression was detected in cancer tissue and localized to prostatic epithelium and stroma by ISH. Thrombin protein was detected in stroma of benign and malignant epithelium (p <.05). CONCLUSIONS: Prostate tissue is a rich reservoir of thrombin. This may have potential for developing antithrombin-based cancer therapy.

Loss of androgen receptor expression is not associated with pathological stage, grade, gender or outcome in bladder cancer: a large multi-institutional study.

OBJECTIVE: • To investigate androgen receptor (AR) expression in a large series of patients with bladder cancer (BC) because data on a limited number of patients showed that loss of AR expression was associated with invasive BC. PATIENTS AND METHODS: • A total of 472 patients with urothelial bladder carcinoma (UBC) from two institutional centres (Toronto and Dallas) were analysed. Tissue microarrays comprising both non-muscle-invasive UBC (n= 167) and muscle-invasive UBC (n= 305) were accrued and immunohistochemical staining for AR was performed. • We used bright-field microscopy imaging coupled with advanced colour detection software to detect, classify and count stained cellular objects and manual scoring. • Results obtained in Dallas were blindly reviewed and validated in Toronto and samples randomly chosen were further analysed in Rochester, NY, USA. RESULTS: • The AR were positively expressed in 61/472 (12.9%) bladder tumours. No statistically significant difference in AR expression between men and women was observed. • Only 9.0% of non-muscle-invasive BC expressed the AR compared with 15.1% of muscle-invasive tumours (P= 0.059). The highest percentage of AR positivity (28.9% of cases) was found in T2 tumours. • There was no statistically significant difference in death from BC, time to death, or time to recurrence between AR-positive and AR-negative cases. CONCLUSION: • In contrast to previous reports, based on our large BC series, we did not observe a decrease in AR protein expression in bladder tumours with increased pathological stage. Our data do not suggest that loss of AR expression is gender-related nor is it associated with invasive BC.

Crawling wave detection of prostate cancer: preliminary in vitro results.

PURPOSE: The focus of this article is to develop signal and imaging processing methods to derive an accurate estimation of local tissue elasticity using the crawling wave (CrW) sonoelastography method. The task is to reduce noise and to improve the contrast of the elasticity map. METHODS: The protocol of the CrW approach was first tested on heterogeneous elastic phantoms as a model of prostate cancers. Then, the contrast-to-noise ratio of the estimation was calculated iteratively with various sequences of algorithms to determine the optimal signal processing settings. Finally, the optimized signal processing was applied to ex vivo prostate cancer detection. The comparison of the segmented elasticity map and the histology tumor outline was made by quadrants to evaluate the diagnostic performance of the protocol. Furthermore, the CrW approach was combined with amplitude-sonoelastography to achieve a higher specificity. RESULTS: This study demonstrated the feasibility of the proposed approach for clinical applications. In the application to ex vivo prostate cancer detection, the established approach was tested on 43 excised prostate glands. The combination of the CrW approach and amplitude-sonoelastography achieved an accuracy of over 80% for finding tumors larger than 4 mm in diameter. The elasticity values and contrast found by the CrW approach were in agreement with the previous results derived from mechanical testing. CONCLUSIONS: Crawling waves can be applied to detect prostate cancer with accuracy approaching 80% and can quantify the stiffness or shear modulus of both cancerous and noncancerous tissues. The technique therefore shows promise for guiding biopsies to suspect regions that are otherwise difficult to identify.

Androgen receptor is a tumor suppressor and proliferator in prostate cancer.

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-AR9) results in decreased invasion in bone lesion assays and in vivo mouse models. Mice lacking the prostate epithelial AR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFbeta1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.

Tissue factor and VEGF expression in prostate carcinoma: a tissue microarray study.

Tissue factor (TF) is the principal physiologic initiator of coagulation. It also plays an important role in tumor growth and metastasis possibly by contributing to angiogenesis. We evaluated the expression of TF in benign and malignant prostate tissue and correlated it with the expression of the pro-angiogenic protein, vascular endothelial growth factor (VEGF). We used a tissue microarray (TMA) constructed from 80 archival prostatectomy specimens. Core samples were collected from benign prostate tissue (BP) (n= 77), high-grade prostatic intraepithelial neoplasia (PIN) (n= 26), and carcinoma (PCa) (n= 93). TMA sections were stained with an immunopurified polyclonal TF antibody and a rabbit polyclonal VEGF. Two pathologists manually scored staining in epithelial cells using the German Immunoreactive Score. Positive staining for TF was seen predominantly in PCa with rare positive glands in BP and PIN. TF expression was significantly lower in BP versus PCa specimens (p< .001) and in PIN versus PCa specimens (p< .001). Positive staining for VEGF was seen in PCa, BP, and PIN. Rates of VEGF expression were also significantly lower in BP versus PCa specimens (p= .003) but not in PCa versus PIN (p= .430). The majority of PCa samples positive for TF were also positive for VEGF (p< .001). Our findings reinforce the link between angiogenesis and TF expression in PCa. We suggest further exploration of TF-mediated pathways leading to increased tumor aggressiveness in PCa, and the possible use of anti-TF agents in PCa.

Loss of BRCA2 promotes prostate cancer cell invasion through up-regulation of matrix metalloproteinase-9.

BRCA2 is a multifunctional tumor suppressor protein which plays critical roles in DNA repair, transcription, and cell proliferation, and the loss of which has been linked to the biology of several types of cancers. Here, on prostate adenocarcinoma specimens from 80 patients, we demonstrate that BRCA2 protein is lost in carcinoma cells compared to normal and hyperplastic prostate epithelium. Using highly metastatic prostate cancer PC-3 cells, we show that while BRCA2 depletion by small-interfering RNA promoted migration onto the extracellular matrix proteins fibronectin, laminin, and collagens, as well as invasion through the reconstituted basement membrane matrix Matrigel by more than 140%, recombinant BRCA2 overexpression decreased both phenomena by 57-80% and changed cell morphology from angular and spindle to round and compact. The BRCA2 inhibitory effect on cancer cell migration and invasion resulted from down-regulation of matrix metalloproteinase (MMP)-9 protein levels due to increased MMP-9 proteolysis, and was signaled through inhibition of PI3-kinase/AKT and activation of MAPK/ERK pathway. In BRCA2-overexpressing PC-3 cells, transient transfection with a constitutively active PI3-kinase mutant or treatment with the MAPK/ERK inhibitor PD98059 rescued MMP-9 levels and restored the migratory and invasive capabilities. Consistently, PI3-kinase inhibition with a dominant-negative mutant or MAPK/ERK activation with a gain-of-function mutant reduced MMP-9 levels and prevented migration and invasion in wild-type PC-3 cells. These results provide novel evidence showing that a functional BRCA2 protein may limit the metastatic potential of neoplastic cells by down-regulating MMP-9 production through inhibition of PI3-kinase/AKT and activation of MAPK/ERK, effectively hindering cancer cell migration and invasion.

Transgelin functions as a suppressor via inhibition of ARA54-enhanced androgen receptor transactivation and prostate cancer cell growth.

The androgen receptor (AR) requires coregulators for its optimal function. However, whether AR coregulators further need interacting protein(s) for their proper function remains unclear. Here we describe transgelin as the first ARA54-associated negative modulator for AR. Transgelin suppressed ARA54-enhanced AR function in ARA54-positive, but not in ARA54-negative, cells. Transgelin suppressed AR transactivation via interruption of ARA54 homodimerization and AR-ARA54 heterodimerization, resulting in the cytoplasmic retention of AR and ARA54. Stable transfection of transgelin in LNCaP cells suppressed AR-mediated cell growth and prostate-specific antigen expression, whereas this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Results from tissue surveys showing decreased expression of transgelin in prostate cancer specimens further strengthened the suppressor role of transgelin. Our findings reveal the novel mechanisms of how transgelin functions as a suppressor to inhibit prostate cancer cell growth. They also demonstrate that AR coregulators, like ARA54, might have dual in vivo roles functioning as both a direct coactivator and as an indirect mediator in AR function. The finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach, with fewer side effects, to battle prostate cancer by targeting AR indirectly.

Androgenic dependence of exophytic tumor growth in a transgenic mouse model of bladder cancer: a role for thrombospondin-1.

BACKGROUND: Steroid hormones influence mitogenic signaling pathways, apoptosis, and cell cycle checkpoints, and it has long been known that incidence of bladder cancer (BC) in men is several times greater than in women, a difference that cannot be attributed to environmental or lifestyle factors alone. Castration reduces incidence of chemically-induced BC in rodents. It is unclear if this effect is due to hormonal influences on activation/deactivation of carcinogens or a direct effect on urothelial cell proliferation or other malignant processes. We examined the effect of castration on BC growth in UPII-SV40T transgenic mice, which express SV40 T antigen specifically in urothelium and reliably develop BC. Furthermore, because BC growth in UPII-SV40T mice is exophytic, we speculated BC growth was dependent on angiogenesis and angiogenesis was, in turn, androgen responsive. METHODS: Flat panel detector-based cone beam computed tomography (FPDCT) was used to longitudinally measure exophytic BC growth in UPII-SV40T male mice sham-operated, castrated, or castrated and supplemented with dihydrotestosterone (DHT). Human normal bladder and BC biopsies and mouse bladder were examined quantitatively for thrombospondin-1 (TSP1) protein expression. RESULTS: Mice castrated at 24 weeks of age had decreased BC volumes at 32 weeks compared to intact mice (p = 0.0071) and castrated mice administered DHT (p = 0.0233; one-way ANOVA, JMP 6.0.3, SAS Institute, Inc.). Bladder cancer cell lines responded to DHT treatment with increased proliferation, regardless of androgen receptor expression levels. TSP1, an anti-angiogenic factor whose expression is inhibited by androgens, had decreased expression in bladders of UPII-SV40T mice compared to wild-type. Castration increased TSP1 levels in UPII-SV40T mice compared to intact mice. TSP1 protein expression was higher in 8 of 10 human bladder biopsies of normal versus malignant tissue from the same patients. CONCLUSION: FPDCT allows longitudinal monitoring of exophytic tumor growth in the UPII-SV40T model of BC that bypasses need for chemical carcinogens, which confound analysis of androgen effects. Androgens increase tumor cell growth in vitro and in vivo and decrease TSP1 expression, possibly explaining the therapeutic effect of castration. This effect may, in part, explain gender differences in BC incidence and implies anti-androgenic therapies may be effective in preventing and treating BC.

Predominantly cystic clear cell renal cell carcinoma and multilocular cystic renal neoplasm of low malignant potential form a low-grade spectrum.

Multilocular cystic renal cell carcinoma has been recently excluded from clear cell renal cell carcinoma (CCRCC) category and re-designated as multilocular cystic renal neoplasm of low malignant potential (MCRNLMP) due to its uniformly good outcomes. While strict distinction between MCRNLMP from predominantly cystic CCRCC (pc-CCRCC) is being emphasized, the significance of extensive true cystic component in CCRCC has not been investigated. Herein, we analyzed 57 MCRNLMP, 69 pc-CCRCC, and 46 non-cystic CCRCC. There were no statistically significant differences between the three subtypes in age, gender, and laterality. ISUP grades were 1 (73%) or 2 (27%) for MCRNLMP; for pc-CCRCC were 1 (31%), 2 (60%), and 3 (9%); and for non-cystic CCRCC were 1 (9%), 2 (52%), 3 (26%), and 4 (13%). MCRNLMP were either pT stage 1 (91%) or 2 (9%), pT stages for pc-CCRCC were 1 (92.5%), 2 (1.5%), and 3 (6%) and for non-cystic CCRCC were 1 (58.7%), 2 (6.5%), and 3 (34.8%). None of MCRNLMP patients developed recurrences or metastases, and only 1 contralateral kidney tumor and 1 metastasis developed in pc-CCRCC. In contrast, 19 patients with non-cystic CCRCC developed metastases (5-year PFS 58%, CI 38.3-73.5%), and 1 patient died of disease. Monosomy 3 was common in both MCRNLMP (3/3) and pc-CCRCC (6/7). This large series of MCRNLMP confirms its indolent behavior, shows that pc-CCRCC has significantly better prognosis than non-cystic CCRCC and may define the lower grade spectrum of CCRCC. We recommend that the presence and extent of CCRCC cystic component should be documented in the pathology report.

Suppression of androgen receptor transactivation and prostate cancer cell growth by heterogeneous nuclear ribonucleoprotein A1 via interaction with androgen receptor coregulator ARA54.

The androgen receptor (AR) requires coregulators for its optimal transactivation. Whether AR coregulators also need interacting proteins to modulate their function remains unclear. Here we describe heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as an associated negative modulator for the AR coregulator ARA54. hnRNP A1 selectively suppressed ARA54-enhanced wild-type and mutant AR transactivation via interruption of AR-ARA54 interaction and ARA54 homodimerization. Stable transfection of hnRNP A1 in the LNCaP cells suppressed AR-mediated cell growth and the expression of prostate-specific antigen, and this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Small interfering RNA knockdown of endogenous hnRNP A1 enhanced cell growth and prostate-specific antigen expression in LNCaP cells. These results not only suggest that the loss of hnRNP A1 expression might activate the ARA54-enhanced cell growth and contribute to the prostate cancer progression, but also demonstrate the dual functional roles for ARA54 as an AR coregulator directly and as a mediator for the suppressive effect of hnRNP A1 indirectly. The novel finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach to battle prostate cancer by targeting AR indirectly with fewer side effects.