Anne R. Simoneau

Expression of Frzb/Secreted Frizzled-Related Protein 3, a Secreted Wnt Antagonist, in Human Androgen-Independent Prostate Cancer PC-3 Cells Suppresses Tumor Growth and Cellular Invasiveness

The ability of Frzb/secreted Frizzled-related protein 3 (sFRP3) to inhibit Wnt signaling and the localization of Frzb/sFRP3 on chromosome 2q to a region frequently deleted in cancers have led some investigators to hypothesize that Frzb/sFRP3 is a tumor suppressor gene. Here, we examined the biological effects of Frzb/sFRP3 on an androgen-independent prostate cancer cell model. We showed that expression of Frzb/sFRP3 in PC-3 cells resulted in decreased colony formation in soft agar and a dramatic inhibition of tumor growth in a xenograft mouse model. When cellular morphology was examined, PC-3 cells expressing Frzb/sFRP3 exhibited an increase in cell-cell contact formation accompanied by a pronounced induction of epithelial markers E-cadherin and keratin-8 and down-regulation of mesenchymal markers N-cadherin, fibronectin, and vimentin. This phenomenon suggested a reversal of epithelial-to-mesenchymal transition and a less invasive phenotype. Indeed, further in vitro studies with a Matrigel assay showed that Frzb/sFRP3 decreased the invasive capacity of PC-3 cells. These changes in the biology of PC-3 cells are associated with a decrease in the expression and activities of both matrix metalloproteinase (MMP)-2 and MMP-9 as well as decreases in AKT activation, cytosolic beta-catenin levels, T-cell factor transcription activity, and expression of Slug and Twist. In addition, transfection of PC-3 with a dominant-negative low-density lipoprotein receptor-related protein 5 (DN-LRP5) coreceptor showed similar biological effects as Frzb/sFRP3 transfection. Together, these data suggest that Frzb/sFRP3 and DN-LRP5 exhibit antitumor activity through the reversal of epithelial-to-mesenchymal transition and inhibition of MMP activities in a subset of prostate cancer.

Transitional cell carcinoma involving the prostate with a proposed staging classification for stromal invasion

PURPOSE We investigated the effect on survival of transitional cell carcinoma of the prostatic urethra, ducts and stroma, and determined the difference between prostatic stromal involvement occurring via direct extension through the bladder wall versus stromal invasion arising intraurethrally. MATERIALS AND METHODS Between August 1971 and December 1989, 489 men underwent radical cystoprostatectomy for transitional cell carcinoma, including 143 (29.2%) identified with prostate involvement by transitional cell carcinoma, in the cystectomy specimen. Patients were separated into 2 groups: 1-19 in whom the primary bladder tumor extended full thickness through the bladder wall to invade the prostate (classified as P4a) and 2-124 in whom prostate involvement arose from within the prostatic urethra. RESULTS Five-year recurrence-free and overall survival rates were 25 and 21%, respectively, in group 1 versus 64 and 55%, respectively, in group 2. In the 124 patients in group 2 survival rates were similar for those with prostatic urethral tumors or carcinoma in situ and ductal tumors (no stromal invasion). Five-year overall survival rates without and with stromal invasion were 71 and 36%, respectively (p < 0.0001). Transitional cell carcinoma of the prostatic urethra or ducts does not alter survival predicted by primary bladder stage alone. Prostatic stromal invasion arising intraurethrally significantly decreases survival, which varies based on primary bladder stage (64.6% in stage P1, 30.8% in stages P2/P3a and 13.6% in stage P3b, p = 0.0001). P1 bladder tumors with prostatic stromal invasion arising intraurethrally had a significantly higher survival rate than P4a tumors (64.6 versus 21%, p = 0.0001). P3b bladder tumors with stromal invasion had a survival rate similar to that of P4a tumors (p = 0.78). CONCLUSIONS Prostatic urethral or ductal transitional cell carcinoma does not alter survival determined by primary bladder stage alone and it should not be classified as P4a. Prostatic stromal involvement arising intraurethrally significantly decreases survival predicted by primary bladder stage alone. P1 bladder tumors with prostatic stromal invasion arising intraurethrally have a significantly higher survival rate than P4a tumors and they should be separately classified as P1str. Muscle invasive (P2/P3a) bladder tumors with stromal invasion have a higher survival rate than P4a tumors (no statistical significance) and they should be designated separately (that is P2str). P3b bladder tumors with prostatic stromal invasion arising intraurethrally are indistinguishable from P4a tumors.

Flavokawain A, a Novel Chalcone from Kava Extract, Induces Apoptosis in Bladder Cancer Cells by Involvement of Bax Protein-Dependent and Mitochondria-Dependent Apoptotic Pathway and Suppresses Tumor Growth in Mice

Consumption of the traditional kava preparation was reported to correlate with low and uncustomary gender ratios (more cancer in women than men) of cancer incidences in three kava-drinking countries: Fiji, Vanuatu, and Western Samoa. We have identified flavokawain A, B, and C but not the major kavalactone, kawain, in kava extracts as causing strong antiproliferative and apoptotic effect in human bladder cancer cells. Flavokawain A results in a significant loss of mitochondrial membrane potential and release of cytochrome c into the cytosol in an invasive bladder cancer cell line T24. These effects of flavokawain A are accompanied by a time-dependent decrease in Bcl-x(L), a decrease in the association of Bcl-x(L) to Bax, and an increase in the active form of Bax protein. Using the primary mouse embryo fibroblasts Bax knockout and wild-type cells as well as a Bax inhibitor peptide derived from the Bax-binding domain of Ku70, we showed that Bax protein was, at least in part, required for the apoptotic effect of flavokawain A. In addition, flavokawain A down-regulates the expression of X-linked inhibitor of apoptosis and survivin. Because both X-linked inhibitor of apoptosis and survivin are main factors for apoptosis resistance and are overexpressed in bladder tumors, our data suggest that flavokawain A may have a dual efficacy in induction of apoptosis preferentially in bladder tumors. Finally, the anticarcinogenic effect of flavokawain A was evident in its inhibitory growth of bladder tumor cells in a nude mice model (57% of inhibition) and in soft agar.

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G1 arrest. The G1 arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G1 arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and β-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G1 arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer. [Mol Cancer Ther 2009;8(2):458–68]

The Wnt inhibitory factor 1 restoration in prostate cancer cells was associated with reduced tumor growth, decreased capacity of cell migration and invasion and a reversal of epithelial to mesenchymal transition

BackgroundAberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3.ResultsThe WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues.ConclusionThese data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

Flavokawain B, a kava chalcone, induces apoptosis via up-regulation of death-receptor 5 and Bim expression in androgen receptor negative, hormonal refractory prostate cancer cell lines and reduces tumor growth.

Limited success has been achieved in extending the survival of patients with metastatic and hormone‐refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. We have shown that flavokawain B (FKB), a kava chalcone, is about 4‐ to 12‐fold more effective in reducing the cell viabilities of androgen receptor (AR)‐negative, HRPC cell lines DU145 and PC‐3 than AR‐positive, hormone‐sensitive prostate cancer cell lines LAPC4 and LNCaP, with minimal effect on normal prostatic epithelial and stromal cells. FKB induces apoptosis with an associated increased expression of proapoptotic proteins: death receptor‐5, Bim and Puma and a decreased expression of inhibitors of apoptosis protein: XIAP and survivin. Among them, Bim expression was significantly induced by FKB as early as 4 hr of the treatment. Knockdown of Bim expression by short‐hairpin RNAs attenuates the inhibitory effect on anchorage‐dependent and ‐independent growth and caspase cleavages induced by FKB. These findings suggest that the effect of FKB, at least in part, requires Bim expression. In addition, FKB synergizes with TRAIL for markedly enhanced induction of apoptosis. Furthermore, FKB treatment of mice bearing DU145 xenograft tumors results in tumor growth inhibition and increases Bim expression in tumor tissues. Together, these results suggest robust mechanisms for FKB induction of apoptosis preferentially for HRPC and the potential usefulness of FKB for prevention and treatment of HRPC in an adjuvant setting.

The Effect of Difluoromethylornithine on Decreasing Prostate Size and Polyamines in Men: Results of a Year-Long Phase IIb Randomized Placebo-Controlled Chemoprevention Trial

Background: Prostate cancer is a major health issue, and prevention of prostate cancer and/or its progression will yield benefits for men. Difluoromethylornithine (DFMO) is an antiproliferative agent, inhibiting ornithine decarboxylase, the first enzyme in the polyamine pathway, and has been studied as a therapeutic and chemopreventive agent. The prostate has high levels of tissue polyamines and has shown sensitivity to DFMO both in vitro and in vivo. Methods: Eighty-one men participated in a 1-year randomized trial of placebo or DFMO. Prostate volume determination and biopsy of the prostate for histology and polyamine content were done at baseline and after 12 months. Other biomarker variables were assessed, including total and free prostate-specific antigen and prostate-specific antigen doubling time. Results: Compared with baseline, men receiving DFMO had a smaller increase in prostate volume (0.14 cm3) than those on placebo (2.95 cm3; P = 0.0301) at 1 year. In addition, DFMO caused a 60.8% reduction of prostate putrescine levels compared with a 139.5% increase in the placebo arm (P = 0.0014). Stratification by ornithine decarboxylase genotype showed that DFMO reduced prostate volume (P = 0.029) and putrescine levels (P = 0.0053) in the AA + GA group but not in the GG group. There were no grade 3 or 4 toxicities. There was no clinical ototoxicity, with one subclinical grade 2 hearing decline on audiogram. Conclusion: In this randomized placebo-controlled trial, DFMO induced a decrease of prostate putrescine levels and rate of prostate growth. The potential of this compound for prostate cancer or hyperplasia should be further studied. (Cancer Epidemiol Biomarkers Prev 2008;17(2):292–9)

Rhodiola rosea extracts and salidroside decrease the growth of bladder cancer cell lines via inhibition of the mTOR pathway and induction of autophagy

The incidence of human urinary bladder cancer increases markedly with age, suggesting a mechanistic connection between aging and bladder carcinogenesis and a potential use of anti‐aging agents in bladder cancer chemoprevention. Rhodiola rosea, growing in high altitude or cold regions of the world, has been reported to have anti‐aging effects in Drosophila. We demonstrated that a R. rosea extract and one of its bioactive components, salidroside, inhibited the growth of bladder cancer cell lines with a minimal effect on nonmalignant bladder epithelial cells TEU‐2. Interestingly, the R. rosea extract and salidroside component exhibited a selective ability to inhibit the growth of p53 knockout primary mouse embryo fibroblasts (p53−/− MEFs) compared to their wild‐type counterparts. The growth inhibitory effects of the R. rosea extract and salidroside were, however, attenuated in TSC2 and p53 double knock MEFs (TSC2−/−, p53−/− MEFs), suggesting that TSC2 protein is, at least in part, required for the growth inhibitory effects of the R. rosea extract and salidroside. The R. rosea extract and salidroside treatment of UMUC3 cells resulted in an increase of AMP‐activated protein kinase (AMPK)‐α phosphorylation and a decrease of 4E‐BP1 phosphorylation, leading to increased binding of 4E‐BP1 to m7 GTP. These results indicate that the R. rosea extract and salidroside inhibit translation initiation. Furthermore, both the R. rosea extract and salidroside treatment of UMUC3 cells caused a significant percentage of cells undergoing autophagy. Therefore, the R. rosea extract and salidroside deserve further study as novel agents for chemoprevention of bladder carcinogenesis.

Impact of Moderate Dose of Postoperative Radiation on Urinary Continence and Potency in Patients with Prostate Cancer Treated with Nerve Sparing Prostatectomy

PURPOSE We analyzed the impact on potency and urinary continence of moderate doses of radiation (45 to 54 Gy.) given postoperatively after nerve sparing prostatectomy. MATERIALS AND METHODS Between 1983 and 1992, 294 of 762 prostate cancer patients were selected to undergo nerve sparing prostatectomy. Subjective patient reports regarding potency and urinary continence status were obtained preoperatively, 1 year postoperatively or 1 year after completion of radiation. RESULTS Of the 294 patients 105 received postoperative radiotherapy (45 to 54 Gy.) to the prostatic bed. There were patients with more advanced stages of disease in the irradiated group, including 89% with stages C and D1 (pT3N0 and pT1 to 3, N1 to 3), compared to 14% with stages C and D1 (pT3N0 and pT1 to 3, N1 to 3) in the nonirradiated group (p < 0.001). No difference in urinary continence was noted in the irradiated (94%) compared to the nonirradiated group (92%, p = 0.64). Of the patients who underwent bilateral nerve sparing prostatectomy 44% who received and 48% who did not receive radiation had recovered potency at 1 year (p = 0.76). Of those who underwent unilateral nerve sparing prostatectomy 10% who received and 33% who did not receive radiation had recovered potency at 1 year (p = 0.14). Using multivariate analysis patient age younger than 63 years and bilateral versus unilateral nerve sparing procedures were significant predictors of potency. CONCLUSIONS Our retrospective study suggests that at 1 year after treatment moderate doses of postoperative radiotherapy did not have a significant impact on the recovery of urinary continence and potency after nerve sparing prostatectomy. However, longer followup is required to determine the impact of this radiation protocol on long-term preservation of potency after nerve sparing prostatectomy.

SPHINCTERIC STENT VERSUS EXTERNAL SPHINCTEROTOMY IN SPINAL CORD INJURED MEN: PROSPECTIVE RANDOMIZED MULTICENTER TRIAL

PURPOSE In a prospective randomized multicenter trial we compared the treatment results of conventional external sphincterotomy with those of UroLume sphincteric stent prosthesis placement in men with spinal cord injury and external detrusor-sphincter dyssynergia. MATERIALS AND METHODS We randomized 57 men with spinal cord injury in whom urodynamics verified external detrusor-sphincter dyssynergia into 2 groups to undergo either sphincter defeating procedure. We compared the primary urodynamic parameter of maximum detrusor pressure, and secondary urodynamic parameters of bladder capacity and post-void residual urine volume in men who underwent sphincterotomy or sphincteric stent placement. Parameters were measured preoperatively, and 3, 6, 12 and 24 months postoperatively. Patients completed questionnaires regarding voiding sensation and quality of life issues at each followup visit. RESULTS Demographic data of the 26 patients treated with sphincterotomy and the 31 treated with sphincteric stent placement were statistically similar. Preoperatively mean maximum detrusor pressure plus or minus standard deviation in sphincterotomy and stent cases was 98.3 +/- 27.6 and 95.7 +/- 27.7 cm. water, respectively (p = 0.73). At 12 months mean maximum detrusor pressure decreased to 48.9 +/- 16.4 and 52.6 +/- 31.6 cm. water in the sphincterotomy and stent groups, respectively (p = 0). Preoperatively mean bladder capacity in sphincterotomy and stent cases was 245 +/- 158 and 251 +/- 145 ml., respectively (p = 0.87). Bladder capacity did not change significantly in either treatment group throughout followup. Preoperatively mean post-void residual urine volume in the sphincterotomy and stent groups was 212 +/- 163 and 168 +/- 114 ml., respectively (p = 0.33). Residual urine volume decreased in each group at some but not all followup evaluations. The duration of hospitalization was greater for sphincterotomy than stenting (p = 0.036). Six stents required explantation. CONCLUSIONS The UroLume stent is as effective as conventional external sphincterotomy for treating external detrusor-sphincter dyssynergia. However, sphincteric stent placement is advantageous because it involves shorter hospitalization and is potentially reversible.