Karen L. Ferrari

Intravesical bacillus Calmette-Guérin Efficiently Reduces p70S6K1 but Not 4E-BP1 Phosphorylation in Nonmuscle Invasive Bladder Cancer

PURPOSE We characterized the functional consequences of intravesical bacillus Calmette-Guérin on the molecular mechanism of the AKT/mTOR signaling pathway in nonmuscle invasive bladder cancer. To our knowledge this has not been reported previously. MATERIALS AND METHODS At age 7 weeks female Fischer 344 rats received 1.5 mg/kg MNU intravesically every other week for 6 weeks. They were randomized at 10 per group to MNU (0.2 ml vehicle), bacillus Calmette-Guérin (10(6) cfu Connaught strain), rapamycin (15 μg/ml) and bacillus Calmette-Guérin plus simultaneous rapamycin, each intravesically for 6 weeks. At week 15 the bladders were collected for histopathology, immunohistochemistry and immunoblot to determine p-AKT, Rictor, Raptor, p-4E-BP1, p-p70S6K1, p-AMPK-α, p-mTOR and p-p53. RESULTS Papillary carcinoma (pTa) and high grade intraepithelial neoplasia (pTis) predominated in the MNU group while normal urothelium, papillary and flat hyperplasia were more common in treated groups. Nonmuscle invasive bladder cancer treated with bacillus Calmette-Guérin showed suppression of p70S6K1 but not 4E-BP1 phosphorylation. This suggests that 4E-BP1 is regulated differently than p70S6K1, escaping the bacillus Calmette-Guérin action that occurs in a mTOR independent manner. The association of bacillus Calmette-Guérin with rapamycin but not rapamycin monotherapy affected p70S6K1 and 4E-BP1 phosphorylation with no features of in situ carcinoma (pTis). CONCLUSIONS The activation status of p70S6K1 and 4E-BP1 might be used to stratify patients who could benefit from targeting such molecular elements with multitarget/multidrug intravesical therapy. In the future 4E-BP1 might be a worthwhile new target for bacillus Calmette-Guérin refractory nonmuscle invasive bladder cancer.

Intravesical Immunomodulatory Imiquimod Enhances Bacillus Calmette-Guérin Downregulation of Nonmuscle-invasive Bladder Cancer

&NA; The Toll‐like receptor (TLR)2/4 agonist bacillus Calmette‐Guérin (BCG) is the most efficient immunomodulatory treatment of nonmuscle‐invasive bladder cancer (NMIBC). BCG modulation by the TLR7 agonist imiquimod was evaluated in an immunocompetent animal model. We found that upregulation of TLR7/4 and downregulation of P70S6K1 boost the BCG effect in NMIBC. Background: The Toll‐like receptor (TLR)2/4 agonist bacillus Calmette‐Guérin (BCG), although not failure proof, has been the most efficient immunomodulatory treatment of immunogenic nonmuscle‐invasive bladder cancer (NMIBC) for > 40 years. We investigated the role of the immunomodulatory molecule TLR7 agonist imiquimod through the BCG key receptors TLR2/4 and the main downstream molecules of the mammalian target of rapamycin pathway in NMIBC treatment. Materials and Methods: A total of 40 Fischer‐344 rats, 7 weeks old, received 4 doses of 1.5 mg/kg N‐methyl‐N‐nitrosourea intravesically on weeks 0, 2, 4, and 6 for cancer induction. At week 8, the rats were randomized into 4 groups (10 per group) and treated intravesically once a week for 6 weeks: control (0.2 mL of vehicle); BCG (2 × 106 colony‐forming units Connaught strain in 0.2 mL); imiquimod (20 mg/kg in 0.2 mL), and associated treatment BCG plus imiquimod in 0.2 mL. The bladders were extracted and analyzed for histopathology, immunohistochemistry, cell proliferation (Ki‐67), apoptosis (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling [TUNEL]), and immunoblotting for TLR2, TLR4, p‐P70S6K, and p‐4E‐BP1 proteins. Results: The histopathology results showed that BCG and imiquimod decreased bladder tumorigenesis compared with the control group, with a proliferation decrease (Ki‐67) and an apoptosis increase (TUNEL). BCG upregulated TLR2/4, imiquimod upregulated TLR4, and both downregulated P70S6K1. Conclusion: Imiquimod is able to efficiently decrease bladder carcinogenesis through upregulation of TLR7/4 and downregulation of P70S6K1 protein, generating new perspectives to boost BCG effects in the future.

MP65-17 INTRAVESICAL THALIDOMIDE BOOSTS BACILLUS CALMETE-GUÉRIN (BCG) IN NON-MUSCLE INVASIVE BLADDER CANCER TREATMENT

The aim of this study was to explore the efficacy of intravesical Thalidomide (immunomodulatory, anti-inflammatory and anti-angiogenic) added to BCG using an immune competent autochthonous orthotopic NMIBC animal model. Female Fischer 344 rats, 7 weeks of age, received every 2 weeks for four times, a dose of 1.5 mg/kg of N-methyl-N-nitrosourea (MNU) intravesically. The rats were randomized into four groups (n = 10 per group) to receive intravesical treatment once a week for 6 weeks as follows: control (0.2 ml vehicle), BCG (2 × 106 CFU of Connaught strain in 0.2 ml), Thalidomide (20 mg/kg in 0.2 ml) and BCG-Thalidomide in 0.2 ml. At week 15, bladders were collected for histopathology, cell turnover index by immunohistochemistry and immunoblotting quantification of 4E-BP1 and p70S6K1 for downstream mTOR proliferation signaling and HIF and VEGF for angiogenesis pathway. Thalidomide-BCG association showed a trend for normal histopathology and down-regulation of cell turnover, p70S6K1, HIF-1 and VEGF. 4E-BP1 was up-regulated by treatment, especially in the Thalidomide groups, supporting that its regulation occurs independently of p70S6K1 on mTOR pathway in NMIBC. Intravesical BCG-Thalidomide might represent a significant increment in NMIBC treatment, suggesting p70S6K1, HIF-1 and VEGF as potential molecular target candidates in a clinically relevant immune competent NMIBC model.

MP70-19 LOCAL IMMUNE MODULATION BY INCREASING T CYTOTOXIC / T HELPER RATIO AFTER PROSTATE CANCER HEMI-CRYOABLATION

CONCLUSIONS: Prior interventional therapy for BPH is associated with increased risks of urinary retention and incontinence after whole gland prostate cryoablation. Nevertheless, in properly selected patients, prior bladder outlet procedures are not an absolute contraindication to cryotherapy. Consideration should be given to management protocol in those men including but not limited to time for catheter removal postoperatively and continuing BPH medical treatment