Purpose: We previously showed that nuclear localization of the actin-binding protein, filamin A (FlnA), corresponded to hormone-dependence in prostate cancer. Intact FlnA (280 kDa, cytoplasmic) cleaved to a 90 kDa fragment which translocated to the nucleus in hormone-naI« ve cells, whereas in hormone-refractory cells, FlnA was phosphorylated, preventing its cleavage and nuclear translocation. We have examined whether FlnA localization determines a propensity to metastasis in advanced androgen-independent prostate cancer. Experimental Design: We examined, by immunohistochemistry, FlnA localization in paraffinembedded human prostate tissue representing different stages of progression. Results were correlated with in vitro studies in a cell model of prostate cancer. Results: Nuclear FlnA was significantly higher in benign prostate (0.6612 F 0.5888), prostatic intraepithelial neoplasia (PIN; 0.6024 F 0.4620), and clinically localized cancers (0.69134 F 0.5686) compared with metastatic prostate cancers (0.3719 F 0.4992, P = 0.0007). Cytoplasmic FlnA increased from benign prostate (0.0833 F 0.2677), PIN (0.1409 F 0.2293), localized cancers (0.3008 F 0.3762, P = 0.0150), to metastases (0.7632 F 0.4414, P < 0.00001). Logistic regression of metastatic versus nonmetastatic tissue yielded the area under the receiver operating curve as 0.67 for nuclear-FlnA, 0.79 for cytoplasmic-FlnA, and 0.82 for both, indicating that metastasis correlates with cytoplasmic to nuclear translocation. In vitro studies showed that cytoplasmic localization of FlnA induced cell invasion whereas nuclear translocation of the protein inhibited it. FlnA dephosphorylation with the protein kinaseA inhibitor H-89 facilitated FlnA nuclear translocation, resulting in decreased invasiveness and AR transcriptional activity, and induced sensitivity to androgen withdrawal in hormone-refractory cells. Conclusions:The data presented in this study indicate that in prostate cancer, metastasis correlates with cytoplasmic localization of FlnA and may be prevented by cleavage and subsequent nuclear translocation of this protein.Filamins are a family of cytoskeletal proteins that organize filamentous actin into networks and stress fibers (1). Filamin A (FlnA) is a 280 kDa non -muscle actin binding protein, the appropriate function of which is essential for development (2, 3). FlnA dimerization forms a V-shaped flexible structure which can induce high-angle orthogonal branching and efficiently gather actin filaments into a three-dimensional gel in vitro by cross-linking actin filaments at the leading edge of migrating cells. Hence, filamins are essential for mammalian cell locomotion, anchoring of transmembrane proteins including integrins, and also act as interfaces for protein-protein interaction (4). More than 30 proteins of great functional diversity are known to interact with filamins which function as a signaling scaffold by connecting and coordinating a large variety of cellular processes (4).In prostate cancer, a role for FlnA was identified in prost...

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra-and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 µg of DNA. This sensitivity may allow the method to be used for clinical applications.

BackgroundThe overarching goal of this project is to establish a patient-derived bladder cancer xenograft (PDX) platform, annotated with deep sequencing and patient clinical information, to accelerate the development of new treatment options for bladder cancer patients. Herein, we describe the creation, initial characterization and use of the platform for this purpose.Methods and FindingsTwenty-two PDXs with annotated clinical information were established from uncultured unselected clinical bladder cancer specimens in immunodeficient NSG mice. The morphological fidelity was maintained in PDXs. Whole exome sequencing revealed that PDXs and parental patient cancers shared 92–97% of genetic aberrations, including multiple druggable targets. For drug repurposing, an EGFR/HER2 dual inhibitor lapatinib was effective in PDX BL0440 (progression-free survival or PFS of 25.4 days versus 18.4 days in the control, p = 0.007), but not in PDX BL0269 (12 days versus 13 days in the control, p = 0.16) although both expressed HER2. To screen for the most effective MTT, we evaluated three drugs (lapatinib, ponatinib, and BEZ235) matched with aberrations in PDX BL0269; but only a PIK3CA inhibitor BEZ235 was effective (p<0.0001). To study the mechanisms of secondary resistance, a fibroblast growth factor receptor 3 inhibitor BGJ398 prolonged PFS of PDX BL0293 from 9.5 days of the control to 18.5 days (p<0.0001), and serial biopsies revealed that the MAPK/ERK and PIK3CA-AKT pathways were activated upon resistance. Inhibition of these pathways significantly prolonged PFS from 12 day of the control to 22 days (p = 0.001). To screen for effective chemotherapeutic drugs, four of the first six PDXs were sensitive to the cisplatin/gemcitabine combination, and chemoresistance to one drug could be overcome by the other drug.ConclusionThe PDX models described here show good correlation with the patient at the genomic level and known patient response to treatment. This supports further evaluation of the PDXs for their ability to accurately predict a patient’s response to new targeted and combination strategies for bladder cancer.

The mTOR (mammalian target of rapamycin) inhibitor rapamycin caused growth arrest in both androgendependent and androgen-independent prostate cancer cells; however, long-term treatment induced resistance to the drug. The aim of this study was to investigate methods that can overcome this resistance. Here, we show that rapamycin treatment stimulated androgen receptor (AR) transcriptional activity, whereas suppression of AR activity with the antiandrogen bicalutamide sensitized androgen-dependent, as well as AR-sensitive androgenindependent prostate cancer cells, to growth inhibition by rapamycin. Further, the combination of rapamycin and bicalutamide, but not the individual drugs, induced significant levels of apoptosis in prostate cancer cells. The net effect of rapamycin is determined by its individual effects on the mTOR complexes mTORC1 (mTOR/ raptor/GbL) and mTORC2 (mTOR/rictor/sin1/GbL). Inhibition of both mTORC1 and mTORC2 by rapamycin-induced apoptosis, whereas rapamycin-stimulation of AR transcriptional activity resulted from the inhibition of mTORC1, but not mTORC2. The effect of rapamycin on AR transcriptional activity was mediated by the phosphorylation of the serine/threonine kinase Akt, which also partially mediated apoptosis induced by rapamycin and bicalutamide. These results indicate the presence of two parallel cell-survival pathways in prostate cancer cells: a strong Akt-independent, but rapamycin-sensitive pathway downstream of mTORC1, and an AR-dependent pathway downstream of mTORC2 and Akt, that is stimulated by mTORC1 inhibition. Thus, the combination of rapamycin and bicalutamide induce apoptosis in prostate cancer cells by simultaneously inhibiting both pathways and hence would be of therapeutic value in prostate cancer treatment.