Gissou Azabdaftari

Parkin controls dopamine utilization in human midbrain dopaminergic neurons derived from induced pluripotent stem cells

Parkinsons disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Here we generate induced pluripotent stem cells from normal subjects and PD patients with parkin mutations. We demonstrate that loss of parkin in human midbrain DA neurons greatly increases the transcription of monoamine oxidases and oxidative stress, significantly reduces DA uptake and increases spontaneous DA release. Lentiviral expression of parkin, but not its PD-linked mutant, rescues these phenotypes. The results suggest that parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of DA neurotransmission and suppressing dopamine oxidation. Thus, the study provides novel targets and a physiologically relevant screening platform for disease-modifying therapies of PD.

Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer

Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer. [Mol Cancer Ther 2008;7(9):2746–57]

Intratumoral and Intertumoral Genomic Heterogeneity of Multifocal Localized Prostate Cancer Impacts Molecular Classifications and Genomic Prognosticators

BACKGROUND Next-generation sequencing is revealing genomic heterogeneity in localized prostate cancer (CaP). Incomplete sampling of CaP multiclonality has limited the implications for molecular subtyping, stratification, and systemic treatment. OBJECTIVE To determine the impact of genomic and transcriptomic diversity within and among intraprostatic CaP foci on CaP molecular taxonomy, predictors of progression, and actionable therapeutic targets. DESIGN, SETTING, AND PARTICIPANTS Four consecutive patients with clinically localized National Comprehensive Cancer Network intermediate- or high-risk CaP who did not receive neoadjuvant therapy underwent radical prostatectomy at Roswell Park Cancer Institute in June-July 2014. Presurgical information on CaP content and a customized tissue procurement procedure were used to isolate nonmicroscopic and noncontiguous CaP foci in radical prostatectomy specimens. Three cores were obtained from the index lesion and one core from smaller lesions. RNA and DNA were extracted simultaneously from 26 cores with ≥90% CaP content and analyzed using whole-exome sequencing, single-nucleotide polymorphism arrays, and RNA sequencing. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Somatic mutations, copy number alternations, gene expression, gene fusions, and phylogeny were defined. The impact of genomic alterations on CaP molecular classification, gene sets measured in Oncotype DX, Prolaris, and Decipher assays, and androgen receptor activity among CaP cores was determined. RESULTS AND LIMITATIONS There was considerable variability in genomic alterations among CaP cores, and between RNA- and DNA-based platforms. Heterogeneity was found in molecular grouping of individual CaP foci and the activity of gene sets underlying the assays for risk stratification and androgen receptor activity, and was validated in independent genomic data sets. Determination of the implications for clinical decision-making requires follow-up studies. CONCLUSIONS Genomic make-up varies widely among CaP foci, so care should be taken when making treatment decisions based on a single biopsy or index lesions. PATIENT SUMMARY We examined the molecular composition of individual cancers in a patients prostate. We found a lot of genetic diversity among these cancers, and concluded that information from a single cancer biopsy is not sufficient to guide treatment decisions.

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knockdown sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC.

A Regulatory Feedback Loop Between Ca2+/Calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) and the Androgen Receptor in Prostate Cancer Progression

Background: Defining molecular mechanisms that regulate AR activity is critical for understanding prostate cancer progression. Results: CaMKK2 increases during disease progression, is transcriptionally regulated by the AR, promotes proliferation, and is required for optimal AR transcriptional activity. Conclusion: CaMKK2 is in a feedback circuit to maintain AR activity. Significance: The CaMKK2 pathway is a promising target for prostate cancer therapy. The androgen receptor (AR) plays a critical role in prostate cancer (PCa) progression, however, the molecular mechanisms by which the AR regulates cell proliferation in androgen-dependent and castration-resistant PCa are incompletely understood. We report that Ca2+/calmodulin-dependent kinase kinase 2 (CaMKK2) expression increases and becomes nuclear or perinuclear in advanced PCa. In the TRAMP (transgenic adenocarcinoma of mouse prostate) model of PCa, CaMKK2 expression increases with PCa progression with many cells exhibiting nuclear staining. CaMKK2 expression is higher in human castration-resistant tumor xenografts compared with androgen-responsive xenografts and is markedly higher in the AR-expressing, tumorigenic cell line LNCaP compared with cell lines that are AR-nonexpressing and/or nontumorigenic. In LNCaP cells, dihydrotestosterone induced CaMKK2 mRNA and protein expression and translocation of CaMKK2 to the nucleus. Conversely, androgen withdrawal suppressed CaMKK2 expression. Knockdown of CaMKK2 expression by RNAi reduced LNCaP cell proliferation and increased percentages of cells in G1 phase, whereas correspondingly reducing percentages in S phase, of the cell cycle. CaMKK2 knockdown reduced expression of the AR target gene prostate-specific antigen at both mRNA and protein levels, AR transcriptional activity driven by androgen responsive elements from the prostate-specific probasin gene promoter and levels of the AR-regulated cell cycle proteins, cyclin D1 and hyperphosphorylated Rb. Our results suggest that in PCa progression, CaMKK2 and the AR are in a feedback loop in which CaMKK2 is induced by the AR to maintain AR activity, AR-dependent cell cycle control, and continued cell proliferation.

Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme

Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population.

Establishment and characterization of a highly tumorigenic African American prostate cancer cell line, E006AA-hT.

Genuine racial differences in prostate cancer (PCa) biology have been considered among the potential reasons to explain PCa disparities. There is no animal model to represent all aspects of human PCa and, more specifically, to be used for PCa disparity research. The lack of a spontaneously transformed in vitro cell-based model system has been a significant impediment to investigating and understanding potential molecular mechanisms, and the hormonal, genetic, and epigenetic factors underlying the biological and clinical aggressiveness of PCa in African American (AA) men. In this study, we established and characterized the E006AA-hT cell line as a highly tumorigenic subline of the previously characterized primary AA-PCa cell line, E006AA. Extensive characterization of the E006AA-hT cell line was accomplished using cytodifferentiation and prostate-specific markers, spectral karyotyping, cell line authentication assays, cell proliferation and migration assays, and in vitro tumorigenesis assays. Spectral karyotyping of E006AA-hT showed a hypertriploid chromosome complement and shared cytogenetic changes similar to its parental cells such as diploid X, absence of Y-chromosomes, numerical gains in chromosomes 5,6,8,10,17,20,21, and marker chromosomes of unknown origin. In addition, E006AA-hT also presented numerous clonal and structural aberrations such as insertion, deletion, duplication, and translocations in chromosomes 1-5, 8, 9, 11, 13, 14, 17, and 18. The E006AA-hT cell line was shown to be highly tumorigenic and produced tumors at an accelerated growth rate in both athymic nude and triple-deficient SCID mice. Silencing the mutated androgen receptor (AR-599 Ser>Gly) did not affect proliferation (loss-of-function), but decreased migration (gain-of-function) in E006AA-hT and its parental cell type. These data support that AR-point mutations may lead simultaneously to different “loss-of-function” and “gain-of-function” phenotypes in PCa cells. E006AA-Par and its subline as the only available spontaneously transformed low- and highly-tumorigenic primary AA-PCa cell lines could be used for basic and translational research aimed in supporting prostate cancer disparity research.

Vascular Disruption in Combination with mTOR Inhibition in Renal Cell Carcinoma

Renal cell carcinoma (RCC) is an angiogenesis-dependent and hypoxia-driven malignancy. As a result, there has been an increased interest in the use of antiangiogenic agents for the management of RCC in patients. However, the activity of tumor-vascular disrupting agents (tumor-VDA) has not been extensively examined against RCC. In this study, we investigated the therapeutic efficacy of the tumor-VDA ASA404 (DMXAA, 5,6-dimethylxanthenone-4-acetic acid, or vadimezan) in combination with the mTOR inhibitor everolimus (RAD001) against RCC. In vitro studies were carried out using human umbilical vein endothelial cells and in vivo studies using orthotopic RENCA tumors and immunohistochemical patient tumor-derived RCC xenografts. MRI was used to characterize the vascular response of orthotopic RENCA xenografts to combination treatment. Therapeutic efficacy was determined by tumor growth measurements and histopathologic evaluation. ASA404/everolimus combination resulted in enhanced inhibition of endothelial cell sprouting in the 3-dimensional spheroid assay. MRI of orthotopic RENCA xenografts revealed an early increase in permeability 4 hours posttreatment with ASA404, but not with everolimus. Twenty-four hours after treatment, a significant reduction in blood volume was observed with combination treatment. Correlative CD31/NG2 staining of tumor sections confirmed marked vascular damage following combination therapy. Histologic sections showed extensive necrosis and a reduction in the viable rim following combination treatment compared with VDA treatment alone. These results show the potential of combining tumor-VDAs with mTOR inhibitors in RCC. Further investigation into this novel combination strategy is warranted. Mol Cancer Ther; 11(2); 383–92. ©2011 AACR.

Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity

IntroductionHigh expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations.MethodsIn the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDHLow), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation.ResultsThe percentage of ALDHHi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDHHi cells was 0.6 to 4%. Recombinants using ALDHHi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDHLow counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDHHi cells and non-side population cells. ABCG2 expression was only enriched in side population cells.ConclusionsThe percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared to cells with high ABCG2 activity, identified with the side population assay. The expression of the stem and differentiation markers indicates the ALDHHi recombinants contained cells with self-renewal and multipotency activity. When the two assays were directly compared, cells with the side population phenotype demonstrated more stem cell potential in the tissue recombination assay compared to ALDHHi cells. The increased stem cell potential of side population cells in the tissue recombination assay and the decrease in sphere formation when ABCG2 is inhibited indicates that the side population enriches for prostate stem cells.

Predictors of Complete Pathologic Response (pT0) to Neoadjuvant Chemotherapy in Muscle-invasive Bladder Carcinoma

UNLABELLED No predictors of a complete pathologic response (pT0) to neoadjuvant chemotherapy (NAC) in muscle-invasive bladder carcinoma have been established. We performed a retrospective analysis of 50 patients to identify potential predictors. Our results showed that the presence of additional transitional cell variants on pathologic examination (mixed tumors) predicted against pT0, suggesting the avoidance of NAC and its morbidity in these patients with mixed tumors. BACKGROUND Randomized trials have supported the use of cisplatin-based neoadjuvant chemotherapy (NAC) in muscle-invasive bladder carcinoma (MIBC) owing to the survival advantage, which has correlated with downstaging of the cancer to pT0. Only 30% to 40% of patients receiving NAC have attained a pT0 response at cystectomy; the remaining have either residual disease or progression. We aimed to identify the factors that could predict a pT0 response to NAC. PATIENTS AND METHODS Of 336 patients who had undergone robotic cystectomy at our institute from May 2007 to March 2014, we identified 50 patients who had undergone NAC for MIBC. We conducted a retrospective study, dividing these 50 patients into 2 groups, those with and without a pT0. Factors, including age, histologic features, hydronephrosis at initial presentation, and chemotherapy type, were examined by both univariate and multivariate logistic regression analysis. RESULTS Of the 50 patients, 14 (28%) had pT0 at cystectomy, 20 (40%) had progressive disease, and 16 (32%) had residual disease. The median age was 67.5 years, the median glomerular filtration rate at presentation was 87.5 mL/min, the patients had undergone a median of 3 NAC cycles, and the median time from the end of chemotherapy to surgery was 4 weeks. The odds of a pT0 response for pure urothelial carcinoma (UC) were approximately 11 times greater relative to cancers with transitional cell variant histologic features or mixed tumors (odds ratio 0.09, 95% confidence interval 0.021-0.380; P = .0011), including squamous, glandular differentiation, small cell, micropapillary, sarcomatoid, nested component, lymphoepithelioma-like, and plasmacytoid variants. CONCLUSION The presence of pure UC favored a pT0 response to NAC compared with those with variant histologic features or mixed tumors. These potential predictors warrant prospective validation to allow the ideal selection of patients for NAC.