Genomic Profiling of Metastatic Castration-Resistant Prostate Cancer Samples Resistant to Androgen Receptor Pathway Inhibitors.

PURPOSE: The androgen receptor axis inhibitors (ARPI; e.g., enzalutamide, abiraterone acetate) are administered in daily practice for men with metastatic castration-resistant prostate cancer (mCRPC). However, not all patients respond, and mechanisms of both primary and acquired resistance remain largely unknown. EXPERIMENTAL DESIGN: In the prospective trial MATCH-R (NCT02517892), 59 patients with mCRPC underwent whole-exome sequencing (WES) and/or RNA sequencing (RNA-seq) of samples collected before starting ARPI. Also, 18 patients with mCRPC underwent biopsy at time of resistance. The objectives were to identify genomic alterations associated with resistance to ARPIs as well as to describe clonal evolution. Associations of genomic and transcriptomic alterations with primary resistance were determined using Wilcoxon and Fisher exact tests. RESULTS: WES analysis indicated that no single-gene genomic alterations were strongly associated with primary resistance. RNA-seq analysis showed that androgen receptor (AR) gene alterations and expression levels were similar between responders and nonresponders. RNA-based pathway analysis found that patients with primary resistance had a higher Hedgehog pathway score, a lower AR pathway score and a lower NOTCH pathway score than patients with a response. Subclonal evolution and acquisition of new alterations in AR-related genes or neuroendocrine differentiation are associated with acquired resistance. ARPIs do not induce significant changes in the tumor transcriptome of most patients; however, programs associated with cell proliferation are enriched in resistant samples. CONCLUSIONS: Low AR activity, activation of stemness programs, and Hedgehog pathway were associated with primary ARPIs' resistance, whereas most acquired resistance was associated with subclonal evolution, AR-related events, and neuroendocrine differentiation. See related commentary by Slovin, p. 4323.

Development of Novel Models of Aggressive Variants of Castration-resistant Prostate Cancer.

BACKGROUND: Genomic studies have identified new subsets of aggressive prostate cancer (PCa) with poor prognosis (eg, neuroendocrine prostate cancer [NEPC], PCa with DNA damage response [DDR] alterations, or PCa resistant to androgen receptor pathway inhibitors [ARPIs]). Development of novel therapies relies on the availability of relevant preclinical models. OBJECTIVE: To develop new preclinical models (patient-derived xenograft [PDX], PDX-derived organoid [PDXO], and patient-derived organoid [PDO]) representative of the most aggressive variants of PCa and to develop a new drug evaluation strategy. DESIGN, SETTING, AND PARTICIPANTS: NEPC (n = 5), DDR (n = 7), and microsatellite instability (MSI)-high (n = 1) PDXs were established from 51 patients with metastatic PCa; PDXOs (n = 16) and PDOs (n = 6) were developed to perform drug screening. Histopathology and treatment response were characterized. Molecular profiling was performed by whole-exome sequencing (WES; n = 13), RNA sequencing (RNA-seq; n = 13), and single-cell RNA-seq (n = 14). WES and RNA-seq data from patient tumors were compared with the models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Relationships with outcome were analyzed using the multivariable chi-square test and the tumor growth inhibition test. RESULTS AND LIMITATIONS: Our PDXs captured both common and rare molecular phenotypes and their molecular drivers, including alterations of BRCA2, CDK12, MSI-high status, and NEPC. RNA-seq profiling demonstrated broad representation of PCa subtypes. Single-cell RNA-seq indicates that PDXs reproduce cellular and molecular intratumor heterogeneity. WES of matched patient tumors showed preservation of most genetic driver alterations. PDXOs and PDOs preserve drug sensitivity of the matched tissue and can be used to determine drug sensitivity. CONCLUSIONS: Our models reproduce the phenotypic and genomic features of both common and aggressive PCa variants and capture their molecular heterogeneity. Successfully developed aggressive-variant PCa preclinical models provide an important tool for predicting tumor response to anticancer therapy and studying resistance mechanisms. PATIENT SUMMARY: In this report, we looked at the outcomes of preclinical models from patients with metastatic prostate cancer enrolled in the MATCH-R trial (NCT02517892).

Reformation of the chondroitin sulfate glycocalyx enables progression of AR-independent prostate cancer.

Lineage plasticity of prostate cancer is associated with resistance to androgen receptor (AR) pathway inhibition (ARPI) and supported by a reactive tumor microenvironment. Here we show that changes in chondroitin sulfate (CS), a major glycosaminoglycan component of the tumor cell glycocalyx and extracellular matrix, is AR-regulated and promotes the adaptive progression of castration-resistant prostate cancer (CRPC) after ARPI. AR directly represses transcription of the 4-O-sulfotransferase gene CHST11 under basal androgen conditions, maintaining steady-state CS in prostate adenocarcinomas. When AR signaling is inhibited by ARPI or lost during progression to non-AR-driven CRPC as a consequence of lineage plasticity, CHST11 expression is unleashed, leading to elevated 4-O-sulfated chondroitin levels. Inhibition of the tumor cell CS glycocalyx delays CRPC progression, and impairs growth and motility of prostate cancer after ARPI. Thus, a reactive CS glycocalyx supports adaptive survival and treatment resistance after ARPI, representing a therapeutic opportunity in patients with advanced prostate cancer.

Enzalutamide response in a panel of prostate cancer cell lines reveals a role for glucocorticoid receptor in enzalutamide resistant disease.

Representative in vitro model systems that accurately model response to therapy and allow the identification of new targets are important for improving our treatment of prostate cancer. Here we describe molecular characterization and drug testing in a panel of 20 prostate cancer cell lines. The cell lines cluster into distinct subsets based on RNA expression, which is largely driven by functional Androgen Receptor (AR) expression. KLK3, the AR-responsive gene that encodes prostate specific antigen, shows the greatest variability in expression across the cell line panel. Other common prostate cancer associated genes such as TMPRSS2 and ERG show similar expression patterns. Copy number analysis demonstrates that many of the most commonly gained (including regions containing TERC and MYC) and lost regions (including regions containing TP53 and PTEN) that were identified in patient samples by the TCGA are mirrored in the prostate cancer cell lines. Assessment of response to the anti-androgen enzalutamide shows a distinct separation of responders and non-responders, predominantly related to status of wild-type AR. Surprisingly, several AR-null lines responded to enzalutamide. These AR-null, enzalutamide-responsive cells were characterized by high levels of expression of glucocorticoid receptor (GR) encoded by NR3C1. Treatment of these cells with the anti-GR agent mifepristone showed that they were more sensitive to this drug than enzalutamide, as were several of the enzalutamide non-responsive lines. This is consistent with several recent reports that suggest that GR expression is an alternative signaling mechanism that can bypass AR blockade. This study reinforces the utility of large cell line panels for the study of cancer and identifies several cell lines that represent ideal models to study AR-null cells that have upregulated GR to sustain growth.

Combining inhibition of galectin-3 with and before a therapeutic vaccination is critical for the prostate-tumor-free outcome.

BACKGROUND: Prostate cancer (PCa) is a major health problem worldwide. Taxol derivatives-based chemotherapies or immunotherapies are usually proposed depending on the symptomatic status of the patient. In the case of immunotherapy, tumors develop robust immune escape mechanisms that abolish any protective response, and to date why prostate cancer is one of the most resistant diseases remains unresolved. METHODS: By using a combination of clinical data to study the transcriptome of metastasis samples from patients with castration-refractory prostate cancer, and state of the art cellular and molecular biology assays in samples from tumor-bearing mice that have been submitted to surgical resection of the tumor before receiving a vaccination, we answered several essential questions in the field of immunotherapy for prostate cancer. We also used two different methods to inhibit the expression of galectin-3 (Gal-3) in tumor cells: a stable RNA interference method to control the expression of this galectin efficiently only in tumor cells, and low and non-cytotoxic doses of docetaxel to easily transfer our findings to clinical settings. RESULTS: Herein, we show for the first time that Gal-3 expressed by prostate tumor cells is the main immune checkpoint responsible for the failure of vaccine-based immunotherapy. Our results show that low and non-cytotoxic doses of docetaxel lead to the inhibition of Gal-3 expression in PCa cells as well as in clinical samples of patients with metastatic and castration-resistant PCa promoting a Th1 response. We thus optimized a prostate cancer animal model that undergoes surgical resection of the tumor to mimic prostatectomy usually performed in patients. Importantly, using Gal-3-knocked down-PCa cells or low and non-cytotoxic doses of taxane before vaccination, we were able to highly control tumor recurrence through a direct impact on the proliferation and infiltration of CD8+ cytotoxic T. CONCLUSIONS: Thus, Gal-3 expression by PCa cells is a crucial inhibitor for the success of immunotherapy, and low doses of docetaxel with non-cytotoxic effect on leukocyte survival could be used before immunotherapy for all patients with PCa to reduce the expression of this critical negative immune checkpoint, pre-conditioning the tumor-microenvironment to activate an antitumor immune response and promote tumor-free outcome.

Regulation of eIF4F Translation Initiation Complex by the Peptidyl Prolyl Isomerase FKBP7 in Taxane-resistant Prostate Cancer.

PURPOSE: Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance.Experimental Design: We established 4 chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by in vitro and in vivo silencing, and mass spectrometry analysis was used to identify its downstream effectors. RESULTS: We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulated in human prostate cancers and its expression correlated with the recurrence observed in patients receiving docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with eIF4G, a component of the eIF4F translation initiation complex, to mediate the survival of chemoresistant cells. Using small-molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to kill docetaxel- and cabazitaxel-resistant cells. CONCLUSIONS: Targeting FKBP7 or the eIF4G-containing eIF4F translation initiation complex could be novel therapeutic strategies to eradicate taxane-resistant prostate cancer cells.

Compound Functional Prediction Using Multiple Unrelated Morphological Profiling Assays.

Phenotypic cell-based assays have proven to be efficient at discovering first-in-class therapeutic drugs mainly because they allow for scanning a wide spectrum of possible targets at once. However, despite compelling methodological advances, posterior identification of a compound's mechanism of action (MOA) has remained difficult and highly refractory to automated analyses. Methods such as the cell painting assay and multiplexing fluorescent dyes to reveal broadly relevant cellular components were recently suggested for MOA prediction. We demonstrated that adding fluorescent dyes to a single assay has limited impact on MOA prediction accuracy, as monitoring only the nuclei stain could reach compelling levels of accuracy. This observation suggested that multiplexed measurements are highly correlated and nuclei stain could possibly reflect the general state of the cell. We then hypothesized that combining unrelated and possibly simple cell-based assays could bring a solution that would be biologically and technically more relevant to predict a drug target than using a single assay multiplexing dyes. We show that such a combination of past screen data could rationally be reused in screening facilities to train an ensemble classifier to predict drug targets and prioritize a possibly large list of unknown compound hits at once.

Stable and high expression of Galectin-8 tightly controls metastatic progression of prostate cancer.

Two decades ago, Galectin-8 was described as a prostate carcinoma biomarker since it is only expressed in the neoplastic prostate, but not in the healthy tissue. To date, no biological function has been attributed to Galectin-8 that could explain this differential expression. In this study we silenced Galectin-8 in two human prostate cancer cell lines, PC3 and IGR-CaP1, and designed a pre-clinical experimental model that allows monitoring the pathology from its early steps to the long-term metastatic stages. We show for the first time that the natural and conserved expression of Gal-8 in tumour cells is responsible for the metastatic evolution of prostate cancer. In fact, Gal-8 controls the rearrangement of the cytoskeleton and E-Cadherin expression, with a major impact on anoikis and homotypic aggregation of tumour cells, both being essential processes for the survival of circulating tumour cells during metastasis. While localized prostate cancer can be cured, metastatic and advanced disease remains a significant therapeutic challenge, urging for the identification of prognostic markers of the metastatic process. Collectively, our results highlight Galectin-8 as a potential target for anti-metastatic therapy against prostate cancer.

A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector.

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making.

Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening.

Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis.

Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors.

Targeting CDC25C, PLK1 and CHEK1 to overcome Docetaxel resistance induced by loss of LZTS1 in prostate cancer.

Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance.

A unique galectin signature in human prostate cancer progression suggests galectin-1 as a key target for treatment of advanced disease.

Galectins, a family of glycan-binding proteins, influence tumor progression by modulating interactions between tumor, endothelial, stromal, and immune cells. Despite considerable progress in identifying the roles of individual galectins in tumor biology, an integrated portrait of the galectin network in different tumor microenvironments is still missing. We undertook this study to analyze the "galectin signature" of the human prostate cancer microenvironment with the overarching goal of selecting novel-molecular targets for prognostic and therapeutic purposes. In examining androgen-responsive and castration-resistant prostate cancer cells and primary tumors representing different stages of the disease, we found that galectin-1 (Gal-1) was the most abundantly expressed galectin in prostate cancer tissue and was markedly upregulated during disease progression. In contrast, all other galectins were expressed at lower levels: Gal-3, -4, -9, and -12 were downregulated during disease evolution, whereas expression of Gal-8 was unchanged. Given the prominent regulation of Gal-1 during prostate cancer progression and its predominant localization at the tumor-vascular interface, we analyzed the potential role of this endogenous lectin in prostate cancer angiogenesis. In human prostate cancer tissue arrays, Gal-1 expression correlated with the presence of blood vessels, particularly in advanced stages of the disease. Silencing Gal-1 in prostate cancer cells reduced tumor vascularization without altering expression of other angiogenesis-related genes. Collectively, our findings identify a dynamically regulated "galectin-specific signature" that accompanies disease evolution in prostate cancer, and they highlight a major role for Gal-1 as a tractable target for antiangiogenic therapy in advanced stages of the disease.

The IGR-CaP1 xenograft model recapitulates mixed osteolytic/blastic bone lesions observed in metastatic prostate cancer.

Bone metastases have a devastating impact on quality of life and bone pain in patients with prostate cancer and decrease survival. Animal models are important tools in investigating the pathogenesis of the disease and in developing treatment strategies for bone metastases, but few animal models recapitulate spontaneous clinical bone metastatic spread. In the present study, IGR-CaP1, a new cell line derived from primary prostate cancer, was stably transduced with a luciferase-expressing viral vector to monitor tumor growth in mice using bioluminescence imaging. The IGR-CaP1 tumors grew when subcutaneously injected or when orthotopically implanted, reconstituted the prostate adenocarcinoma with glandular acini-like structures, and could disseminate to the liver and lung. Bone lesions were detected using bioluminescence imaging after direct intratibial or intracardiac injections. Anatomic bone structure assessed using high-resolution computed tomographic scans showed both lytic and osteoblastic lesions. Technetium Tc 99m methylene diphosphonate micro single-photon emission computed tomography confirmed the mixed nature of the lesions and the intensive bone remodeling. We also identified an expression signature for responsiveness of IGR-CaP1 cells to the bone microenvironment, namely expression of CXCR4, MMP-9, Runx2, osteopontin, osteoprotegerin, ADAMTS14, FGFBP2, and HBB. The IGR-CaP1 cell line is a unique model derived from a primary tumor, which can reconstitute human prostate adenocarcinoma in animals and generate experimental bone metastases, providing a novel means for understanding the mechanisms of bone metastasis progression and allowing preclinical testing of new therapies.

Ligand-dependent degradation of SRC-1 is pivotal for progesterone receptor transcriptional activity.

The progesterone receptor (PR), a ligand-activated transcription factor, recruits the primary coactivator steroid receptor coactivator-1 (SRC-1) gene promoters. It is known that PR transcriptional activity is paradoxically coupled to its ligand-dependent down-regulation. However, despite its importance in PR function, the regulation of SRC-1 expression level during hormonal exposure is poorly understood. Here we report that SRC-1 expression level (but not other p160 family members) is down-regulated by the agonist ligand R5020 in a PR-dependent manner. In contrast, the antagonist RU486 fails to induce down-regulation of the coactivator and impairs PR agonist-dependent degradation of SRC-1. We show that SRC-1 proteolysis is a proteasome- and ubiquitin-mediated process that, predominantly but not exclusively, occurs in the cytoplasmic compartment in which SRC-1 colocalizes with proteasome antigens as demonstrated by confocal imaging. Moreover, SRC-1 was stabilized in the presence of leptomycin B or several proteasomal inhibitors. Two degradation motifs, amino-acids 2-16 corresponding to a PEST motif and amino acids 41-136 located in the basic helix loop helix domain of the coactivator, were identified and shown to control the stability as well as the hormone-dependent down-regulation of the coactivator. SRC-1 degradation is of physiological importance because the two nondegradable mutants that still interacted with PR as demonstrated by coimmunoprecipitation failed to stimulate transcription of exogenous and endogenous target genes, suggesting that concomitant PR/SRC-1 ligand-dependent degradation is a necessary step for PR transactivation activity. Collectively our findings are consistent with the emerging role of proteasome-mediated proteolysis in the gene-regulating process and indicate that the ligand-dependent down-regulation of SRC-1 is critical for PR transcriptional activity.

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer.

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2ß1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.

Bioresponsive hyperbranched polymers for siRNA and miRNA delivery.

This work presents the novel use of reducible hyperbranched (rHB) polymers for delivery of RNA interference (RNAi) therapeutics. Cationic poly(amido amine) hyperbranched polymers that contain different contents of reducible disulfide to nonreducible linkages (0%, 17%, 25%, and 50%) were used to form interpolyelectrolyte polyplexes with siRNA and precursor miRNA (pre-miRNA). Atomic force microscopy (AFM) revealed rHB complexes of ∼100 nm in size, which exhibited redox-activated disassembly in the presence of dithiothreitol (DTT). The complexes were avidly internalized and showed no cellular toxicity in an endogenous enhanced green fluorescence protein (EGFP) expressing H1299 human lung cancer cell line. The highest specific EGFP gene silencing (∼75%) was achieved with rHB (17%)/siRNA complexes at a weight-to-weight (w/w) ratio of 40 that correlated with the ability for this polymer to successfully transfect pre-miRNA. Evaluation of temporal silencing levels over 72 h revealed incremental knockdown that reached a maximum at 72 h for the rHB (50%) complexes, in contrast to maximum knockdown at 24 h that remained relatively consistent, thereafter, for the rHB (17%), rHB (25%), and non-rHB complexes. The role of particle disassembly for intracellular targeting and modulation of gene silencing addressed in this work are important considerations in the development of this and other next-generation delivery systems.

The interval from the last cycle of docetaxel-based chemotherapy to progression is associated with the efficacy of subsequent docetaxel in patients with prostate cancer.

There is currently no standard treatment after first-line docetaxel-based chemotherapy for patients with castration-refractory prostate cancer (CRPC). Some patients are likely to discontinue first-line docetaxel-based chemotherapy because of either completed treatment or the occurrence of manageable side-effects. The aim of this study was to determine whether a rechallenge with docetaxel might be appropriate in patients with CRPC previously treated with docetaxel. Between December 2004 and July 2009, 39 patients diagnosed with metastatic cancer prostate at the Institut Gustave Roussy were administered subsequent docetaxel after front-line docetaxel-based chemotherapy. The medical records of these patients were extracted from the database. The PSA response rate (PSA decline > or =30% and > or =50%), progression-free survival (PFS) and overall survival (OS) of patients receiving docetaxel as a subsequent line of therapy were evaluated using consensus criteria. The effect of pre-treatment variables on efficacy was studied. A PSA decline > or =30% and > or =50% was observed in 64% and 38% of patients, respectively, median PFS was 4.3 months [confidence interval (CI) 95%: 3.6-4.9] and median OS was 15.8 months (CI 95%: 11.7-20.3) in 39 patients who received subsequent docetaxel. The interval between the last cycle of first-line docetaxel and progression [median: 3.0 months; range: 1-30 months] was associated with PFS: median PFS was 3.4 months (CI 95%: 2.6-4.1) and 6.3 months (CI 95%: 3.0-5.6), respectively, in patients with an interval <3.0 months and an interval > or =3.0 months, (p=0.04). Tolerance of re-treatment with docetaxel was acceptable with no toxicity-related death. Re-treatment with subsequent docetaxel in patients with CRPC pretreated with first-line docetaxel is safe and demonstrates some activity. The interval from the last cycle of first-line docetaxel-based chemotherapy to progression is associated with the efficacy of subsequent docetaxel.

E2F3 is a mediator of DNA damage-induced apoptosis.

The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.