Josep Domingo-Domenech

Targeting cancer stem cells to suppress acquired chemotherapy resistance

Acquired resistance has curtailed cancer survival since the dawn of the chemotherapy age more than half a century ago. Although the application of stem cell (SC) concepts to cancer captured the imagination of scientists for many years, only the last decade has yielded substantial evidence that cancer SCs (CSCs) contribute to chemotherapy resistance. Recent studies suggest that the functional and molecular properties of CSCs constitute therapeutic opportunities to improve the efficacy of chemotherapy. Here we review how these properties have stimulated combination strategies that suppress acquired resistance across a spectrum of malignancies. The clinical implementation of these strategies promises to rejuvenate the effort against an enduring challenge.

Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression.

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.

Predictive Value of Microtubule Associated Proteins Tau and Stathmin in Patients With Nonmuscle Invasive Bladder Cancer Receiving Adjuvant Intravesical Taxane Therapy

PURPOSE After encouraging results from 2 clinical trials performed at our institution to test intravesical taxane based chemotherapy for bacillus Calmette-Guérin refractory, nonmuscle invasive bladder cancer we designed a study to identify molecular markers linked to the optimal response to such treatment modality. MATERIALS AND METHODS Included in the institutional review board approved study were 32 patients with nonmuscle invasive, bacillus Calmette-Guérin refractory bladder cancer who received intravesical taxane chemotherapy, that is docetaxel or nanoparticle albumin-bound paclitaxel. Immunophenotype analysis on tissue samples obtained before intravesical taxane therapy was done using a panel of molecular markers, including Ki-67, p53, and the microtubule associated proteins tau and stathmin. RESULTS Increased total tau (cytoplasmic and nuclear) and stathmin expression before intravesical taxane therapy was significantly associated with decreased recurrence-free survival (p <0.0001 and 0.007, respectively). A tau positive phenotype was an independent prognostic factor for recurrence-free survival on multivariate analysis (HR 15.66, 95% CI 2.68-91.71, p = 0.002). Neither the proliferation index assessed by Ki-67 expression nor p53 status was significantly associated with recurrence-free survival. CONCLUSIONS Assessment of tau and stathmin protein expression should be considered to select patients before intravesical taxane based chemotherapy for nonmuscle invasive, bacillus Calmette-Guérin refractory bladder cancer since those who have tumors with low tau/stathmin protein expression show a better response to therapy.

A Common MicroRNA Signature Consisting of miR-133a, miR-139-3p, and miR-142-3p Clusters Bladder Carcinoma in Situ with Normal Umbrella Cells

miRNAs are small noncoding RNAs with critical roles in a large variety of biological processes such as development and tumorigenesis. miRNA expression profiling has been reported to be a powerful tool to classify tissue samples, including cancers, based on their developmental lineage. In this study, we have profiled the expression of miRNAs in bladder carcinoma in situ (CIS) and distinct cell compartments of the normal bladder, namely umbrella and basal-intermediate urothelial cells, as well as the muscularis propria. We identified several miRNAs differentially expressed between umbrella and basal-intermediate cells (miR-133a, miR-139-3p, miR-142-3p, miR-199b-5p, and miR-221). In situ hybridization confirmed the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. Strikingly, miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells. Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Again, this analysis revealed the significant expression of umbrella-specific markers in CIS when compared to non-CIS lesions. Overall, our studies represent a comprehensive and accurate description of the different miRNAs expressed in CIS tumors and three distinct histological areas of the urinary bladder. Notably, this study provides evidence of the possible origin relationship between CIS and normal umbrella cells.

PAX7-FKHR fusion gene inhibits myogenic differentiation via NF-kappaB upregulation

ObjectiveAlveolar rhabdomyosarcomas (ARMS) are characterised by a PAX3/7-FKHR translocation, which is presumed to promote a differentiation arrest in the myogenic lineage, in which setting secondary genetic events occur, resulting in sarcomagenesis. The aim of this study was to identify the mechanism by which PAX3/7-FKHR expression results in a myogenic differentiation block, as discrete from the secondary genetic events that complete the sarcomagenic process.MethodsWe performed a novel differential gene expression analysis comparing normal mesenchymal stem cells with previously generated non-tumorigenic mesenchymal stem cells expressing the PAX7-FKHR fusion gene, as well as with a known tumorigenic, PAX7-FKHR-expressing ARMS cell line, CW9019.ResultsThis novel analysis uncovered the upregulation of the NF-kappaB pathway as a function of PAX3/7-FKHR expression, but distinct from the secondary sarcomagenic process; thus implicating NF-kappaB as a mediator of the PAX3/7-FKHR differentiation block. We further show that NF-kappaB activity is upregulated in PAX7-FKHR cells when compared to parental MSCs due to upregulation of the PI3K/AKT pathway. In addition we show that NF-kappaB inhibits myogenesis via activation of cyclinD1/cdk4 complexes, which sequester MyoD1, a key myogenic transcription factor.ConclusionsOur results highlight the importance of the NF-kappaB pathway in myogenesis and sarcomagenesis and suggest that this pathway may be one of the potential therapeutic targets in the treatment of ARMS.

Phase 2 Trial of Gemcitabine, Cisplatin, plus Ipilimumab in Patients with Metastatic Urothelial Cancer and Impact of DNA Damage Response Gene Mutations on Outcomes

BACKGROUND Chemotherapy may exert immunomodulatory effects, thereby combining favorably with the immune checkpoint blockade. The pharmacodynamic effects of such combinations, and potential predictive biomarkers, remain unexplored. OBJECTIVE To determine the safety, efficacy, and immunomodulatory effects of gemcitabine and cisplatin (GC) plus ipilimumab and explore the impact of somatic DNA damage response gene alterations on antitumor activity. DESIGN, SETTING, AND PARTICIPANTS Multicenter single arm phase 2 study enrolling 36 chemotherapy-naïve patients with metastatic urothelial cancer. Peripheral blood flow cytometry was performed serially on all patients and whole exome sequencing of archival tumor tissue was performed on 28/36 patients. INTERVENTION Two cycles of GC followed by four cycles of GC plus ipilimumab. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The primary endpoint was 1-yr overall survival (OS). Secondary endpoints included safety, objective response rate, and progression-free survival. RESULTS AND LIMITATIONS Grade ≥3 adverse events occurred in 81% of patients, the majority of which were hematologic. The objective response rate was 69% and 1-yr OS was 61% (lower bound 90% confidence interval: 51%). On exploratory analysis, there were no significant changes in the composition and frequency of circulating immune cells after GC alone. However, there was a significant expansion of circulating CD4 cells with the addition of ipilimumab which correlated with improved survival. The response rate was significantly higher in patients with deleterious somatic DNA damage response mutations (sensitivity=47.6%, specificity=100%, positive predictive value=100%, and negative predictive value=38.9%). Limitations are related to the sample size and single-arm design. CONCLUSIONS GC+ipilimumab did not achieve the primary endpoint of a lower bound of the 90% confidence interval for 1-yr OS of >60%. However, within the context of a small single-arm trial, the results may inform current approaches combining chemotherapy plus immunotherapy from the standpoint of feasibility, appropriate cytotoxic backbones, and potential predictive biomarkers. TRIAL REGISTRATION ClinicalTrials.gov NCT01524991. PATIENT SUMMARY Combining chemotherapy and immune checkpoint blockade in patients with metastatic urothelial cancer is feasible. Further studies are needed to refine optimal combinations and evaluate tests that might identify patients most likely to benefit.

The role of GATA2 in lethal prostate cancer aggressiveness

Advanced prostate cancer is a classic example of the intractability and consequent lethality that characterizes metastatic carcinomas. Novel treatments have improved the survival of men with prostate cancer; however, advanced prostate cancer invariably becomes resistant to these therapies and ultimately progresses to a lethal metastatic stage. Consequently, detailed knowledge of the molecular mechanisms that control prostate cancer cell survival and progression towards this lethal stage of disease will benefit the development of new therapeutics. The transcription factor endothelial transcription factor GATA-2 (GATA2) has been reported to have a key role in driving prostate cancer aggressiveness. In addition to being a pioneer transcription factor that increases androgen receptor (AR) binding and activity, GATA2 regulates a core subset of clinically relevant genes in an AR-independent manner. Functionally, GATA2 overexpression in prostate cancer increases cellular motility and invasiveness, proliferation, tumorigenicity, and resistance to standard therapies. Thus, GATA2 has a multifaceted function in prostate cancer aggressiveness and is a highly attractive target in the development of novel treatments against lethal prostate cancer.

Isolation of cancer stem cells from human prostate cancer samples.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

Nuclear Pores Promote Lethal Prostate Cancer by Increasing POM121-Driven E2F1, MYC, and AR Nuclear Import

Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.