Geraldine Gueron

Critical role of endogenous heme oxygenase 1 as a tuner of the invasive potential of prostate cancer cells.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Inflammation has been recognized as a risk factor for this disease. Heme oxygenase 1 (HO-1), the inducible isoform of the rate-limiting enzyme in heme degradation, counteracts oxidative and inflammatory damage. Here, we investigated the regulated expression of HO-1 and its functional consequences in PCa. We studied the effect of genetic and pharmacologic disruption of HO-1 in the growth, invasion, and migration in androgen-sensitive (MDA PCa2b and LNCaP) and androgen-insensitive (PC3) PCa cell lines. Our results show that HO-1 levels are markedly decreased in PC3 compared with MDA PCa2b and LNCaP. Hemin treatment increased HO-1 at both protein and mRNA levels in all cell lines and decreased cell proliferation and invasion. Furthermore, overexpression of HO-1 in PC3 resulted in markedly reduced cell proliferation and migration. Accordingly, small interfering RNA–mediated silencing of HO-1 expression in MDA PCa2b cells resulted in increased proliferation and invasion. Using reverse transcription-quantitative PCR–generated gene array, a set of inflammatory and angiogenic genes were upregulated or downregulated in response to HO-1 overexpression identifying matrix metalloprotease 9 (MMP9) as a novel downstream target of HO-1. MMP9 production and activity was downregulated by HO-1 overexpression. Furthermore, PC3 cells stably transfected with HO-1 (PC3HO-1) and controls were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth and MMP9 expression was significantly reduced in PC3HO-1 tumors compared with control xenografts. Taken together, these results implicate HO-1 in PCa cell migration and proliferation suggesting its potential role as a therapeutic target in clinical settings. (Mol Cancer Res 2009;7(11):1745–55)

Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5β1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports a key role of HO-1 as a modulator of the angiogenic switch in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.

Advanced prostate cancer: reinforcing the strings between inflammation and the metastatic behavior.

It is currently estimated that inflammatory responses are linked to 15–20% of all deaths from cancer worldwide. Although many studies point to an important role of inflammation in prostate growth, the contribution of inflammation to castration-resistant prostate cancer is not completely understood. The presence of inflammatory mediators in tumor microenvironment raises the question whether genetic events that participate in cancer development and progression are responsible for the inflammatory milieu inside and surrounding tumors. Activated oncogenes, cytokines, chemokines and their receptors, sustained oxidative stress and antioxidant imbalance share the capacity to orchestrate these pro-inflammatory programs; however, the diversity of the inflammatory cell components will determine the final response in the prostate tissue. These observations give rise to the concept that early genetic events generate an inflammatory microenvironment promoting prostate cancer progression and creating a continuous loop that stimulates a more aggressive stage. It is imperative to dissect the molecular pathologic mechanism of inflammation involved in the generation of the castration-resistant phenotype in prostate cancer. Here, we present a hypothesis where molecular signaling triggered by inflammatory mediators may evolve in prostate cancer progression. Thus, treatment of chronic inflammation may represent an important therapeutic target in advanced prostate cancer.

Identification of new Rel/NF-kappaB regulatory networks by focused genome location analysis

NF-κB is an inducible transcription factor that controls kinetically complex patterns of gene expression. Several studies reveal multiple pathways linking NF-κB to the promotion and progression of various cancers. Despite extensive interest and characterization, many NF-κB controlled genes still remain to be identified. We used chromatin immunoprecipitation combined with microarray technology (ChIP/Chip) to investigate the dynamic interaction of NF-κB with the promoter regions of 100 genes known to be expressed in mitogen-induced T-cells. Six previously unrecognized NF-κB controlled genes (ATM, EP300, TGFβ, Selectin, MMP-1, and SFN) were identified. Each gene is induced in mitogen-stimulated T-cells, repressed by pharmacological NF-κB blockade, reduced in cells deficient in the p50 NF-κB subunit and dramatically repressed by RNAi specifically designed against cRel. A coregulatory role for Ets transcription factors in the expression of the NF-κB controlled genes was predicted by comparative promoter analysis and confirmed by ChIP and by functional disruption of Ets. NF-κB deficiency produces a deficit in ATM function and DNA repair indicating an active role for NF-κB in maintaining DNA integrity. These results define new potential targets and transcriptional networks governed by NF-κB and provide novel functional insights for the role of NF-κB in genomic stability, cell cycle control, cell-matrix and cell-cell interactions during tumor progression.

BRCA1 loss induces GADD153-mediated doxorubicin resistance in prostate cancer.

BRCA1 plays numerous roles in the regulation of genome integrity and chemoresistance. Although BRCA1 interaction with key proteins involved in DNA repair is well known, its role as a coregulator in the transcriptional response to DNA damage remains poorly understood. In this study, we show that BRCA1 plays a central role in the transcriptional response to genotoxic stress in prostate cancer. BRCA1 expression mediates apoptosis, cell-cycle arrest, and decreased viability in response to doxorubicin treatment. Xenograft studies using human prostate carcinoma PC3 cells show that BRCA1 depletion results in increased tumor growth. A focused survey of BRCA1-regulated genes in prostate carcinoma reveals that multiple regulators of genome stability and cell-cycle control, including BLM, FEN1, DDB2, H3F3B, BRCA2, CCNB2, MAD2L1, and GADD153, are direct transcriptional targets of BRCA1. Furthermore, we show that BRCA1 targets GADD153 promoter to increase its transcription in response to DNA damage. Finally, GADD153 depletion significantly abrogates BRCA1 influence on cell-cycle progression and cell death in response to doxorubicin treatment. These findings define a novel transcriptional pathway through which BRCA1 orchestrates cell fate decisions in response to genotoxic insults, and suggest that BRCA1 status should be considered for new chemotherapeutic treatment strategies in prostate cancer. Mol Cancer Res; 9(8); 1078–90. ©2011 AACR.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor-reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.

Combinatorial Antileukemic Disruption of Oxidative Homeostasis and Mitochondrial Stability by the Redox Reactive Thalidomide 2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) and Flavopiridol

2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a recently identified class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin-dependent kinases and also shows selective lethality against leukemic cells. The purpose of this study is to explore the efficacy and mechanism of action of the combinatorial use of the redox-reactive thalidomide CPS49 and the cyclin-dependent kinase inhibitor flavopiridol as a selective antileukemic therapeutic strategy. In combination, CPS49 and flavopiridol were found to induce selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. Highest synergy was observed at the level of ROS generation with a strong correlation between cell-specific cytotoxicity and reciprocal coupling of drug-induced ROS elevation with glutathione depletion. Examination of the transcriptional targeting of CPS49 and flavopiridol combinations reveals that the drugs act in concert to initiate a cell specific transcriptional program that manipulates nuclear factor-κB (NF-κB), E2F-1, and p73 activity to promote enhanced mitochondrial instability by simultaneously elevating the expression of the proapoptotic factors BAX, BAD, p73, and PUMA while depressing expression of the antiapoptotic genes MCL1, XIAP, BCL-xL, SURVIVIN, and MDM2. The coadministration of CPS49 and flavopiridol acts through coordinate targeting of transcriptional pathways that enforce selective mitochondrial dysfunction and ROS elevation and is therefore a promising new therapeutic combination that warrants further preclinical exploration.

Key Questions in Metastasis: New Insights in Molecular Pathways and Therapeutic Implications

The metastatic cascade and colonization remains a major challenge in clinical therapeutics. The formation of metastasis has many rate limiting steps. The expression of metastases initiation genes in primary tumors is driven by the need for cell motility, invasiveness, handling the shear stress in the vasculature and lymphatic circulation, and the survival and persistent growth in the distant organ. However, the expression of the progression genes in the primary tumors has a more complex basis. These metastasis-prone genes support primary tumor growth through one particular effect, whereas they enhance distant metastasis through another effect. The boundaries between metastasis initiation and metastasis progression genes are not rigid. In this review, we examine novel gene signatures identified in metastases, address key inflammatory factors mastering homing selection, gain further mechanistic insights into cell plasticity and evaluate the role of microRNAs. Moreover, we also describe the recent progress in developing nanoparticle imaging substantiating a promising theranostic platform for future cancer diagnostics and treatment, and assess the relevance of the bioinformatic analysis of metastasis-related proteins with an eye toward the metastatic niche. All these tools will provide valuable biological information of the progression of the disease, helping find potential therapeutic targets and improving surgical procedures. In a near future the understanding of the molecular mechanisms in tumor dissemination will be pivotal for the translation of these methods to the clinic and will help to overcome the barriers in clinical therapy of metastases.

Association of HO-1 and BRCA1 Is Critical for the Maintenance of Cellular Homeostasis in Prostate Cancer

Prostate cancer is the second leading cause of cancer-related death in men worldwide. Many factors that participate in the development of prostate cancer promote imbalance in the redox state of the cell. Accumulation of reactive oxygen species causes injury to cell structures, ultimately leading to cancer development. The antioxidant enzyme heme oxygenase 1 (HMOX1/HO-1) is responsible for the maintenance of the cellular homeostasis, playing a critical role in the oxidative stress and the regulation of prostate cancer development and progression. In the present study, the transcriptional regulation of HO-1 was investigated in prostate cancer. Interestingly, the tumor suppressor BRCA1 binds to the HO-1 promoter and modulates HO-1, inducing its protein levels through both the increment of its promoter activity and the induction of its transcriptional activation. In addition, in vitro and in vivo analyses show that BRCA1 also controls HO-1–negative targets: MMP9, uPA, and Cyclin D1. HO-1 transcriptional regulation is also modulated by oxidative and genotoxic agents. Induction of DNA damage by mitoxantrone and etoposide repressed HO-1 transcription, whereas hydrogen peroxide and doxorubicin induced its expression. Xenograft studies showed that HO-1 regulation by doxorubicin also occurs in vivo. Immunofluorescence analysis revealed that BRCA1 overexpression and/or doxorubicin exposure induced the cytoplasmic retention of HO-1. Finally, the transcription factor NRF2 cooperates with BRCA1 protein to activate HO-1 promoter activity. In summary, these results show that the activation of BRCA1–NRF2/HO-1 axis defines a new mechanism for the maintenance of the cellular homeostasis in prostate cancer. Implications: Oxidative and genotoxic stress converge on HO-1 transcriptional activity through the combined actions of BRCA1 and NRF2. Mol Cancer Res; 13(11); 1455–64. ©2015 AACR.

Improving risk stratification of patients with childhood acute lymphoblastic leukemia: Glutathione-S-Transferases polymorphisms are associated with increased risk of relapse.

The inclusion of genotype at Acute Lymphoblastic Leukemia (ALL) diagnosis as a genetic predictor of disease outcome is under constant study. However, results are inconclusive and seem to be population specific. We analyzed the predictive value of germline polymorphisms for childhood ALL relapse and survival. We retrospectively recruited 140 Argentine patients with de novo ALL. Genotypes were analyzed using PCR-RFLP (GSTP1 c.313A > G, MDR1 c.3435T > C, and MTHFR c.665C > T) and multiplex PCR (GSTT1 null, GSTM1 null). Patients with the GSTP1 c.313GG genotype had an increased risk for relapse in univariate (OR = 2.65, 95% CI = 1.03–6.82, p = 0.04) and multivariate (OR = 3.22, 95% CI = 1.17–8.83, p = 0.02) models. The combined genotype slightly increased risk for relapse in the univariate (OR = 2.82, 95% CI = 1.09–7.32, p = 0.03) and multivariate (OR = 2.98, 95% CI = 1.14–7.79, p = 0.03) models for patients with 2/3-risk-genotypes (GSTT1 null, GSTM1 null, GSTP1 c.313GG). The Recurrence-Free Survival (RFS) was shorter for GSTP1 c.313GG (p = 0.025) and 2/3-risk-genotypes (p = 0.021). GST polymorphisms increased the risk of relapse and RFS of patients with childhood ALL. The inclusion of these genetic markers in ALL treatment protocols might improve risk stratification and reduce the number of relapses and deaths.