Belen Elguero

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.

Low doses of CPS49 and flavopiridol combination as potential treatment for advanced prostate cancer.

Prostate cancer (PCa) still ranks as the second most frequently diagnosed cancer and metastatic castration resistant prostate cancer (CRPC) is a foremost cause of men cancer death around the world. The aim of this work was to investigate the selectivity and efficacy of new drug combinations for CRPC. We combined three compounds: paclitaxel (PTX: taxane that inhibits microtubule polymerization); 2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole- 1,3(2H)-dione (CPS49; redox-reactive thalidomide analog with anti-angiogenic properties) and flavopiridol (flavo: semisynthetic flavonoid that inhibits cyclin dependent kinases). We assessed CPS49-flavo or -PTX combinations cytotoxicity in a panel of PCa cell lines and PC3 xenografts. We found that CPS49 enhanced flavo or PTX cytotoxicity in human PCa cell lines while showed resistance in a non-tumor cell line. Furthermore, xenografts generated by inoculation of human prostate carcinoma PC3 cells in nu/nu mice showed that CPS49/flavo administration reduced tumor growth both after 2 weeks of co-treatment and after 1 week of pretreatment with a low dose of flavo followed by 2 weeks of co-treatment. PTX and CPS49 combination did not significantly reduce tumor growth in PC3 xenografts. Histological analysis of xenograft PC3 tumor samples from CPS49/flavo combination showed extensive areas of necrosis induced by the treatment. RT-qPCR array containing 23 genes from PC3 cells or PC3 xenografts exposed to CPS49/flavo combination showed that this treatment shut down the expression of several genes involved in adhesion, migration or invasion. In summary, the antitumor activity of CPS49 or flavopiridol was improved by the combination of these compounds and using half dose of that previously reported. Hence, CPS49-flavo combination is a promising new alternative for PCa therapy.

Abstract 207: Rearrangements of the E-cadherin/B-catenin-based adherens junctions caused by forced-expression of heme oxygenase 1 (HO-1) in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Prostatic tumor cell plasticity involves cooperative changes in the interactions between tumoral cells. The stable E-cadherin-based Adherence Junctions (AJs) play a pivotal role in the integrity of the epithelium and the maintenance of tissue homeostasis. Heme oxygenase-1 (HO-1) acts as a cell rheostat counteracting oxidative and inflammatory damage. Given that inflammation is critical for the development and progression of PCa, we sought to determine whether HO-1 could regulate the adhesive properties of PCa cells, towards the acquisition of an epithelial-like phenotype. We previously showed that HO-1 over-expression impairs tumor growth and represses angiogenesis in vivo. Here we demonstrate that HO-1 over-expression significantly increased PCa cell adhesion, remodeling the AJs. To better understand the nature of alterations of cell-cell interactions during neoplastic evolution, we examined the expression levels of E-cadherin and B-catenin. Treatment with hemin, a potent and specific inducer of HO-1, increased E-cadherin and b-catenin protein and mRNA levels in PCa cell lines, shown by Western Blot, RT-qPCR and flow cytometry analyses. These epithelial markers were also significantly induced when PCa cells were stably transfected with HO-1. Immunofluorescence, confocal microscopy together with a three-dimensional (3D) acquisition process leading to the collection of image stacks, confirmed the augmented levels of these proteins under forced-expression of HO-1 and also revealed a striking rearrangement of their localization pattern towards the cell membrane. Moreover, quantitative image analysis detected a significant increase in the percentage of cell-to-cell contact among tumoral cells under HO-1 overexpression. The enhanced levels of E-cadherin and B-catenin in PCa cells under hemin treatment coincided with a markedly different morphology compared to untreated cells, observed by bright field microscopy. While untreated cells displayed an elongated and fibroblastic-like shape, with increased number of lamellipodia, HO-1-overexpressing cells showed a spherical cobblestone epithelial shape. These results define a novel role for HO-1 in modulating the cellular morphology and the architecture of cell-to-cell interactions, favoring a less aggressive phenotype and further supporting its anti-tumoral function in PCa. Citation Format: Geraldine Gueron, Belen Elguero, Alejandra Paez, Jimena Giudice, Martin Toscani, Felipe Jaworski, Daiana Leonardi, Federico Coluccio-Leskow, Adriana De Siervi, Nora Navone, Elba Vazquez. Rearrangements of the E-cadherin/B-catenin-based adherens junctions caused by forced-expression of heme oxygenase 1 (HO-1) in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 207. doi:10.1158/1538-7445.AM2013-207

Abstract 2340: CPS49 and Flavopiridol: a new selective drug combination for advanced prostate cancer

Prostate cancer (PCa) still ranks as the second most commonly diagnosed cancer and metastatic castration-resistant prostate cancer (CRPC) is a leading cause of cancer death in men around the world. The common treatment for patients with CRPC is chemotherapy based on docetaxel. While there are currently seven agents approved for CRPC and four regimens have shown some survival benefit, those survival prolongations have been modest and unfortunately all patients will eventually progress. Thus, there is a need for new agents and regimens for this disease. 2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a 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 provokes lethality against leukemic cells. We previously found that CPS49 and flavopiridol combination induced selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. The goal of this study was to investigate the selectivity and efficacy of CPS49-flavopiridol combination in prostate cancer preclinical models. Our results showed that flavopiridol enhanced CPS49 cytotoxicity in all human prostate tumor cell lines analyzed (PC3, C4-2, LNCaP and 22RV1); however non tumor cell lines (293HEK and MCF10) were resistant to the tested doses. Furthermore, it was previously reported, high doses of flavopiridol (10 μM) or CPS49 (12 μM) were needed to inhibit tumor growth in PC3 xenograft mice compared with vehicle treated mice. Here, we demonstrated that combining these two agents, antitumor activity was synergistically enhanced with low doses. Injecting subcutaneously PC3 cells in nu/nu mice, we found that CPS49-flavopiridol administration reduced tumor volume approximately 83% after 2 weeks of co-treatment and 54% after 1 week of low dose flavopiridol pretreatment and 2 weeks of drug combination. In addition, we performed RT-qPCR array containing 26 genes from PC3 cells exposed to CPS49 and flavopiridol combination. We determined that this treatment shut down the expression of several genes involved in cell cycle, DNA damage and tumor progression. Histological analysis of xenograft PC3 tumor samples showed extensive areas of necrosis induced by the treatment. Furthermore, we assessed the efficacy of CPS49 in combination with paclitaxel (docetaxel analog). All the prostate tumor cell lines tested were highly sensitive to this combination. However, this combination did not reduce the tumor volume in PC3 xenografts. These results indicate that the CPS49 and flavopiridol is a promising new alternative for the treatment of CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2340. doi:1538-7445.AM2012-2340

Abstract 5260: Heme oxygenase-1 is involved in prostate cancer cells-bone interaction

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prostate cancer metastasis is well characterized by its tropism to the bone with metastatic lesions being typically osteoblastic. Osteoblasts produce factors that promote growth and survival of prostate cancer cells, but the molecular nature of this interaction is still unknown. Previous reports from our laboratory documented that heme oxygenase-1 (HO-1) is expressed in primary prostate carcinomas and is localized in the nucleus. Recently, the involvement of HO-1 in bone metabolism has been reported. The aim of this study is to investigate the role of HO-1 in the interaction between prostate cancer cells and bone. We analyzed the expression of HO-1 by immunohistochemistry using a tissue microarray generated from 12 castrate-resistant prostate cancer specimens (MDA Anderson Cancer Center), growing as xenografts in male CB17 SCID wild or castrated mice. Each of these tumors led to clinically relevant biological models to study human prostate cancer progression. All samples showed cytoplasmic HO-1 staining of varying intensity. Intense positive nuclear staining was also detected in 3/5 tumors with neuroendocrine morphology. HO-1 expression was correlated with other markers such as PSA, AR and Ki-67 expression. Co-cultures of the osteolytic PC3 cancer cell line with PMO (primary mice osteoblasts) demonstrated that HO-1 induction in PC3 cells abolished the PC3-induced loss of osteoblasts and significantly increased Dickkopf-1 expression in tumor cells. Furthermore, using PC3-PMO co-cultures, we found that HO-1 induction in prostate cancer cells increased mRNA levels of Runx-2 and Cyclin D1 and diminished β-catenin protein expression in PMO. These results clearly indicate the involvement of HO-1 in the interaction between prostate cancer cells and bone. The study of the molecular mechanisms of this interaction will provide effective therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5260. doi:10.1158/1538-7445.AM2011-5260

Abstract LB-245: Nuclear translocation of HO-1 in prostate cancer. Role beyond heme degradation

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. The metastatic spread of PCa cells and the ability to survive when reaching the metastatic nitch is affected by growth factors, chemokines, angiogenic factors and hormones. Heme oxygenase-1 (HO-1), the inducible isoform of the rate-limiting enzyme in heme degradation, counteracts oxidative and inflammatory damage. Here we investigated its nuclear translocation in androgen sensitive (MDAPCa2b and LNCaP) and insensitive (PC3) PCa cell lines. Our results show that endogenous levels of HO-1 are markedly lower in PC3 compared to MDAPCa2b and LNCaP. Hemin treatment, a potent and specific inducer of HO-1, increased protein levels with a resulting nuclear translocation in PCa cell lines. Stable transfection of HO-1 in PC3 (PC3HO-1) also showed a significant nuclear translocation. Furthermore, human prostate cancer PC3HO-1 cells growing subcutaneously in athymic nude mice showed significant HO-1 nuclear staining (detected by immunohistochemsitry and immunofluorescence) compared to PC3pcDNA3 xenografts. Using RT-qPCR-generated gene array we observed a set of inflammatory and angiogenic genes up- or down-regulated in response to HO-1 overexpression, identifying VEGFA as a novel downstream target of HO-1. HO-1 modulation limited the metastatic potential of neoplastic cells by down-regulating VEGFA production. An in vivo angiogenic assay also demonstrated that PC3HO-1 tumors presented less neovascularization than tumors derived from control cells, giving further evidence to support HO-1 anti-angiogenic function. These results implicate for the first time HO-1 in the regulation of key proinflammatory factors, involved in PCa progression. Taken together our results suggest that HO-1 may be a potential target for therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-245.