HEME OXYGENASE-1: A RHEOSTAT OF TUMORAL GROWTH IN PROSTATE CANCER. SPATIOTEMPORAL REGULATION AND FUNCTIONAL SIGNIFICANCE

ELBA VAZQUEZ

Mar del Plata

Congreso; LVII REUNION CIENTIFICA DE LA SOCIEDAD ARGENTINA DE INVESTIGACIONES CLINICAS; 2012

Sociedad Argentina de Investigación ( SAIC)

HEME OXYGENASE-1: A RHEOSTAT OF TUMORAL GROWTH IN PROSTATE CANCER. SPATIOTEMPORAL REGULATION AND FUNCTIONAL SIGNIFICANCE Elba Vazquez – Departamento de Química Biológica, FCEN – UBA, IQUIBICEN - CONICET Prostate cancer (PCa) is a leading cause of death among males. It is currently estimated that inflammatory responses are linked to 15-20% of all deaths from cancer worldwide. The presence of inflammatory mediators in tumor microenvironment, epidemiologically unrelated to inflammation, raises the question whether genetic events that participate in cancer development and progression are responsible of the inflammatory milieu inside and surrounding tumors. Hence, it is imperative to dissect the molecular pathologic mechanism of inflammation involved in prostate cancer. Heme Oxygenase 1 (HO-1) the inducible isoform of the rate-limiting enzyme in heme degradation, counteracts oxidative damage and inflammation. Therefore modulation of HO-1 expression may play a pivotal role in this disease. We previously documented the nuclear expression of HO-1 in human primary prostate carcinomas naïve of treatment. We also showed that HO-1 is implicated in PCa, demonstrating that endogenous HO-1 inhibits bone derived-prostate cancer cell migration, invasion and proliferation. MMP9 was identified as a novel downstream target of HO-1. Moreover, a striking finding of this study came from the in vivo experiments. Tumor growth receded in mice bearing HO-1 overexpressing tumors (PC3HO-1) compared to controls. In addition, other mechanisms including a direct effect on angiogenic factors may account for the HO-1 anti-tumoral role described above. Clinical studies reported that high VEGF levels in PCa are associated with poor prognosis and that plasma levels of VEGF increase with PCa progression to skeletal metastases. In PCa tumors, the switch to an angiogenic phenotype is known to be critical for its progression. Although it is well accepted that PCa dissemination and its capacity to survive in a distant organ is affected by angiogenic factors, the earliest molecular events that dictate the angiogenic switch remain elusive in PCa, probably because they occur before a clinical diagnosis can be established. To further assess HO-1 properties, we investigated its potentiality to modulate PCa associated-angiogenesis. 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 also indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. Moreover, an in vivo angiogenic assay showed that intradermal inoculation of the PC3HO-1 cell line generated tumors less vascularized 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. Considering the cross talk between inflammation, angiogenesis and cancer progression, our next step sought to identify a signaling pathway by which HO-1 could be operating, with an eye toward the NFκB axis. Of note, NFκB stimulates angiogenic genes, it is constitutively activated in PCa, it correlates with disease progression and its blockade results in decreased angiogenesis in several PCa models. Our results show that HO-1 down-modulated the NFB signaling axis, repressing NF-κB-mediated transcription from an NFκB responsive luciferase reporter construct, up-regulating IκB and down regulating the IKK, strongly suggesting that HO-1 may modulate angiogenesis through this signaling cascade. Moreover, STAT3 is a downstream target of NFκB in several angiogenic-signaling pathways in cancer. Activation of the androgen receptor (AR) is a key step in the development of PCa and several mechanisms have been identified in AR activation, among them STAT3 signaling. Hence, disruption of STAT3 activity has been associated to cancer progression. We then evaluated a further effect of HO-1 on STAT3 signaling. Our data demonstrated for the first time that HO-1 induction represses PSA transcription in PCa cells, indicating that HO-1 down-modulates AR signaling. By co-immunoprecipitation we showed that HO-1 and STAT3 interact and forced-expression of HO-1 increased STAT3 cytoplasmic retention. Confocal microscopy images exhibited a decreased rate of AR/STAT3 nuclear co-localization under hemin treatment, a potent HO-1 inducer. Interestingly, in vivo studies confirmed that STAT3 nuclear delimitation was significantly decreased in PC3HO-1 tumors. These results provide a novel function for HO-1 down-modulating AR transcriptional activity in PCa, interfering with NFκB/STAT3 signaling, evidencing its role beyond heme degradation. Altogether, these data support a key anti-tumoral role of HO-1 in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.