Cancer incidence is rising fast in Qatar, including in particular breast cancer. The expression of the oncogenic protein HER-2/neu is associated with an aggressive phenotype, and agents targeting this molecule have been successful in prolonging survival of breast cancer patients. Despite treatment progresses, metastatic breast cancer is still a fatal condition, and mechanisms of HER-2/neu regulation/resistance are not fully explained. Therefore a better knowledge of the dynamic of HER-2/neu regulation would enable to define new generation targeted therapy agents. The cAMP responsive element-binding protein (CREB) is involved in tumor initiation as well as tumor progression, but a link between HER-2/neu-induced transformation, CREB activation and alterations of the tumor micromilieu including hypoxia-mediated increased angiogenesis has not yet been established. Therefore, the effect of CREB activation on angiogenesis and its modulation by hypoxia was analysed in CREBhigh and CREBlow HER-2/neu+ murine fibroblasts in vitro as well as in vivo. Human tumor cells transfected with HER-2/neu or a mutant deficient in HER-2/neu signalling served as controls. By determination the number and density of blood vessels, necrosis and hypoxic areas a decreased tumorigenicity coupled with a reduced angiogenesis, but increased necrotic cells and hypoxic areas was found upon injection of CREBlow vs. CREBhigh HER-2/neu-transformed cells. In vitro, a time-dependent increase of pCREBSer133, but not of pCREBSer121 in the presence of hypoxia was detected, which was dependent on HER-2/neu signalling and accompanied by an upregulation of HIF-1α, GLUT1 and VEGF as well as of cell migration and invasion mediated by an increased expression of metalloproteinases. Under normoxic conditions, CREBhigh, but not CREBlow HER-2/neu+ cells showed a low intracellular pH, which was restored by CREB silencing. This was at least partially increased in the presence of hypoxia and correlated with an upregulation of lactate in the cellular supernatant of all cells. Furthermore, a hypoxia-mediated post-transcriptional modification due to an increased ubiquitination of CREB and altered cellular distribution of CREB was found. Under hypoxia CREB was translocated into mitochondria, which was accompanied by an altered expression of mitochondrial genes and enhanced apoptosis sensitivity. Thus, HER-2/neu-mediated CREB activation and localization is modulated by the hypoxic and acidic tumor microenvironment, thereby affecting the tumorigenic phenotype. In conclusion a link between HER-2/neu-mediated CREB activation and changes in the microenvironment and CREB localization was found, which contributes to the pathogenesis of HER-2/neu-expressing tumors, including mammary carcinoma. HER-2/neu-mediated CREB activation stimulates angiogenesis and hypoxia and is increased during hypoxia thereby causing translocation of CREB to the mitochondria, which enhances the malignant phenotype. Thus, targeting molecules downstream of HER-2/neu, like CREB, appear to be promising targets for the development of novel innovative therapies of HER-2/neu overexpressing tumors.


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