Fibroblast growth factor signaling confers estrogen-independent and antiestrogen resistant in vitro and in vivo growth phenotypes to estrogen receptor positive (ER+) breast cancer cell lines. This occurs independently of estrogen receptor activation. Thus, activation of this signaling pathway may represent an underlying mechanism for the refractoriness of some ER+ tumors to primary and second line antihormonal therapies. A better understanding of the signaling pathway(s) that is activated in response to FGFs is needed to provide the conceptual basis for novel combination therapies that would abrogate autocrine growth factor signaling and thereby restore sensitivity to new """"""""pure"""""""" antiestrogens that are incapable of acting as agonists of ER. It might also increase the effectiveness of tamoxifen by providing new insights into possible predictive indicators capable of identifying those patients most likely to respond.
Three specific aims are proposed to dissect the autocrine or intracrine signaling pathways that are activated by FGF in ER+ breast cancer cells.
In aim one, pharmacologic inhibitors of signaling pathway intermediates will be used to determine if FGF dependent in vitro growth phenotypes can be specifically affected in a dose dependent manner. Transient transfection reporter assays will be used in combination with these inhibitors to explore which of the potential transcriptional targets of FGF signaling may be linked to growth.
In aim 2, the effect of dominant negative mutants of the Ras/Raf/MEK/MAPK, Rac/MEKK1/SEK/JNK and ras/PI3K/AKT signaling cascades on FGF dependent transcription and growth will be examined. Dominant negative mutants of Src, SHP-2, SNT-1, and ERK5/BMK, which appear to be critical components of growth factor signaling in other systems, will also be used. A tetracycline inducible expression system will be further developed to allow the assessment of the effect of controlled expression of dominant negative mutants on the tumorigenicity of FGF-1 overexpressing MCF-7 breast cancer cells in ovariectomized or antiestrogen treated nude mice.
In aim three, a ribozyme targeting strategy will be used to specifically and individually ablate the expression of each of the four members of the FGFR family present in MCF-7 breast cancer cells. Active ribozymes in constitutive and inducible expression vectors will be used to transfect FGF-1 overexpressing MCF-7 cells. Expression of individual FGFRs will be monitored and the effects of reduced or absent expression on FGF dependent in vitro and in vivo growth phenotypes and transcriptional activation will be assessed.
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