It is now certain that oncogenic signaling and cytoskeleton function are directly involved in breast cancer progression. Our laboratory has determined that overexpression of CD44 variant (CD44v) isoforms [hyaluronan (HA) receptors] appears to confer the malignant properties of increased growth, migration and invasion on breast epithelial cells. In this continuation research proposal, we plan to test the hypothesis that the interaction between CD44v isoforms (CD44v3, CD44v10 and CD44v6/7) and specific signaling activators [e.g. RhoGEFs/RacGEF (Tiam1) and c-Src kinase] plays an important role in HA-mediated oncogenic signaling such as the RhoA-activated ROK pathway, Tiam1-regulated Rac1-PKNgamma kinase pathway and c-Src-induced cortactin and Gab-1/PI3 kinase-AKT pathways. These activation events induce various tumor cell-specific behaviors (e.g. cellular acidification, ECM degradation, cytoskeleton function, tumor cell growth/survival, migration and invasion) leading to breast cancer progression. To test this hypothesis, we plan to use a variety of biochemical, molecular biological techniques and immunohistochemical staining to elucidate HA-CD44v isoform interaction with the various signaling molecules. We will evaluate the functional ramifications of these interactions with respect to specific downstream effector functions (e.g. NHE1-related acidic pH enzyme activation, ankyrin/cortactin-actin binding and PI3 kinase/AKT-mediated biological activities) required for ECM degradation, cytoskeleton function and metastatic tumor cell properties. In addition, we plan to analyze the co-expression of CD44v isoforms and various signaling molecules (e.g. RhoGEF, Tiam1, ROK, PKNgamma and c-Src) in human breast carcinoma tissues obtained from breast cancer patients using immunocytochemistry. We will also employ novel signaling perturbation strategies to impair HA-CD44v isoform-mediated oncogenic signaling by constructing dominant-negative mutants of certain signaling molecules (e.g. ROK, PKNgamma, c-Src, and cortactin). Finally, we will develop therapeutic antisense and small interference RNAs (siRNAs) specifically targeting CD44 in order to effectively block CD44 expression, inhibit HA-mediated downstream oncogenic signaling events and block breast tumor progression. We believe that the information obtained from this proposal has particularly important clinical utility and could establish CD44v isoforms and associated signaling molecules (e.g. RhoGEF, Tiam1, ROK, PKNgamma and c-Src) as important tumor markers for early detection and evaluation of oncogenic potential, as well as allow the development of new drug targets to inhibit breast tumor metastasis and cancer progression. ? ? ? ? ?
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