In this continuation research proposal, we plan to test the hypothesis that transmembrane interaction between CD44 variants (CD44V) and the cytoskeletal protein, ankyrin, plays an important role in promoting onocongenic [e.g. Tiam1-catalyzed RhoA activation and Rho-Kinase (ROK)-mediated cytoskeleton function] leading to tumor cell adhesion growth. invasion and migration during breast cancer progression. The results of this research will definitely provide a better understanding of the cellular and molecular mechanisms involved in human breast cancer metastasis and progression. Specifically, we plan to use a variety of biochemical, cell biological and molecular biological techniques to elucidate the binding site diversity on the cytoskeletal protein, ankyrin, required for CD44 variant (CD44V) association. An emphasis will be placed on the structural and functional relationships between the CD44V isoforms and the ankyrin repeat domain (ARD) in metastatic breast tumor cells. We will also explore a new CD44V and ankyrin-linked oncogenic signaling pathway with a special focus on the guanine nucleotide exchange factor, Tiam1 (T lymphoma invasion and metastasis)-catalyzed RhoA signaling and Rho-Kinase (ROK) activation in metastatic breast tumor cells. In addition, we will employ a novel signaling perturbation strategy to impair Tiam1-RhoA signaling and/or ROK-specific cytoskeleton function by first constructing various Tiam1 and/or ROK deletion and site-directed mutants lacking specific functional domains (e.g. dominant negative mutants). These dominant-negative Tiam1 and/or ROK mutant polypeptides will then be expressed in metastatic breast epithelial cells to test their ability to downregulate CD44V isoform-mediated metastatic tumor cell behaviors (e.g. tumor cell adhesion growth, migration and invasion). In the second part of the proposal, we will use immunohistochemistry to analyze the coexpression of CD44 variants (e.g. CD44V3 and CD44V10-containing isoforms) with ankyrin and certain oncogenic signaling molecules (e.g. Tiam1 or ROK) in human breast carcinomas in order to establish useful structure/function-related markers for breast cancer detection and prognosis. Finally, we plan to establish a therapeutic antisense strategy to specifically inhibit the mRNA encoding for certain CD44 isoforms (e.g. CD44V3/V10) in order to effectively block the expression of CD44V isoforms and their downstream oncogenic signaling events which lead to metastatic breast tumor progression. We believe the results of these proposed experiments will provide a much better understanding of the CD44V isoform interaction with ankyrin which regulates Tiam1-RhoA signaling and ROK activation required for cytoskeleton function and various metastatic tumor cell properties (e.g. tumor cell adhesion, growth, invasion and migration). The information obtained from this proposal may establish the coexpression of CD44V (e.g. CD44V3/CD44V 10-containing isoforms) with ankyrin and certain oncogenic signaling molecules (e.g. Tiam1 or ROK) as an important tumor marker for early detection and evaluation of oncogenic potential. Most importantly, our proposed new strategy using a novel Tiam1/ROK-related signaling perturbation, and specific antisense constructs of CD44V3/V10 isoforms together with certain oncogenic molecules, may identify new drug targets for the inhibition of breast tumor metastasis and cancer progression.
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