As breast and ovarian carcinoma progress the tumor cells develop invasive structures, which provide the cells with a mechanism to cross tissue barriers and metastasize. Activation of the tyrosine kinase cSrc is known to occur in breast and ovarian cancers, will stimulate the formation of invasive structures, and promote invasion. Although cSrc is activated in these tumors, it is not mutated. Rather, cSrc is activated in response to input signals. One example of a cSrc-activating protein is AFAP-110, which has two known functions ? it cross-links actin filaments and can bind to and activate cSrc. AFAP-110 is a substrate of PKCa. Phosphorylation by PKCa results in a conformational change in AFAP-110 that releases 'autoinhibition'and enables it to efficiently cross-link stress filaments and direct cSrc activation. This signaling cascade results in the formation of invasive structures. The question to be addressed in this application is, 'can increased expression of AFAP-110 in mouse breast promote cSrc activation, and if so, how?' Our preliminary data indicate that knockdown of AFAP-110 in breast cancer cells results in reduced cell adhesion. This effect is linked with reduced stress filament cross-linking and decreased invasive potential. Upon MDA-231 cell adhesion to fibronectin, AFAP-110 becomes phosphorylated on Tyr94, a Src target. Thus, in Aim 1, we hypothesize that AFAP-110 promotes cell adhesion by activating cSrc. We also find that the ability of AFAP-110 to activate cSrc requires that the PH1 domain bind to phosphatidic acid (PA). There are two grooves in the PH1 domain that can bind phospholipids.
In Aim 2, we predict that binding of PA and PtdIns4P are required for AFAP-110 to activate cSrc and that they bind to separate grooves and work cooperatively to dock AFAP-110 on membranes and subsequently activated cSrc. Interestingly, we identified a polymorphic variant of AFAP-110 that has a nonsynomous SNP in the carboxy terminal pleckstrin homology (PH2) domain. This Ser403?Cys403 change enables AFAP-110403C to independently activate cSrc, but only under conditions of overexpression. We hypothesize that the Cys403 SNP may result in a less efficiently stabilized AFAP-110 multimer, which could weaken autoinhibition and result in cSrc activation.
In Aim 3, we will determine how the PH2 domain fosters self-association and if mutations in the opposing PH2 binding site within AFAP-110 also destabilize the multimer and enable cSrc activation. Lastly, we find that AFAP-110 and cSrc are overexpressed together in the same cells in breast and ovarian cancer tissues. We hypothesize that AFAP-110 has the capacity to activate cSrc in breast tissue.
In Aim 4, we will create a transgenic mouse and determine if, under conditions of overexpression in mouse mammary tissue, AFAP-110 has the potential to activate cSrc. The significance of these studies is that AFAP-110 may be one protein responsible for activating cSrc. Thus, it may be a legitimate drug target, with a 'drug-able'site in the PH1 domain that could be targeted to disrupt cSrc activation and invasive potential.
This project is relevant to breast cancer in that it will address a mechanism by which cSrc becomes activated in breast cancer. This will be the first time anyone has attempted to determine if cSrc activating proteins (AFAP-110) can direct cSrc activation in mouse breast tissues. As AFAP-110 has a phospholipid binding pocket that appears to be crucial for this function, it may be a drug-able target.
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