Two types of alterations of SYK expression are consistently associated with breast malignancy: (a) In a subset of breast cancers, SYK expression is lost due to aberrant SYK gene promoter hypermethylation that leads to silencing of SYK expression. The loss of expression is associated with increased invasiveness of breast cancer cells. (B) Altered SYK expression is also reflected by the addition of short form Syk [Syk(S)] in breast tumors in contrast to only the long form Syk [Syk(L)] expression in normal mammary tissues. Syk(S) is generated by alternative splicing that results in an in-frame deletion of 23 residues within the interdomain B (IDB) of Syk. Expression of Syk(L) but not Syk(S) inhibits tumor cell invasion and tumorigenicity. The tyrosine kinase activity of Syk is responsible for the invasion suppression function of Syk(L). Consistent with their differential effects on invasion, Syk(L) but not Syk(S) is able to induce tyrosine phosphorylation in breast cancer cells. These studies lead us to postulate that the downstream phosphorylation targets for Syk(L) mediate Syk(L) anti-invasion functions. Also, the inability of Syk(S) to phosphorylate in vivo targets despite an intact kinase domain suggests a critical role of IDB in mediating the process of phosphorylation by Syk(L). In this proposal, we will define the biological roles of Syk in control of tumor-associated phenotypes. A potential oncogenic activity of Syk(S) will also be assessed (Aim 1.1). We will further analyze the interplay of the two Syk isoforms (Aim 1.2). We will also establish an association of Syk isoform expression and the pathological features of breast tumors (Aim 1.3). Phosphorylation targets for Syk that are responsible for Syk(L)-inducible tumor (invasion) suppression will be searched for. We will purify these proteins by immuno-affinity method coupled with mass spectrometry sequencing (Aim 2.1). The participation of these targets in SYK signaling and Syk(L) phenotypic responses will then be analyzed (Aim 2.2). To elucidate the mechanism of differential in vivo phosphorylation and biological responses by Syk(L) and Syk(S), we will analyze the role IDB in SYK signaling in breast cancers. We will search for proteins that interact with the IDB of Syk(L) in mammary epithelial cells (Aim 3.1). How these IDBinteracting proteins assist in Syk(L)-mediated phosphorylation and the resultant phenotypes will be analyzed (Aim 3.2). Taken together, these studies should clarify the mechanisms by which the loss of Syk(L) and aberrant expression of Syk(S) promotes breast cancer progression. ? ?
