Abstract 3131: A novel tyrosine kinase signaling pathway in human basal breast cancer

Basal-type breast cancers account for up to 25 % of breast cancer cases, depending on the patient population. These cancers usually lack estrogen, progesterone and erbB2 receptors and are therefore resistant to endocrine or trastuzumab therapy, and markers that stratify this subset according to clinical behaviour have yet to be identified. Consequently, basal breast cancers present a major treatment dilemma. In order to identify potential therapeutic targets and prognostic markers for this disease subgroup, we are currently undertaking a detailed characterization of tyrosine kinase signaling networks in basal breast cancer cells. Mass spectrometry (MS)-based global profiling of the tyrosine phosphoproteome of basal breast cancer cells identified a prominent Src family kinase (SFK) signaling network that featured high expression of the SFK Lyn (Hochgräfe et al, Cancer Res 2010 doi: 10.1158/0008-5472.CAN-10-0911). In order to identify Lyn substrates, we used stable isotope labelling with amino acids in cell culture (SILAC) in combination with immunoaffinity-based phosphotyrosine enrichment and LC-MS/MS to profile cellular tyrosine phosphorylation following transient siRNA-mediated Lyn knockdown in MDA-MB-231 and BT-549 basal breast cancer cells. This identified several Lyn substrates characteristic of both cell lines, which included annexin A2 (Y42), 40S ribosomal protein S10 (Y12) and SgK269 (Y635). The latter is a relatively understudied kinase that contains several tyrosine phosphorylation sites that are likely to act as recruitment sites for SH2/PTB domain-containing signalling proteins, and a C-terminal kinase-like domain that lacks the consensus DFG motif usually regarded as essential for kinase activity. Protein expression of SgK269 was markedly higher in basal, versus luminal, breast cancer cells, while mRNA transcript levels in the two cell types were similar, indicating post-transcriptional regulation of SgK269 expression. Phosphorylation of SgK269 on Y635 could be detected readily in basal breast cancer cells. Overexpression of SgK269 in MCF-10A immortalized mammary epithelial cells led to a more elongated morphology in monolayer and perturbed acinar morphogenesis in Matrigel, resulting in large, multilobular acini that failed to undergo proliferative suppression in long-term culture. SgK269-overexpressing cells also exhibited enhanced tyrosine phosphorylation of Stat3. In addition, we undertook stable shRNA-mediated knockdown of SgK269 in BT-549