Abstract Focal Adhesion Kinase (FAK) is an intracellular kinase known to mediate integrin signalling following cell adhesion to the extracellular matrix. FAK is also implicated in invasive signalling pathways downstream of growth factor receptors including the HER2. Cross talk between tumor and stroma can result in the activation of growth factor signalling pathways and thus may play a significant role in promoting tumor progression and spread. In this study, we have investigated the metastatic responses of HER2+ breast cancer cells to fibroblasts and whether FAK is involved in these events. Conditioned medium derived from human lung fibroblast MRC-5 cells was used to stimulate Her2+ SKBr3 breast cancer cells in the presence or absence of FAK inhibitor, PF271, or after FAK gene knockdown, prior to determining changes in migration (Boyden Chamber assay), 3d matrix invasion, growth and FAK-mediated signalling. To compare the role of FAK with HER2 in these cells, the HER2 targeted agent, trastuzumab was additionally employed. Whilst fibroblast-conditioned medium (FCM) did not affect the growth of SKBr3 cells or their HER2- negative, ER+ counterparts MCF7 cells, FCM significantly stimulated SKBr3 cell migration (∼10 fold vs control) and promoted invasive dissociation of 3d SKBr3 cultures. FCM treatment of SKBr3 cells resulted in rapid (within 5 minutes) activation of MAPK and AKT and increased phosphorylation of the HER4 receptor and FAK (Y861). FCM also promoted an increase in STAT3 (Y704) activity with an accompanying re-distribution to sites of membrane ruffling and to the nucleus. Pharmacological inhibition of FAK, or FAK siRNA, in SKBr3 cells attenuated FCM-mediated FAK activity and suppressed STAT3 activation and subcellular re-distribution and increased the expression of E-Cadherin. Correspondingly, FCM treatment of PF271 or FAKsiRNA- treated SKBr3 cells poorly elicited migratory and invasive responses. Importantly, suppression of HER2 using trastuzumab did not significantly antagonise these FCM responses. Taken together, these results suggest that FAK plays a role in the signalling stimulated by fibroblast- derived stimulators of migration and invasion through a mechanism involving STAT3. These highlight the importance of understanding tumor-stromal interactions and support the potential of FAK as a novel therapeutic target in HER2+ breast cancer particularly in the context of tumor stroma interactions where it may suppress stromal-induced tumor signalling that can contribute to progression and spread. Citation Format: Glorianne Lazaro, Chris Smith, Stephen Hiscox. Focal adhesion kinase (FAK) mediates fibroblast-induced HER2+ breast cancer cell migration and invasion through a mechanism involving Stat3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1165. doi:10.1158/1538-7445.AM2014-1165