Background: Kinases represent one of the main therapeutic targets in cancer treatment as their impairing relates to tumor growth and progression. Despite increasing evidence that the tumor microenvironment (TME) signaling influences the behavior of surrounding cancer epithelial cells, still relatively little is known about what changes in stromal cells influence tumor cells' behavior and how they affect their response to target therapy. TNBC patients are still lacking an effective therapy, as not much is known about the biology of this BC tumor subtype. Clinical trials focus mainly on targeting epithelial cancer cells by using a combination of kinase inhibitors and standard chemotherapy however these therapeutic regimens are not considering the action of stromal cells that may influence response to treatment.
Aims: • Identify fibroblasts-expressed kinases that modulate tumor cells' invasion • Characterize the mechanism by which these kinases promote/reduce tumor cell invasion.
Methods: Human Mammary Fibroblast (HMF) and normal lung fibroblast (MRC5) cells were used in a high-throughput siRNA kinome screening targeting 710 Kinases. 24 hours after transfection, fibroblasts were co-cultured with MDA-MB-231 in 3D for spheroid formation. Matrigel and chemoattractants were then added to promote invasion. Spheroids pictures taken at different time-points were analyzed and results were expressed as changes in spheroid surface formation (ΔRatio= ΔCtrl/ΔKinase).
Results: PIK3Cδ was among the most prominent targets, whose silencing decreased TNBC invasion. PIK3Cδ is essential for regulating chemokine production in leukocytes and promotes migration during inflammation, while PIK3Cδ inhibitors (CAL -101) interfere in tumour-stroma interactions without directly killing cancer cells. Despite PIK3Cδ being expressed mainly in leucocytes, we detected high PIK3Cδ protein expression in fibroblast cell lines and primary fibroblasts derived from TNBC patients; still, PIK3Cδ was hardly detectable in breast cancer cell lines. CAL-101 treatment affected fibroblasts viability, while it had limited/no effects on breast cancer cells. Moreover, CAL-101 resulted in reduced phosphorylation of AKT, a PIK3Cδ target. Pretreatment of fibroblasts with CAL-101 significantly decreased TNBC cells' invasion in 2D & 3D co-culture experiments. Interestingly, using transwell systems we saw that TNBC co-culturing increased PIK3Cδ expression in fibroblasts, suggesting a feedback loop that ‘fuels' tumor progression. Additional proteomic/genomic studies, provided us with evidence about the PIK3Cδ-mediated paracrine signaling leading to TNBC invasion.
Conclusion: Using a novel 3D co-culture invasion assay, we identified stromal PIK3Cδ as a key mediator of TNBC invasion. Our results suggest that targeting PIK3Cδ in the TME may represent a novel transformative strategy for TNBC therapy.
Citation Format: Teresa Gagliano, Viviana Vella, Kamila Bienkowska, Angeliki Ditsiou, Georgios Giamas. Fibroblast-kinome siRNA screening identifies PIK3Cδ as a mediator of Triple-negative breast cancer (TNBC) invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2087.