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The role of the tumour microenvironement (TME) in cancer progression and resistance to therapies is now widely recognized. The most prominent non-immune cell type in the microenvironment of oral cancer (OSCC) is cancer-associated fibroblasts (CAF). Although CAF are a poorly characterised and heterogenous cell population, those with an “activated” myofibroblastic phenotype have been shown to support OSCC progression, promoting growth, invasion and numerous other “hallmarks of malignancy.” CAF also confer broad resistance to different types of therapy, including chemo/radiotherapy and EGFR inhibitors; consistent with this, CAF-rich OSCC are associated with poor prognosis. In recent years, much CAF research has focused on their immunological role in the tumour microenvironment, showing that CAF shield tumours from immune attack through multiple mechanisms, and particularly on their role in promoting resistance to anti-PD-1/PD-L1 checkpoint inhibitors, an exciting development for the treatment of recurrent/metastatic oral cancer, but which fails in most patients. This review summarises our current understanding of CAF subtypes and function in OSCC and discusses the potential for targeting these cells therapeutically.

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As there is growing evidence for the tumor microenvironment's (TME) role in tumorigenesis, we sought to investigate the role of fibroblast-expressed kinases in triple negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of progression. Although PIK3Cδ has been mainly described in leucocytes, we detected high expression in primary fibroblasts derived from TNBC patients, while PIK3Cδ was undetectable in cancer epithelial cell lines. Genetic and pharmacologic gain-and loss-of functions experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. By employing an integrated secretomics and transcriptomics analysis, we revealed a paracrine mechanism via which f-PIK3Cδ confers its pro-tumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, which subsequently led to upregulation of NR4A1 in TNBC cells where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, in addition to a decrease on tumor metastasis emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA datasets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease free survival, highlighting it as a therapeutic target for TNBC.

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.