PIK3Cδ expression by fibroblasts promotes triple-negative breast cancer progression
Teresa Gagliano, … , Justin Stebbing, Georgios Giamas
Teresa Gagliano, … , Justin Stebbing, Georgios Giamas
Published March 10, 2020
Citation Information: J Clin Invest. 2020;130(6):3188-3204. https://doi.org/10.1172/JCI128313.
View: Text | PDF
Research Article Cell biology Oncology

PIK3Cδ expression by fibroblasts promotes triple-negative breast cancer progression

Abstract

As there is growing evidence for the tumor microenvironment’s role in tumorigenesis, we investigated 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 cancer progression. Although PIK3Cδ was expressed in primary fibroblasts derived from TNBC patients, it was barely detectable in breast cancer (BC) cell lines. Genetic and pharmacological gain- and loss-of-function experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. Integrated secretomics and transcriptomics analyses revealed a paracrine mechanism via which f-PIK3Cδ confers its protumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, that 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, along with a decrease in tumor metastasis, emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA data sets 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.

Authors

Teresa Gagliano, Kalpit Shah, Sofia Gargani, Liyan Lao, Mansour Alsaleem, Jianing Chen, Vasileios Ntafis, Penghan Huang, Angeliki Ditsiou, Viviana Vella, Kritika Yadav, Kamila Bienkowska, Giulia Bresciani, Kai Kang, Leping Li, Philip Carter, Graeme Benstead-Hume, Timothy O’Hanlon, Michael Dean, Frances M.G. Pearl, Soo-Chin Lee, Emad A. Rakha, Andrew R. Green, Dimitris L. Kontoyiannis, Erwei Song, Justin Stebbing, Georgios Giamas

×

Figure 1

Experimental design of siRNA kinome screening and identification of fibroblast-expressed kinases affecting TNBC invasion.

Options: View larger image (or click on image) Download as PowerPoint
Experimental design of siRNA kinome screening and identification of fibr...
(A) Step 1: Silencing of 710 kinases in HMF and MRC5 cells using a siRNA kinome library. Step 2: 3D coculture of HMF or MRC5 with MDA-MB-231 in the presence of Matrigel and chemoattractants to promote invasion. A representative image of cells stained with different fluorescent lipophilic tracers is shown: MDA-MB-231 (red/DiRDiIC18) and MRC5 (green/DiOC6). Step 3: The invasive potential of MDA-MB-231 cells was used as a readout tool. Results are expressed as changes in spheroid surface between day 6 and day 3 (Δratio = ΔCT/ΔK). The Δratio values were used to calculate the Z scores based on the formula Z = (x – μ)/σ, where μ is Δratio mean of 710 kinases, σ is standard deviation (SD), and x is Δratio value for each kinase. For HMF, the Δratio Z score color code refers to SD, as the screening was performed twice, while for MRC5 the Δratio Z score color code refers to P value. (B) Step 4: The Z scores for HMF and MRC5 are shown. Kinases were divided depending on their effects on MDA-MB-231 invasion. Invasion-promoting: Δratio ≤ 0.5, P < 0.01 (as well as SD < 0.5 for HMF). Invasion-inhibiting: Δratio > 2, P > 0.05 (as well as SD > 0.5 for HMF).