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.
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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

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Figure 5

Effects of fibroblast PIK3Cδ expression on NR4A1-mediated invasion of TNBC cells.

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Effects of fibroblast PIK3Cδ expression on NR4A1-mediated invasion of TN...
(A) 2D invasion assay: MDA-MB-231 cells were seeded on the Matrigel-coated upper chamber of the Transwell insert and were treated with DMSO or 5 μM cytosporone B. After 24 hours, migrated MDA-MB-231 cells were fixed, stained, and counted (n = 2 independent experiments, minimum 9 technical replicates). Significance was calculated using unpaired t test. Results are expressed as mean SEM; ****P < 0.0001 vs. DMSO-treated cells. (B) 2D invasion assay: MDA-MB-231 cells transfected with control or NR4A1 siRNAs were seeded as above. After 24 hours, migrated MDA-MB-231 cells were fixed, stained, and counted (n = 2 independent experiments, minimum 9 technical replicates). Significance was calculated using unpaired t test. Results are expressed as mean ± SEM; ***P < 0.001 vs. siRNA control–transfected cells. (C) Effects of PIK3Cδ overexpression in MRC5 on MDA-MB-231 invasion following pretreatment of MDA-MB-231 with 5 μM cytosporone B (n = 2 independent experiments, minimum 9 technical replicates). Significance was calculated using 2-way ANOVA and Tukey’s multiple-comparisons tests. Results are expressed as mean ± SEM; ***P < 0.001, ****P < 0.0001 vs. the samples indicated in the graph. (D) Left and middle: MRC5 cells were treated with CAL-101 or DMSO. NR4A1 siRNA MDA-MB-231 or control siRNA cells were seeded on the Matrigel-coated upper chamber of a Transwell and cocultured with fibroblasts. After 24 hours, migrated MDA-MB-231 cells were fixed, stained, and counted (n = 3 independent experiments, minimum 6 technical replicates). Significance was calculated using 2-way ANOVA and Tukey’s multiple-comparisons tests. Results are expressed as mean ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the samples indicated in the graph. Right: NR4A1 levels were evaluated in siRNA-transfected MDA-MB-231 cells before and after coculture with CAL-101–treated MRC5. Significance was calculated using 1-way ANOVA followed by Dunnett’s tests. Results are expressed as mean ± SEM; *P < 0.05 vs. control. Scale bars: 400 μm.