DNA Damage Signaling and Cell Senescence in BRCA1 Mutated Adipose Stem Cells Leads to Breast Cancer Progression
Ruya Zhao, BS1, Irene J. Pien, MD2, Victoria L. Seewaldt, MD3, Chuan-yuan Li, PhD4, Scott T. Hollenbeck, MD4.
1Duke University School of Medicine, Durham, NC, USA, 2UCLA, Los Angeles, CA, USA, 3City of Hope, Duarte, CA, USA, 4Duke Medical Center, Durham, NC, USA.
Introduction: The tumor microenvironment can influence the progression of breast cancer. Targeting active pathways within the tumor microenvironment may be especially beneficial for triple negative breast cancer (TNBC). Our study focuses on the phenotype of human adipose-derived stem cells (ASCs) with the BRCA1 mutation and the resulting effects on breast cancer cells. We hypothesize that the mutation of BRCA1, a DNA damage response protein, induces cell senescence in ASCs and promotes the secretion of inflammatory cytokines, which leads to increased tumor invasion and metastasis.
Methods: CRISPR/Cas9 was used to generate de novo BRCA1-knockdown in human ASCs (BRCA1-KD). We have previously generated a human breast ASC cell line repository from mastectomy specimens. Breast ASCs from patients who have BRCA1 mutation (BRCA1 -/-) and normal BRCA1 expression (WT) were also evaluated. Tumor proliferation was examined by co-culturing ASCs and luciferase-labeled TNBC cell line MDA-MB-231 in 5% FBS media over 8 days. Tumor invasion was evaluated using transwell assays, in which the CRISPR/Cas9 control and BRCA1-KD ASCs served as feeders. To investigate DNA damage in ASCs, immunofluorescence staining was performed for phosphorylated ATM, an established marker for DNA double strand breaks. The level of cell senescence was tested using beta-galactosidase assays. Finally, conditioned media was obtained from ASC lines after 48hr incubation and the inflammatory cytokine levels were evaluated using quantitative ELISA.
Results: We demonstrated 79.1% knockdown expression of the BRCA1 gene in our CRISPR/Cas9-mediated knockdown; there are 52.1% and 94.9% decreased BRCA1 expressions in the two patients with BRCA1 mutation, respectively (Figure 1A). Tumor growth in vitro was significantly faster when co-cultured with BRCA1-KD ASCs (Figure 1B, 7.43% vs 5.16% increase at day4 comparing to day0, P=0.0074; 10.17% vs 6.57% at day6, P=0.036; 13.39% vs 8.02% at day8, P=0.045). Serving as feeders, BRCA1-KD ASCs induced significantly higher level of tumor cell migration (Figure 1C, 151±17.39 vs 66±13.17 cells migrated. P=0.0171). Immunofluorescence staining revealed higher level of phosphorylated ATM activation in BRCA1-KD cells compared to controls (Figure 1D, 62.73±3.12 vs 45.4±2.91%. P=0.0155). The beta-galactosidase assay demonstrated more cell senescence in both BRCA1-KD and patients with BRCA1 mutation (Figure 1E, 7.9±0.25% vs 0.17±0.17%, P<0.0001 in BRCA1-KD; 41.7±2.30% vs 18.4±2.34%, P<0.0001 in patient samples). By performing ELISA assay on supernatant of BRCA1-KD and control ASCs, we found significantly higher level of IL-8 inflammatory cytokine (Figure1F, 2.57±0.32 fold change, P=0.0049).
Conclusion: We show here for the first time that BRCA1 mutation in adipose-derived stem cells leads to increased inflammatory cytokine production via a DNA damage-mediated cell senescence pathway. The effect from ASCs increases tumor proliferation and invasion. This interaction between ASCs and breast cancer cells can be targeted for more effective anticancer therapies in the setting of high-risk breast cancer.
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