Human Adipose-Derived Stem Cells with BRCA Mutation Promote Breast Tumor Cell Migration
Ruya Zhao, BS1, Irene J. Pien, MD2, Xinjian Liu, PhD3, Victoria L. Seewaldt, MD4, Chuan-yuan Li, PhD5, Scott T. Hollenbeck, MD6.
1Duke University School of Medicine, Durham, NC, USA, 2University of California in Los Angeles Medical Center, Los Angeles, CA, USA, 3Duke University, Durham, NC, USA, 4City of Hope Medical Center, Duarte, CA, USA, 5Duke Cancer Institute, Durham, NC, USA, 6Duke University Department of Surgery, Division of Plastic, Maxillofacial, and Oral Surgery, Durham, NC, USA.
Purpose: We have previously shown that human breast adipose-derived stem cells (ASC) from BRCA mutation carriers behave differently from non-mutation carriers in regards to adipocyte differentiation and cytokine production. It is unknown if the BRCA mutation impacts adipose tissue in a manner that promotes breast cancer. In this study we further investigate the effects that BRCA mutations have on the interaction between ASCs and breast cancer cells.Methods: BRCA1-knockout ASCs were generated using CRISPR/Cas9 system in commercially available human ASC lines. BRCA1 protein expression was assessed with Western blots. Next, conditioned medium was generated using ASC groups with and without BRCA1 knockdown. ASC conditioned media was added to MDA-MB-231/Luciferase cell lines (Luciferase-labeled triple negative breast tumor cell lines). Cancer cell proliferation was evaluated using luciferase assays. Scratch assays were used to monitor migration in MDA-MB-231 cells incubated in ASC conditioned media. In parallel studies, human breast primary ASC lines from our repository were also evaluated (n=8, 2 patients with BRCA1 deleterious mutations, 2 patients with BRCA1 intron mutation or mutation without clinical significance, 1 patient with BRCA2 mutation, and 3 patients without genetic mutations). The luciferase assay and scratch assay were repeated using patient samples. Results: Using CRISPR/Cas9-mediated methods, we were able to generate effective BRCA1 knockdown in human ASCs, as demonstrated by Western blot (Figure 1A). Conditioned media from BRCA1 knockdown ASCs did not significantly alter MDA-MB-231 cell proliferation in comparison to control ASCs (data not shown). However, MDA-MB-231 cells incubated in BRCA1 knockout ASC conditioned media showed significantly higher tumor migration rate in comparison to control ASCs (Figure 1B, p = 0.03 at 48hours, p = 0.008 at 72hours). Conditioned media from our human breast ASC lines with BRCA1 deleterious mutations also demonstrated increased cell migration in 24 hours in vitro (p<0.00001). This effect was not observed for either BRCA1 intron mutations or BRCA2 mutations. Conclusions: We demonstrate here for the first time that human adipose-derived stem cells with BRCA1 mutations have the potential to increase breast tumor cell migration. This is true in both CRISPR/Cas9-mediated BRCA1 knockdown cell lines and in patient samples possessing the BRCA1 deleterious mutation.
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