CRISPR/Cas9-Mediated BRCA1 Knockdown Adipose Stem Cells Promote Breast Cancer Progression

Abstract

Background: The tumor microenvironment within the breast is rich in adipose elements. The interaction between adipose cells and breast cancer is poorly understood, particularly as it pertains to patients with genetic susceptibility to breast cancer. This study focuses on the phenotype of human adipose-derived stem cells with the BRCA1 mutation and the effect they may have on breast cancer cell behavior.

Methods: CRISPR/Cas9 was used to generate de novo BRCA1-knockdown human adipose-derived stem cells. The effect of the BRCA1 knockdown on the adipose-derived stem cell phenotype was compared to wild-type adipose-derived stem cells and patient-derived breast adipose-derived stem cells with known BRCA1 mutations. Interactions between adipose-derived stem cells and the MDA-MB-231 breast cancer cell line were evaluated.

Results: BRCA1-knockdown adipose-derived stem cells stimulated MDA-MB-231 proliferation (1.4-fold increase on day 4; p = 0.0074) and invasion (2.3-fold increase on day 2; p = 0.0171) compared to wild-type cells. Immunofluorescence staining revealed higher levels of phosphorylated ataxia telangiectasia-mutated activation in BRCA1-knockdown cells (72.9 ± 5.32 percent versus 42.9 ± 4.97 percent; p = 0.0147), indicating higher levels of DNA damage. Beta-galactosidase staining demonstrated a significantly higher level of senescence in BRCA1-knockdown cells compared with wild-type cells (7.9 ± 0.25 percent versus 0.17 ± 0.17 percent; p < 0.0001). Using quantitative enzyme-linked immunosorbent assay to evaluate conditioned media, the authors found significantly higher levels of interleukin-8 in BRCA1-knockdown cells (2.57 ± 0.32-fold; p = 0.0049).

Conclusions: The authors show for the first time that the BRCA1 mutation affects the adipose-derived stem cell phenotype. Moreover, CRISPR/Cas9-generated BRCA1-knockdown adipose-derived stem cells stimulate a more aggressive behavior in breast cancer cells than wild-type adipose-derived stem cells. This appears to be related to increased inflammatory cytokine production by means of a DNA damage-mediated cell senescence pathway.

Figure 1.

BRCA1-KD ASC promoted proliferation and invasion of breast tumor cells. A. WB of BRCA1 expression in CRISPR/Cas 9-mediated BRCA1-KD ASCs and primary brASC. B. WB of BRCA1 expression in patient brASCs. The table below shows the patient demographics, cancer details, and BRCA1 mutation status for these patients. C. Tumor growth rate when MDA-MB-231 cells were co-cultured with ASCs (N=3). D&E. Representative images and quantification of tumor cell invasion when ASCs were used as feeders in Transwell assays (N=3).

Figure 2.

DNA Damage in BRCA1-Deficient ASCs. A&B. Representative immunofluorescence images and quantification of phosphorylated ATM. C. WB of pATM expression (N=3, * indicates P<0.05).

Figure 3.

Cell Senescence in BRCA1-deficient ASCs. A&B. Representative images and quantifications of beta-galactosidase assays in both BRCA1-KD ASCs and brASCs (N=3, **** indicates P<0.0001). C. WB of p21 expression.

Figure 4.

IL-8 in BRCA1-KD ASC cell supernatant that promotes cell invasion. A. Quantification of ASC supernatant using ELISA (N=4). B&C. Representative images and quantifications of cell invasion using IL6 and IL8 in transwell assays comparing to control (N=3, * indicates P<0.05, ** indicates P<0.01).

Figure 5.

The mechanism of how BRCA1-deficient ASCs promote breast cancer progression. BRCA1-deficienct ASCs are unable to repair both spontaneous and stress-induced DNA damage. This accumulation of DNA damage leads to more persistently active ATM complex, which activates p21, and induces cellular senescence. In the senescence state, BRCA1- deficient ASCs secrete increased number of inflammatory cytokines, which promotes breast tumor proliferation and invasion.