The Impact of YAP-Mediated Hippo Signaling in Obesity and Triple Negative Breast Cancer

PURPOSE Obesity is one of the most prevalent and modifiable risk factors for chronic diseases such as cancer. Recent meta-analyses suggests that breast cancer-specific death among obese women is elevated 1.3-fold in comparison to normal weight woman. There is also an increased risk of developing triple negative breast cancer (TNBC) in women with a higher waist/hip ratio. However, the underlying pathophysiology linking obesity, adiposity, and TNBC needs further investigation. METHODS Colony formation assays were performed where cells were infected with control or shYAP lentivirus for 48 hours before 400 4T1 cells per well were seeded into a six-well plate. Seeded colonies were treated with stromal conditional medium (SCM) or adipocyte conditional medium (ACM) for 10 days. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by staining with 0.025% crystal violet for 30 minutes. For Western blotting, cells were harvested and lysed in 1x SDS loading buffer. The protein concentration was determined using Bio-Rad protein reagent. Antibodies against YAP and HRP-conjugated goat anti-rabbit secondary antibody (Promega) were used. Signals were detected with ECL reagents (Bio-Rad). Semi-quantification of data was performed using NIH ImageJ. RESULTS This study establishes a mechanistic link between obesity-altered metabolism and dysregulated proteasomal pathways, specifically the YAP-mediated Hippo cascade. Results show that the Hippo signaling pathway is highly regulated in obese mice in comparison to normal controls, as supported by RNA-sequencing. KEGG data also depicts other genes in oncogenic metabolic pathways as present in obese mice. Furthermore, data from clonogenic assays on three TNBC cell lines treated with ACM show increased cancer cell colony formation efficiency, suggesting that adipocyte secretome can increase tumor growth. In addition, YAP expression levels are elevated in the breast cancer cells under ACM treatment, and the knockdown of YAP diminishes the tumor inductive effect driven by ACM. Thus, this study uncovers the clinical relevance of the Hippo-YAP axis in regulating obesity-related breast cancer. CONCLUSION In conclusion, the study suggests that adipocyte secretome affects tumor proliferation and progression, as observed by experimentation on TNBC cell lines using ACM/SCM and testing for YAP expression levels. Future directions would studying the effect of the YAP inhibitor on ACM treated tumor cell growth.