Abstract P5-13-06: Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth

Abstract

Abstract Background: We had identified somatic mutations of the NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) gene from breast tumors of patients with distant metastasis using whole-exome sequencing. This study aimed to investigate the function of NSDHL mutants in breast cancer. Methods: ER-positive and tamoxifen-resistant human breast cancer cell line (ZR75-1) and mouse macrophage cell line (RAW264,7) were used. GFP-tagged NSDHL mutants were generated by subcloning the mutant NDHL into a lentiviral vector yielding an in-frame fusion of 3′ to GFP. ZR75-1 cells expressing the GFP-tagged NSDHL mutants were selected by puromycin with subsequent FACS analysis. The following experiments were performed: western blot, immunofluorescence staining, qRT-PCR, CellTiter-Glo assay, cell cycle assay, transwell migration assay, wound healing assay, tumor spheroid formation assay, and total cellular cholesterol measurement. An orthotopic breast tumor mouse model by injection of ZR75.1 cells into mammary gland fat pad was used in vivo. Results: Four novel NSDHL somatic mutations were discovered in the breast tumor tissues of patients. While total cholesterol levels in NSDHL mutant-transduced cells did not significantly increase, they were notably higher in wild-type NSDHL-transduced cells than in parent ZR75-1 cells. When compared to the parent and wild-type NSDHL-transduced cells, there was a minor increase in growth rate and a large increase in migratory capacities in NSDHL mutant-transduced cells (P< 0.05). The size of tumor spheroids is significantly larger in NSDHL mutant-transduced cells than in wild-type NSDHL-transduced cells. Both EGFR expression and epithelial to mesenchymal transition (EMT) markers were higher in NSDHL mutant-transduced cells than in parent and wild-type NSDHL-transduced cells. The NSDHL mutant-transduced cells’ conditioned media promoted the migratory ability and M2 polarization of RAW264.7 cells. In an orthotopic xenograft mouse model, NSDHL mutant-transduced cells were shown to promote tumor growth better than parent and wild-type NSDHL-transduced cells, suggesting that somatic mutation of the NSDHL contributes to the malignant progression of breast cancer cells. Conclusion: The present data indicate that breast cancer cells harboring somatic mutants of the NSDHL gene seem to display more aggressive behavior by gaining biological worse phenotype both in vitro and in vivo. More investigation is required into the mechanisms behind the function and accumulation of mutant NSDHL proteins to speed the development of therapies for patients with breast cancer harboring mutant NSDHL. Citation Format: Moonjou Baek, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Sangeun Lee, Kyoungsook Park, Han-Byoel Lee, Wonshik Han. Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-06.