Abstract 1980: Nucleophosmin regulates centrosome amplification and is associated with anti-HER2 resistance in HER2-positive breast cancer cells.

Aneuploidy is a hallmark of solid tumors, contributing to tumorigenesis and resistance to chemotherapy. Aneuploidy is prevented by two centrosomes directing a bipolar mitotic spindle. Centrosome amplification (CA), the presence of supranumerary centrosomes within a cell, has been detected in both premalignant lesions and breast tumors, supporting the role of this phenomenon in breast cancer initiation and progression. Moreover, CA has been associated with HER2 overexpression. CA is initiated by abnormal signals from oncogenes or altered tumor suppressors that affect regulatory molecules shared by both cell and centrosome cycles. Elucidating the molecular circuitry and functional consequences of impaired centrosome cycle is therefore important for a complete understanding of its role in breast cancer. Nucleophosmin (NPM) is a widely expressed nucleolar phosphoprotein that has several functions, including the early regulation of centrosome duplication. Recent reports from breast cancer patients indicate the presence of NPM antigens in HER2-negative cases and low NPM expression as a prognostic marker of poor survival in the luminal A subtype. We therefore hypothesized that NPM expression and activity might directly impact the CA in HER2-positive breast cancer cells. To test this hypothesis, we have used genetic alteration of total and phospho-NPM to investigate its function in human mammary epithelial cell lines that includes non-tumorigenic and HER2-positive tumorigenic lines. Our initial findings revealed significantly elevated CA percentages and higher expression level of p-NPM (T199) in the HCC1954, JIMT1, and SKBR3 cells (HER2-positive) compared to other subtypes and non-tumorigenic MCF10A cells. This residue is known to be responsible for regulating the function of NPM in centrosome duplication. Conversely, HER2-positive cells displayed lower protein levels of total NPM compared to the other cell lines investigated. Expression of a deletion mutant phospho-NPM (T199A) reduced the percentage of CA in HCC1954 cells. In addition, an HCC1954 derivative with primary resistance to Herceptin showed augmented frequency of CA versus sensitive parental cells. Ongoing experiments involve the inhibition of p-NPM at T199 in JIMT1 and SKBR3 cells as well as the overexpression of a wild type form in all of the three HER2-positive breast cancer cell lines followed by the assessment of CA. In parallel, the non-tumorigenic MCF10A cells have been transfected with a phospho-mimetic NPM (T199D) and will be subjected to similar investigations. Finally, the correlation between p-NPM-driven CA and Herceptin resistance is being studied in all of our HER2-positive cell models of breast cancer. These results suggest that the high NPM expression could have a protective effect against CA and a more aggressive phenotype displayed by HER2-positive breast cancer cells. Citation Format: Mihaela Marina, Harold Ivan Saavedra. Nucleophosmin regulates centrosome amplification and is associated with anti-HER2 resistance in HER2-positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2013-1980