Abstract P6-03-22: MUC1 oncoprotein confers trastuzumab resistance in HER2-positive breast cancer cells by maintaining cancer stem cell phenotype

Abstract

Abstract Background: Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to the presence of cancer stem-like cells (CSCs). Interestingly, upregulation of the MUC1 gene has been reported to be associated with CSC growth, but its function in regulating CSC proliferation has not been studied in the trastuzumab-resistant (TrR) breast cancer cohort. Purpose: To evaluate the function of MUC1 in regulating cancer stem cell phenotype in TrR HER2-positive breast cancer cohort Methods 20,000 cells of each HER2-positive cell line (BT474R, T47D, HCC1954) were plated in ultralow attachment dishes and cultured in complete Mammocult Media for two weeks. Bright-field images were acquired. For CD44/CD24 staining, cells were incubated with Cy7-conjugated anti-CD44 and CF594-conujugated anti-CD24 per the previously described protocol. Cy7- and CF594-conjugated anti-mouse IgG were used as negative controls. For cell cycle analysis, cells were harvested and fixed with 70% cold ethanol for 24 H, followed by incubation with propidium iodide (PI, 50 μg/ml) and RNase (50 μg/ml) for 30 min, and analyzed by flow cytometry using Flowjo software. To silence MUC1 expression, 1 × 106 cells were transfected using the Neon electroporation system with 5 ng siMUC1 or 5 ng control siRNA vectors. The cells were electroporated for 30 ms at 1100 volts with two pulses. The cells were plated in 6-well plates with the appropriate media, incubated at 37°C for 48 hours and processed for further experiments. Results: Our results show that BT474R, the TrR cell line, forms more mammospheres and has a greater inherent CD44high/CD24low CSC population than BT474, the trastuzumab-sensitive (TrS) cell line, and that MUC1 is significantly upregulated in TrR compare to TrS HER2-positive breast cancer cells. We further demonstrate the significant inhibition of the CSC population upon silencing MUC1 expression by performing both mammosphere formation assay and CD44high/CD24low phenotype characterization and that this regulation is independent of cell cycle arrest or HER2 expression levels. Conclusion: MUC1 oncoprotein confers trastuzumab resistance in HER2-Positive breast cancer cells by maintaining cancer stem cell phenotype, indicating that it could be a potential predictive and targetable biomarker to overcome trastuzumab resistance in HER2-Positive breast cancer patients. Citation Format: Jun Yin, Andrea Sand, Mitchell Piacsek, Chaoyang Sun, Logan S. Friedrich, Richard A. Rovin, Judy A. Tjoe. MUC1 oncoprotein confers trastuzumab resistance in HER2-positive breast cancer cells by maintaining cancer stem cell phenotype [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-22.