Breast cancer cells express Type 1 Insulin-like Growth Factor Receptor (IGF1R) and Insulin Receptor (IR). These receptors have a role in breast cancer biology and mediate metabolism, proliferation, survival, and motility. IR gene undergoes differential splicing that generates two IR isoforms, IRA (exclusion of exon 11) and IRB. High levels of IRA expression in prenatal growth and development are observed, whereas IRB expression is more highly expressed in adult insulin responsive tissues. IRA overexpression is the predominant receptor isoform expressed in breast cancer specimens obtained from patients with endocrine resistant disease. To measure levels of IRA and IRB mRNA expression, we utilized IR isoform specific primers in quantitative reverse transcription PCR (qRT-PCR). The validation of IRA and IRB primers was determined via using IRA and IRB overexpressing clones of MCF7 cells. IRA and IRB mRNA expression was also studied in human adipose and liver tissues that majorly express the adult IRB isoform. Over 40 breast cancer cell lines and 20 ER+ patient tumor samples were studied to determine IRA and IRB mRNA expression. Total IR, IGF1R, IRS1, and IRS2 were also analyzed in cell lines and patient samples. Across the ATCC breast cancer cell lines and ER+ patient tumors, heterogeneity was found among all targeted genes. Using MCF-7 cells as control, we found one of the breast cancer cell lines, Du4475, has very high IRA mRNA expression level (200-fold of that in MCF-7). Using mass spectrometry technology, we quantified levels of IR isoform expression in Du4475 cells. Briefly, IR immunoprecipitates were subjected to SDS-PAGE gels and the IRA protein gel band was submitted for mass spectrometry. Since IRA does not contain exon 11, the identified unique peptide consistent with its expression is TFEDYLHNVVFVPRPS. After this IRA specific peptide was determined, the heavy-labeled (C13- and N15-containing) IRA peptide was used as an internal injection control to quantify the IRA level. Quantitative results of IRA peptode sequence were proportional to the mRNA levels previously detected in Du4475 cell lysate. Using MCF-7 IRB overexpression cell line MCF7L-IRB-C5, we identified two IRB peptides, KTSSGTGAEDPRPSR and TSSGTGAEDPRPSR at exon 11 region by mass spectrometry. Cell membrane and total cell lysates could also be used to detect IR protein isoforms by mass spectrometry analysis. Further measurement of IRA and IRB level on other breast cancer cell lines is ongoing. In summary, we are the first to use mass spectrometry to validate protein expression of IR isoforms at levels that correlate with mRNA expression. IRA mRNA and protein expression levels in breast cancer cell lines may serve as biomarkers to screen patients for targeted therapy.

Citation Format: Xihong Zhang, LeeAnn Higgins, Todd Markowski, Tzu-Yi Yang, Jacob Wragge, Kevin Murray, Bruce Witthuhn, Albert Barrios, Douglas Yee. Detection of insulin receptor isoforms by mass spectrometry in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-03-02.