Abstract 1915: Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling

Abstract

Abstract Introduction The widespread clinical use of monoclonal antibodies such as trastuzumab in personalized medicine, targeting upstream proteins such as receptors with cascading effects on downstream signaling, elicits the importance of studying the phosphoproteome in the investigation of resistance to targeted therapy. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins in key signal transduction pathways, in cellular models of resistance. Method DigiWest®, a Luminex® xMAP® bead-based high-throughput protein profiling method, was utilized to measure 194 proteins and phospho-proteins from 12µg of total protein, obtained from HER2-positive cell lines which were trastuzumab-sensitive (SKBR3), intrinsically resistant (JIMT-1, UACC-893) and with acquired resistance (T15, obtained by long-term culture of SKBR3 in 15µg/mL trastuzumab). The resulting median fluorescence intensities (MFI) were normalized to total protein content. Relative quantification was done against SKBR3 and a log2 fold-change of ±1 was considered to be significant. For each of the three trastuzumab-resistant cell lines, targets which were differentially expressed were organized into established signal transduction pathways, and specific phosphosite expression values were used to determine whether each protein was under- or over-activated. Results A total of 115 proteins were expressed in at least one of the HER2-positive cell lines at a normalized MFI above the set threshold. JIMT-1 showed the most concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β-catenin stabilization, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance – it was over-activated in all the three trastuzumab-resistant cell lines. The overexpression of upstream RAS, however, was confined to the intrinsically resistant cell lines JIMT-1 and UACC-893; meanwhile, in T15, entry into the ERK pathway was mostly mediated by pan-PKC overexpression. PKCs, especially PKCα, were also highly activated in UACC-893. β-catenin stabilization was another mechanism observed only in the intrinsically trastuzumab-resistant cell lines. Conclusion Both distinct and overlapping mechanisms are involved in intrinsic and acquired trastuzumab resistance. Multiple pathways can also co-exist to promote cell survival, as was mostly exemplified by JIMT-1, derived from an aggressive, basal-like tumor. Furthermore, different models of intrinsic resistance (JIMT-1 vs UACC-893) showed significant differences in differentially activated signal transduction pathways. DigiWest® provided a simple and robust multiplexing solution for this phospho-proteomic study. Citation Format: Jeanesse Scerri, Felix Ruoff, Markus Templin, Godfrey Grech, Christian Scerri. Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1915.