Abstract
Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed SNPs. These results contribute to a better understanding of trastuzumab action and resistance mechanisms.
Highlights
The ‘HER2-positive’ subtype of breast cancer overexpresses the human epidermal growth factor receptor 2 (HER2)
We describe the results for the validated gene candidates of the differentially expressed (DE) analysis on BT474 and its trastuzumab treated version (ALPP, CALCOCO1, CAV1, CYP1A2, IGFBP3) as well as the candidates of the DE analysis on BT474 vs. HCC1954 which were already reported in the context of trastuzumab efficacy, namely GDF15, IL8, LCN2 and PTGS2
The validated selection of the candidate genes was differentially expressed between the sensitive cell line BT474 and its trastuzumab treated version, as well as between BT474 and the resistant cell line HCC1954
Summary
The ‘HER2-positive’ subtype of breast cancer overexpresses the human epidermal growth factor receptor 2 (HER2) This receptor tyrosine kinase is part of the epidermal growth factor receptor (EGFR) family, further including HER1 (EGFR), HER3 and HER4 [1]. It is overexpressed in 10–20% of breast tumors, and the related subtype is associated with increased recurrence and mortality rates [2, 3]. The improved outcome by adding trastuzumab to chemotherapy for example is not completely understood It has been associated with different mechanisms of action apart from inhibiting HER2, its dimerization and cleavage [4, 5]. These mechanisms include inhibition of downstream signal transduction pathways like the PI3K pathway, antigendependent cellular cytotoxicity (ADCC), induction of cell cycle arrest and apoptosis or inhibition of tumor angiogenesis
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