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Anti-Cancer Mechanism of Trastuzumab via Blocking Nuclear HER2 Function and Epigenetic Mechanism of Resistance
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- Author / Creator
- Nami Mollalou, Babak
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HER2 receptor tyrosine kinase (encoded by ERBB2 gene) is overexpressed in approximately 25% of all breast cancer tumors (known as HER2-positive breast cancers). Overexpression of HER2 causes overactivation of downstream receptor tyrosine kinase pathways including PI3K/Akt and MAPK pathways and is a poor prognosis factor in breast cancer. Trastuzumab which is a humanized monoclonal antibody designed to target HER2 receptor is approved by FDA to treat patients with early-stage and metastatic HER2-positive breast cancer as an adjuvant in combination with other chemotherapy. However, approximately 60-70% of HER2-positive breast cancer patients develop de novo resistance to trastuzumab, partially due to the loss of HER2 expression on their tumor cells during the treatment. Little is known about the exact mode of action of trastuzumab in inhibiting HER2-positive breast cancer cells and the mechanism of trastuzumab resistance. The overall aim of this thesis was to study the mechanism of action of trastuzumab in inhibiting HER2-positive breast cancer and the mechanism of resistance to trastuzumab. We found that HER2 overexpression in CHO cells had no major effect on the activation of downstream PI3K/Akt and MAPK pathways, however, significantly increased the cell growth. These suggest a non-canonical oncogenic function of HER2. Our results showed that trastuzumab blocks proteolytic cleavage of HER2, production and nuclear localization of a ctHER2 with an approximate molecular weight of 85 kDa (p85HER2). Trastuzumab showed a synergic effect with a proteinase inhibitor in blocking HER2 cleavage that led to cell growth inhibition. This is a new molecular anti-cancer mechanism of trastuzumab. Immunoprecipitation of nuclear p85HER2 followed by mass spectrometry analysis showed that p85HER2 directly interacts with the spliceosome protein complex and transcription factors and mediates in RNA processing, splicing, and gene expression regulation. These results demonstrate that nuclear HER2 mediates in the regulation of RNA processing and gene expression. Gene set enrichment analysis of the mass spectrometry results also revealed that most of the nuclear p85HER2 client proteins are downstream targets for oncogenic/stemness transcription factors which are master regulators of breast cancer stemness and epithelial-mesenchymal transition (EMT). These results demonstrate a novel mechanism of action of trastuzumab in blocking a non-canonical function of HER2 via nuclear p85HER2.In this study, we also hypothesized that EMT abrogates HER2 expression by chromatin-based epigenetic silencing of ERBB2 gene as a mechanism of development of trastuzumab resistance. we found positive and negative correlation of HER2 expression levels with epithelial and mesenchymal phenotypes respectively. This indicates that epithelial-like cells are HER2-high, while mesenchymal-like cells are HER2-low. We found that the correlation is due to active and inactive chromatin dynamics of ERBB2 gene in epithelial-like and mesenchymal-like cells respectively. HER2-low mesenchymal-like breast cancer cell lines revealed less promoter-enhancer interaction and larger chromatin loops compared to the HER2-high epithelial-like breast cancer cell lines. Further, the cell line with higher expression levels of HER2 showed higher numbers of chromatin-chromatin interaction, super-enhancers and topologically associated domains (TADs) at the chromatin of ERBB2 gene and flanking regions. The lower HER2 expression, the higher EMT phenotype, and inactivated chromatin all were found correlated with a lower response to lapatinib. We also demonstrated that inducing EMT of HER2-positive cancer cells results in the downregulation of HER2 expression and lower binding rate of trastuzumab. These results show that the downregulation of HER2 expression in mesenchymal-like cells derived from HER2-positive breast cancer cell lines is due to ERBB2 gene silencing by global epigenetic reprogramming during EMT.We strongly suggest further studying the oncogenic function of p85HER2 through regulation of coding and non-coding RNA processing as well as transcription co-factor function of p85HER2 in breast cancers. We also suggest testing proteinase inhibitors in combination with trastuzumab and lapatinib to prevent the non-canonical pathways of HER2 and development of de novo trastuzumab resistance in HER2-positive breast cancers. We here strongly recommend developing and testing HER2-targeting small molecules inhibitors to inhibit HER2 cleavage as an alternative therapy for trastuzumab to use in combination with lapatinib. Further, we propose targeting EMT and cancer stem cells as an effective approach to inhibit tumor growth and overcome drug resistance in breast cancer.
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- Subjects / Keywords
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- Graduation date
- Spring 2020
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.