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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Allan, Alison L.

Abstract

Resistance to tyrosine kinase inhibitors (TKIs) presents a growing challenge in the development of therapeutic targets for cancers such as triple negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Potential targets in TNBC include the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met, however, targeted anti-EGFR and anti-c-Met therapies have faced challenges in clinical trials due to acquired resistance. We hypothesize that response versus resistance of triple negative breast cancer to the tyrosine kinase inhibitors erlotinib and cabozantinib is mediated by compensatory changes in the kinome and phosphoproteome. To test this, we (1) assessed the sensitivity of MDA-MB-468 and MDA-MB-231 TNBC cell lines to erlotinib and cabozantinib, (2) developed an integrated mass spectrometry proteomics approach to infer changes in kinase activity at kinome-level, and (3) identified mediators of resistance in developed erlotinib- and cabozantinib-resistant TNBC cell lines. First, we observed that MDA-MB-468 cells were sensitive to the anti-proliferative, anti-migratory and anti-invasive effects of erlotinib, whereas MDA-MB-231 cells were sensitive to cabozantinib. Secondly, we used SILAC (Stable Isotope Labeled Amino Acids in Cell Culture)-labeled MDA-MB-468 cells in quantitative mass spectrometry-based kinomics and phosphoproteomics to assess the effects of erlotinib in TKI-responsive cells. Using a modified KSEA analysis, we inferred that erlotinib decreases activity of ERK1/2. Finally, we observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion and activation of EGFR or c-Met downstream signaling (respectively). Using the integrated proteomics approach, we identified upregulation of CDK1, CDK7 and CK2A1 activity in cabozantinib-resistant cells and upregulation in AKT1, CK2A1 and ERK1 activity in erlotinib-resistant cells. Functional validation revealed inhibitor synergy between erlotinib and AKT inhibitor VIII. Using the integrated proteomics approach, we identified several potential kinase mediators of cabozantinib-resistance and confirmed the contribution of AKT1 to erlotinib-resistance in TNBC resistant cell lines. The resistance mediators identified in this study can be used to develop combination therapeutics to apply in further in vivo and clinical studies of resistance to EGFR and c-Met inhibitors in triple negative breast cancer.

Summary for Lay Audience

Breast cancer is the most commonly diagnosed cancer in Canadian women, with over 5,000 people losing their life to the disease each year. In particular, a type of breast cancer called triple negative breast cancer (TNBC) is the most lethal because of a lack of effective cancer treatments that can specifically target it. Researchers have previously discovered new potential protein targets known as receptor tyrosine kinases (RTKs), including proteins called epidermal growth factor receptor (EGFR) and c-Met. Although drugs have been developed to target EGFR and c-Met that show early effectiveness in treating TNBC, patients often develop resistance to these drugs (erlotinib and cabozantinib) over time. The goal of this thesis is to understand the molecular changes that are driving those tumors to become resistant and stop responding to the targeted drugs. We believe that proteins known as kinases are the most likely culprit, because they often lead to cancer growth and spread. To test this, we used TNBC cells to study how RTK-targeting drugs like erlotinib and cabozantinib work to kill cancer cells and how they stop them from spreading. Then we made the cells resistant to those drugs by growing them with the drugs over long periods of time. Before looking for kinases responsible for resistance, we first had to develop an approach that uses mass spectrometry-based proteomics – a technique that allows us to measure changes in thousands of proteins at the same time – to find kinases that are more active in resistant cells. Using this approach, we found many kinases that might be responsible for resistance to erlotinib and cabozantinib and further studied a particular kinase known as AKT1 in cells resistant to erlotinib. To test if AKT1 was responsible, we treated erlotinib-resistant cells with erlotinib and a drug targeting AKT1 known as AKT inhibitor VIII. We found that stopping AKT1 reversed the resistance to erlotinib, and the cells became more responsive to erlotinib again. This suggests that AKT1 may be a useful target to study for future development of clinical treatment strategies and/or biomarkers in resistant TNBC.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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