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Publication Date

2013-01-10

Availability

UM campus only

Embargo Period

2013-01-10

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2012-12-17

First Committee Member

Mark D. Pegram

Second Committee Member

Dorraya El-Ashry

Third Committee Member

Stefan Gluck

Fourth Committee Member

Kerry Burnstein

Fifth Committee Member

Ralf Landgraf

Abstract

Amplification and resulting overexpression of the epidermal growth factor receptor ERBB2 (HER2) is found in ~20% of human breast cancers. Therapies targeting the ERBB2 receptor, including the kinase inhibitor lapatinib, have improved clinical outcome for women with ERBB2-amplified breast cancer. However, de novo and acquired resistance to lapatinib present a significant clinical problem and the mechanisms governing resistance remain poorly understood. We hypothesized that ERBB2-amplified breast tumor cells acquire molecular alterations during lapatinib treatment that may affect the activation of intracellular signaling pathways and lead to a drug resistant phenotype. To identify potential resistance mechanisms we rendered ERBB2-amplified SKBR3 breast cancer cells resistant to lapatinib (SK-lapR cells) via culture in increasing concentrations of the drug up to a clinically relevant concentration of 2.6μM. Comprehensive analysis of intracellular signaling pathway activation revealed constitutive activation of mammalian target of rapamycin complex1 (mTORC1) in SK-lapR cells despite apparent inactivation of the PI3K/AKT signaling pathway and other canonical activating pathways. Using pharmacological mTORC1 inhibitors we were able to demonstrate a role for downstream activation of mTORC1 in acquired lapatinib resistance in SKlapR cells. To identify potential novel regulators of mTORC1 activity and/or molecular alterations that mediate the lapatinib resistant phenotype in SK-lapR cells we characterized parental and sensitive cells through whole exome sequence analysis, high density SNP array and genome wide expression analysis. This approach led to the identification of 37 coding mutations and 13 novel gene fusions, which were validated by (RT-) PCR, as well as 276 differentially expressed genes (fold change > 2.0, p-value < 0.01) and more than thousand genes with assigned copy number changes associated with lapatinib resistance. Individual engineered overexpression of two prioritized candidate drug resistance mutations failed to render SKBR3 cells resistant to lapatinib. Altogether, the complexity of molecular alterations identified in SK-lapR cells suggests the possibility that these cells may employ a combinatorial mechanism rather than a single resistance gene mutation to escape the growth inhibitory effects of lapatinib. However our data suggest a potential role for mTORC1 in acquired lapatinib resistance and supports the rationale of combination or sequential therapy using ERBB2 and mTOR-targeting molecules to prevent or target resistance to lapatinib in ERBB2+ breast cancer provided that mTORC1 activation is found to be a general mechanism observed in clinical samples of lapatinib resistant breast cancer.

Keywords

breast cancer, ERBB2 amplified, lapatinib resistance, mTOR

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