Graduation Date

Fall 12-18-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Hamid Band

Abstract

The epidermal growth factor receptor (EGFR) family member ErbB2 (Her2) is overexpressed in 20 -30% of invasive breast cancers and this overexpression correlates with poor prognosis and shorter overall as well as disease-free survival. Aberrant expression of ErbB2 through gene amplification, transcriptional deregulation and/or altered endocytic trafficking results in overexpression of ErbB2 at the plasma membrane and biases ErbB2 from primarily ligand-driven hetero-dimerization under normal expression conditions to increased ligand-independent homo-dimer and hetero-dimer formation and consequent activation. C-terminus of HSC70-Inteeracting protein (CHIP)/STIP1-homologous U-Box containing protein 1 (STUB1) is an HSP90/HSC70 interacting negative co-chaperone known to promote ubiquitination and degradation of unfolded proteins, playing an essential role in protein quality control. HSP90/HSC70 are required for the stability and function of a variety of signaling proteins, including a number of protein kinases and their downstream signaling components. It is now known that CHIP can function as an E3 ubiquitin ligase towards such HSP90/HSC70 clients to induce their ubiquitination and degradation. ErbB2 is a prominent receptor tyrosine kinase that is dependent on its continuous association with HSP90 for its stability and function as an oncogene. Thus, CHIP, acting as an E3 ubiquitin ligase towards ErbB2 and its downstream signaling proteins has been hypothesized to function as a tumor suppresser. Consistent with this idea, recent work indicates that CHIP mRNA and protein expression is reduced in a subset of breast cancers, primarily those that are estrogen-receptor negative and belonging to ErBB2+ and triple-negative subsets. How CHIP functions as a tumor suppressor in breast cancer in general and in ErbB2+ breast cancer in particular, has not been fully elucidated. In this thesis, we identify two inter-connected and novel mechanisms by which CHIP suppresses ErbB2-mediated breast oncogenesis. First, we demonstrate that CHIP targets newly synthesized HSP90/HSC70-associated ErbB2 for in the Endoplasmic Reticulum and Golgi for ubiquitin/proteasome-dependent degradation to negatively control the levels of cell surface ErbB2. Second, by analyzing CHIP expression in tissue microarrays from a large and well-annotated cohort of breast cancer patient samples, we identified a series of transcription factors whose cognate DNA-binding activity is up- or down-regulated by CHIP. We identify Myeloid Zinc Finger 1 (MZF-1), a transcription factor that transcriptionally upregulates the expression of extracellular matrix degrading enzymes cathepsin B and cathepsin L (CTSB/L) as a target of direct CHIP-dependent ubiquitination and degradation as well as indirect inhibition through reduced transcription. Small hairpin RNA (shRNA)-mediated depletion of CHIP in ER+ breast cancer cells or ectopic overexpression of CHIP in ErbB2 in ErbB2+ and triple-negative breast cancer cells demonstrated that loss of CHIP in the two latter subtypes of breast cancer is a principal determinant of MZF1-dependent upregulation of CTSB/L expression and activity, increased cell migration, invasiveness and matrix degradation, anchorage-independent growth in soft agar, and xenograft tumor formation and metastasis in vivo. Targeting of CTSB using specific chemical inhibition lead to statistically-significant tumor growth inhibition with reduced angiogenesis, correlating with inhibition of matrix degradation in vitro, suggesting altogether a potentially new therapeutic avenue to improve metastatic breast cancer treatment by targeting those tumors with reduced CHIP expression for cathepsin inhibitor therapy in conjunction with conventional and ErbB2-targeted therapeutics.

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