Publication Date

2012-07-30

Availability

Embargoed

Embargo Period

2013-07-30

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2012-06-28

First Committee Member

Joyce M. Slingerland

Second Committee Member

Zafar Nawaz

Third Committee Member

Karoline Briegel

Fourth Committee Member

Dorraya El Ashry

Fifth Committee Member

Tan Ince

Abstract

Accumulating evidence over the past decade has established the existence of a cancer stem cell (CSC) subpopulation within breast cancers that is responsible for tumor initiation, progression and drug resistance. However, few models have characterized phenotypically distinct subsets of cells within CSC populations that may underlie the heterogeneity of tumor responses to standard chemo and radiation therapies. Similarly, despite the widely accepted postulate that CSCs generate metastasis, few studies have demonstrated experimentally the existence of heterogeneity within the CSCs with regard to metastatic potential. The work in this thesis was undertaken to identify whether such discrete subsets exist among CSCs of the most deadly form of breast cancer: that lacking estrogen and progesterone receptors and HER2 amplification (so called triple negative—hereafter TNBC). We postulated that, as for normal stem cells, primary TNBC-derived cultures and immortal lines would have a CSC hierarchy with precursor/progeny populations that differ in molecular pathways conferring self-renewal, tumorigenicity and metastatic potential. We show that CSC-enriched CD44+CD24neg/low breast cancer cells comprise a functional hierarchy: a minor CD44+CD24low+ subpopulation generates CD44+CD24neg progeny with reduced self-renewal and tumorigenicity. In both triple negative breast cancer (TNBC) lines and primary dissociated tumor (DT) cultures, CD44+CD24low+ generate more tumor spheres, while CD44+CD24neg -generated spheres decline with serial passage. Tumor-initiating CSCs were more frequent in CD44+CD24low+: fewer cells yielded orthotopic xenograft tumors with reduced latency. Furthermore, metastasis arose exclusively from CD44+CD24low+ cells which preferentially express embryonic stem cell gene profiles and metastatic gene signatures. CD44+CD24low+ but not CD44+CD24neg cells express Notch-1 intracellular domain (N1-ICD) and a Notch-activated gene profile. N1-ICD transactivates SOX2 mediating Sox2-dependent increases in the percentaged of ALDH1+, CD44+CD24low+ and sphere forming cells. Gamma -Secretase Inhibitors (GSI) that block Notch pathway activation notably reduced serial sphere formation and xenograft growth from CD44+CD24low+ cells while CD44+CD24neg showed no response. While Notch family members are implicated in stem cell self-renewal many cancers, the existence of stem cell hierarchies such as that observed herein could limit potential GSI efficacy.

Keywords

breast cancer stem cells; metastasis; tumor-initiating; CD44; CD24; Notch1; Sox2

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