[log in to unmask]" type="cite">Dissertation
To: The George Mason University Community
Candidate: Virginia Espina
Program: PhD Biosciences
Date: Friday April 19, 2013
Time: 2:30 p.m.
Place: George Mason University
Research Hall, Room 161
Committee Chair: Dr. Lance Liotta
Committee members: Dr. Calvin Carpenter, Dr. Robin Couch,
Dr. Kirsten Edmiston, Dr. Brian D. Mariani
Title: "Killing Pre-Invasive Breast Cancer by Targeting Autophagy:
A New Vision for Chemoprevention"
The dissertation is on reserve in the Johnson Center Library, Fairfax
The doctoral project will not be read at the meeting, but should be
read in advance.
All members of the George Mason University community are invited to
All invasive breast cancer is thought to be preceded by a pre-invasive
state in which cells accumulate within the breast ductal niche. Breast
cancer progression is thought to be a multi-step process involving a
continuum of changes from a normal phenotype through hyperplastic
lesions, carcinoma in situ, invasive carcinoma, to metastatic disease.
Previously it was assumed that the invasive phenotype acquired major
genetic changes during the phenotypic transition from ductal carcinoma
in situ (DCIS) to invasive carcinoma. In direct contradiction to this
previous assumption, herein we demonstrate, for the first time, the
pre-existence of genetically abnormal, tumorigenic carcinoma progenitor
cells within human breast DCIS lesions.
Human DCIS cells were cultivated ex vivo without a priori enzymatic
treatment or sorting. The DCIS organoid cultures induced the emergence
of neoplastic epithelial cells exhibiting the following
characteristics: a) spontaneous generation of hundreds of spheroids and
duct-like 3-D structures in culture within 2-4 weeks, b) tumorigenicity
in NOD/SCID mice, and c) in vitro migration and invasion of autologous
breast stroma. Proteomic characterization revealed that DCIS cells
up-regulate signaling pathways directly, and indirectly, linked to
cellular autophagy. Cells that proliferate and accumulate within the
non-vascular intraductal space are under severe hypoxic and metabolic
stress. Pre-invasive cells must adapt to hypoxic stress within the duct
in order to survive and proliferate. Autophagy was found to be required
for survival and anchorage independent growth, in the patient’s
original DCIS lesion and the mouse xenograft. Molecular karyotyping
demonstrated DCIS cells to be cytogenetically abnormal (copy number
loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to
the normal karyotype of the non-neoplastic cells in the patient’s
To demonstrate the dependence of the cytogenetically abnormal DCIS
cells on autophagy as a survival mechanism, primary human DCIS cell
cultures were treated with chloroquine phosphate, a lysosomotropic
inhibitor of autophagy. Chloroquine treatment completely suppressed the
generation of DCIS spheroids/3-D structures, suppressed ex vivo
invasion of autologous stroma, induced apoptosis, suppressed autophagy
associated proteins including Atg5, AKT/PI3 Kinase, and mTOR,
eliminated cytogenetically abnormal spheroid forming cells from the
organ culture, and abrogated xenograft tumor formation.
With the broad goal of arresting all breast cancer at the non-invasive,
non-lethal stage, a phase I/II clinical trial (PINC; Preventing
Invasive breast Neoplasia with Chloroquine) was established for
clinical evaluation of the safety and efficacy of chloroquine phosphate
as a strategy to treat human breast Ductal Carcinoma in Situ (DCIS).
Therapy that induces regression, or prevents progression, of occult or
overt pre-invasive lesions could comprise a new treatment strategy for
pre-invasive cancers independent of hormone receptor status.