[log in to unmask]" type="cite">Dissertation Defense Announcement
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
            Fairfax Campus
            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 campus.
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 attend.


ABSTRACT:
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 breast tissue.
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.


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