title: Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival.
abstract: BACKGROUND
  While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment.
  
  
  RESULTS
  We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, 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; c) 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 source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy 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.
  
  
  CONCLUSIONS
  Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.
authors.affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, United States of America. vespina@gmu.edu
authors.forename: Virginia
authors.initials: V
authors.lastname: Espina
authors.forename: Brian D
authors.initials: BD
authors.lastname: Mariani
authors.forename: Rosa I
authors.initials: RI
authors.lastname: Gallagher
authors.forename: Khoa
authors.initials: K
authors.lastname: Tran
authors.forename: Stacey
authors.initials: S
authors.lastname: Banks
authors.forename: Joy
authors.initials: J
authors.lastname: Wiedemann
authors.forename: Heather
authors.initials: H
authors.lastname: Huryk
authors.forename: Claudius
authors.initials: C
authors.lastname: Mueller
authors.forename: Luana
authors.initials: L
authors.lastname: Adamo
authors.forename: Jianghong
authors.initials: J
authors.lastname: Deng
authors.forename: Emanuel F
authors.initials: EF
authors.lastname: Petricoin
authors.forename: Lucia
authors.initials: L
authors.lastname: Pastore
authors.forename: Syed
authors.initials: S
authors.lastname: Zaman
authors.forename: Geetha
authors.initials: G
authors.lastname: Menezes
authors.forename: James
authors.initials: J
authors.lastname: Mize
authors.forename: Jasbir
authors.initials: J
authors.lastname: Johal
authors.forename: Kirsten
authors.initials: K
authors.lastname: Edmiston
authors.forename: Lance A
authors.initials: LA
authors.lastname: Liotta
mesh_terms: D000818:Animals; D001343:Autophagy; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000473:pathology*; D002470:Cell Survival; D002869:Chromosome Aberrations; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009361:Neoplasm Invasiveness; D014411:Neoplastic Stem Cells / Q000473:pathology* / Q000637:transplantation; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured