Abstract
BackgroundWhile 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.Methodology and Principal FindingsWe 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.ConclusionsCytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.
Highlights
While the transition from in situ to invasive cancer is central to the origin of the malignant phenotype, very little is known about the time of onset, and the triggering mechanism, that switches in situ neoplastic lesions to overt invasive carcinoma in the human breast
Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human ductal carcinoma in situ (DCIS) and require cellular autophagy for survival
It is generally accepted that women diagnosed with DCIS remain at high risk for subsequent development of invasive carcinoma, with lesion size, degree of nuclear atypia and the presence of comedo necrosis being histopathological factors of DCIS identified as affecting this risk of recurrence [6,7]
Summary
While the transition from in situ to invasive cancer is central to the origin of the malignant phenotype, very little is known about the time of onset, and the triggering mechanism, that switches in situ neoplastic lesions to overt invasive carcinoma in the human breast. DCIS accounts for an estimated 30% of the 185,000 breast cancers detected by mammography each year [4,5] There is both clinical and experimental evidence to suggest that DCIS is a precursor lesion to most, if not all, invasive carcinoma. The critical unanswered biologic questions, addressed in this study, are: Do invasive, cytogenetically abnormal neoplastic cells pre-exist in the pure intraductal DCIS lesion prior to the overt histologic transition to invasive carcinoma? If such precursor carcinoma cells pre-exist in DCIS, does autophagy support their survival in the face of nutrient deprivation and hypoxia?. 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
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