Malignant Precursor Cells Pre-Exist in Human Breast DCIS and Require Autophagy for Survival

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.

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

“…Exposure of cancer cells to an acidic environment plays a crucial function in promoting invasive capacity, metastasis, and resistance to apoptotic-inducing agents. Intriguingly, autophagy was shown to represent a major survival mechanism for cytogenetically abnormal neoplastic ductal carcinoma in situ cells (65). The capacity of these cells to form spheroids and generate tumors in SCID mice was abrogated upon treatment with the autophagy inhibitor chloroquine.…”

Surviving without oxygen: Hypoxia regulation of mammary morphogenesis and anoikis

“…In fact, current research has shown that while DCIS is non-invasive and clinically defined as "pre-cancerous," these types of lesions do indeed possess carcinoma precursor cells. 20,21 In a recent review, Espina and Liotta 22 address the malignant nature of DCIS and postulate that the stressful, hypoxic intraductal niche selects for the survival and expansion of a subset of genetically abnormal malignant progenitor cells. In accordance with this notion, our data offers activation of signaling pathways and suppression of Bim, Bmf and anoikis by hypoxia as means to enhance cell survival in conditions of low adhesion, such conditions exhibit a significant degree of architectural disorganization as compared to structures formed under normoxic conditions.…”

Effect of Increased Extracellular Ca++ on Microvesicle Production and Tumor Spheroid Formation

“…These concentric spheroid regions are generated by the gradients of nutrients, oxygen and growth factors that produce distinctive microenvironments within the tumor mass that are associated with in the build-up of acidic metabolic waste and hypoxic condition in the tumor core. Researchers have suggested that similar biophysical parameters may define the microenvironment of solid tumors in vivo and may account for some aspects of therapeutic sensitivity to chemotherapy and radiation [19].…”

“…Research studies by Espina et al [19] have shown that ex vivo organoid cultures of breast ductal carcinoma in situ (DCIS) spontaneously produce hundreds of spheroids in the first several weeks in culture that are capable of anchorage independent growth, stromal invasion and tumorgenicity in SCID mice. Moreover, these researchers observed that most of the organoid cultures of DCIS displayed microcalcification, which has been linked to the presence of high grade DCIS by mammography [20].…”