Killing Pre-invasive Breast Cancer by Targeting Autophagy: A New Vision for Chemoprevention

Abstract

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 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 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 proliferate and accumulate within the non-vascular intraductal space under hypoxic and metabolic stress. 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 generation of DCIS spheroids, suppressed "ex vivo" invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3K, and mTOR, eliminated cytogenetically abnormal spheroid forming cells, and abrogated xenograft tumor formation. A phase I/II clinical trial (PINC; Preventing Invasive breast Neoplasia with Chloroquine) was established for evaluating safety and efficacy of chloroquine phosphate to treat breast Ductal Carcinoma in Situ. Therapy that induces regression, or prevents progression, of pre-invasive lesions could comprise a new treatment strategy for pre-invasive cancers independent of hormone receptor status.