High-resolution mapping of molecular events associated with immortalization, transformation, and progression to breast cancer in the MCF10 model

Abstract

A comprehensive and consistent picture of the genetic changes that underlie breast cancer initiation, development, and progression remains unresolved. The MCF10 series of cell lines represents many steps in that progression. We performed high resolution mapping of the MCF10 series of cell lines to identify specific gene targets to elucidate the molecular correlates of immortalization, development, and progression of breast cancer at the level of individual genes. We evaluated the initial untransformed outgrowths (MCF-10MS and MCF-10A) with six transformed cell lines with benign proliferations (MCF-10AT1, MCF-10AT1kcl2), carcinoma in situ (MCF-10CA1h cl13), and invasive carcinoma (MCF-10CA1h cl2, MCF-10CA1a cl1, MCF-10CA1d cl1). Losses and gains of loci at 112 unique human genome sites were interrogated using the multiplex ligation-dependent probe amplification assay (MLPA). Cytogenetic alterations in the four benign progenitors that persisted in the CIS and invasive cell lines corresponded to gains and losses of genes by MLPA. MCF-10MS had only normal gene copies. The untransformed MCF-10A had cytogenetic gain of 5q13-qter with corresponding gains of the IL3, IL4 and IL12B genes at 5q31-q33; gain of distal 19q12-qter was reflected in gains in KLK3 and BAX gene loci at 19q13-q13.4. The observed genic gain of cMYC at 8q24.12 was not indicated by cytogenetics. The apparently balanced t(3;9) component of the t(3;9)(p13;p22)t(3;5)(p26;q31) resulted in complete loss of the CDKN2A and CDKN2B genes at 9p21. Additional clonal cytogenetic changes in the DCIS cell line (MCF-10A1h cl13) involving chromosomes 1, 3 and 10 persisted in the invasive progeny, with gain of corresponding genes at 1p13 (BCAR2, BCAR3, NRAS, TGFB2), at 3p12–13 (IL12A), and 3q21–27 (MME, PIK3CA, BCL6). Our study adopted a comprehensive exploration of genetic changes using high resolution molecular probes applied to the MCF10 family of cell lines to identify individual genes in a continuum starting from normal breast epithelial cells and progressing through immortalization, transformation and invasive malignancy. Homozygous loss of CDKN2A and CDKN2B genes and gain of MYC were initiating immortalization events. Transformation and progression to malignancy event were marked by gains of IL13, VEGF, HRAS, TRAF2, and BCAS2, IL12A, and MME, respectively.