Title: Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells

Abstract

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell linesmore » that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.« less

Authors:

Lee, Jonathan K. ^[1]; Garbe, James C. ^[1]; Vrba, Lukas ^[2]; Miyano, Masaru ^[1]; Futscher, Bernard W. ^[2]; Stampfer, Martha R. ^[3]; LaBarge, Mark A. ^[1]

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+ Show Author Affiliations

1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Division
2. Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Univ. of Arizona, Tucson, AZ (United States). College of Pharmacy. Dept. of Pharmacology and Toxicology
3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Division; Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center

Publication Date:

Wed Mar 11 00:00:00 EDT 2015

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

OSTI Identifier:

1629233

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

Frontiers in Cell and Developmental Biology

Additional Journal Information:

Journal Volume: 3; Journal ID: ISSN 2296-634X

Publisher:

Frontiers Media S.A.

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; human mammary epithelial cell; HMEC; breast cancer; immortalization; aging; stasis; intrinsic subtype