Abstract B008: Efficient immortalization of normal human mammary epithelial cells using two pathologically relevant agents does not require gross genomic alterations
Authors: Stampfer Martha , Vrba Lukas , Fuchs Laura , Brothman Arthur , LaBarge Mark , Futscher Bernard , Garbe James 
Published: May 2015

Abstract
Most human carcinomas contain genomically unstable cells with multiple genomic errors, and express telomerase activity. Widespread instability can first be observed in vivo at the pre-malignant stage, such as DCIS, and in vitro as finite lifespan cells with critically shortened telomeres approach replicative senescence. We have proposed that telomere-dysfunction induced genomic instability in pre-malignant finite cells may be needed to generate the errors required for telomerase reactivation and immortalization, while also generating many additional “passenger” errors that will be carried forward into resulting carcinomas. Genomic errors that occur prior to immortalization may influence the cancer phenotype and form the basis for ongoing genomic instability in the malignant cells. Further, the prevalence of many genomic errors in primary human carcinomas makes it difficult to identify the driver errors responsible for immortalization using only in vivo tissues. Although immortalization is crucial for human carcinoma development, little is known about the processes that allow telomerase reactivation during malignant progression. This knowledge gap is due, in part, to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. For example, most murine models lack a significant replicative senescence/immortalization barrier; immortalization employing hTERT precludes examination of the errors responsible for telomerase reactivation during carcinogenesis; a reproducible human mammary epithelial cell immortalization model employing agents implicated in in vivo carcinogenesis has been lacking. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the two main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier, enforced by elevated levels of p16INK4A, was bypassed using shRNA to p16. The replicative senescence barrier due to critically shortened telomeres was bypassed in post-stasis HMEC by c-Myc transduction. These results demonstrate that just two pathologically relevant oncogenic agents are sufficient to immortally transform normal human epithelial cells. The non-clonal immortalized lines generated by direct targeting of the senescence barriers exhibited normal karyotypes at early passages, supporting our hypothesis that the genomic instability commonly present in human carcinomas may not be required per se for immortal transformation, but is needed to generate errors that overcome tumor suppressive barriers. This method of achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression. Additionally, immortalization, associated with telomerase reactivation, is necessary for progression of all subtypes of human beast carcinomas, and could therefore be a valuable therapeutic target. Our reproducible method for immortalization can permit exploration of agents that may prevent or reverse the immortalization process. That transduction of just shRNA to p16 and c-Myc can immortally transform normal HMEC validates our model of the two main tumor-suppressive senescence barriers (Garbe et al, Cancer Res 2009): stasis, a stress-associated arrest independent of telomere length and extent of replication, and replicative senescence due to telomere dysfunction.
Citation Format: Martha Stampfer, Lukas Vrba, Laura Fuchs, Arthur Brothman, Mark LaBarge, Bernard Futscher, James Garbe. Efficient immortalization of normal human mammary epithelial cells using two pathologically relevant agents does not require gross genomic alterations. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B008.