Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16INK4; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional “passenger” errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.

[1]  M. Stampfer,et al.  Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells. , 2014, Mutation research. Genetic toxicology and environmental mutagenesis.

[2]  U. Herbig,et al.  The replicometer is broken: telomeres activate cellular senescence in response to genotoxic stresses , 2014, Aging cell.

[3]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[4]  W. Gu,et al.  Differential effects on ARF stability by normal versus oncogenic levels of c-Myc expression. , 2013, Molecular cell.

[5]  F. Penault-Llorca,et al.  Early Telomere Shortening and Genomic Instability in Tubo-Ovarian Preneoplastic Lesions , 2013, Clinical Cancer Research.

[6]  M. Stampfer,et al.  An Integrated Human Mammary Epithelial Cell Culture System for Studying Carcinogenesis and Aging , 2013 .

[7]  H. Schatten.  Cell and Molecular Biology of Breast Cancer , 2013, Humana Press.

[8]  A. Borowsky,et al.  Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia. , 2012, Cancer research.

[9]  L. Tusell,et al.  Progressive Telomere Dysfunction Causes Cytokinesis Failure and Leads to the Accumulation of Polyploid Cells , 2012, PLoS genetics.

[10]  Chi V Dang,et al.  MYC on the Path to Cancer , 2012, Cell.

[11]  M. Stampfer,et al.  Oncogenes induce senescence with incomplete growth arrest and suppress the DNA damage response in immortalized cells , 2011, Aging cell.

[12]  James M. Roberts,et al.  Separation of telomerase functions by reverse genetics , 2011, Proceedings of the National Academy of Sciences.

[13]  C. Perou,et al.  Defining the cellular precursors to human breast cancer , 2011, Proceedings of the National Academy of Sciences.

[14]  Martha R. Stampfer,et al.  Epigenetic regulation of normal human mammary cell type-specific miRNAs. , 2011, Genome research.

[15]  Christopher D. Putnam,et al.  Aneuploidy Drives a Mutator Phenotype in Cancer , 2011, Science.

[16]  L. Loeb,et al.  Human cancers express mutator phenotypes: origin, consequences and targeting , 2011, Nature Reviews Cancer.

[17]  M. Jackson,et al.  TGF-β signaling engages an ATM-CHK2-p53–independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells , 2011, Proceedings of the National Academy of Sciences.

[18]  E. Lander,et al.  Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate. , 2011, Cell stem cell.

[19]  Victor V Lobanenkov,et al.  BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells , 2010, Nucleic acids research.

[20]  Virginia Espina,et al.  What is the malignant nature of human ductal carcinoma in situ? , 2011, Nature Reviews Cancer.

[21]  A. Ashworth,et al.  BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. , 2010, Cell stem cell.

[22]  D. Carrasco,et al.  PIK3CA mutations in in situ and invasive breast carcinomas. , 2010, Cancer research.

[23]  T. Halazonetis,et al.  Genomic instability — an evolving hallmark of cancer , 2010, Nature Reviews Molecular Cell Biology.

[24]  M. Gönen,et al.  Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. , 2010, The Journal of clinical investigation.

[25]  P. Argani,et al.  The alternative lengthening of telomeres phenotype in breast carcinoma is associated with HER-2 overexpression , 2009, Modern Pathology.

[26]  Heidi S Feiler,et al.  Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. , 2009, Cancer research.

[27]  Robert E. Kingston,et al.  Mechanisms of Polycomb gene silencing: knowns and unknowns , 2009, Nature Reviews Molecular Cell Biology.

[28]  S. Fox,et al.  Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers , 2009, Nature Medicine.

[29]  M. Stampfer,et al.  Stepwise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization. , 2009, Cancer research.

[30]  G. Evan,et al.  Distinct thresholds govern Myc's biological output in vivo. , 2008, Cancer cell.

[31]  K. Catania,et al.  Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan , 2008, Aging cell.

[32]  Angel Porgador,et al.  Cell type-specific DNA methylation patterns in the human breast , 2008, Proceedings of the National Academy of Sciences.

[33]  A. Sparks,et al.  The Genomic Landscapes of Human Breast and Colorectal Cancers , 2007, Science.

[34]  T. Tlsty,et al.  Inactivation of p53 Function in Cultured Human Mammary Epithelial Cells Turns the Telomere-Length Dependent Senescence Barrier from Agonescence into Crisis , 2007, Cell cycle.

[35]  R. Young,et al.  A Chromatin Landmark and Transcription Initiation at Most Promoters in Human Cells , 2007, Cell.

[36]  J. Herman,et al.  hTERT is expressed in cancer cell lines despite promoter DNA methylation by preservation of unmethylated DNA and active chromatin around the transcription start site. , 2007, Cancer research.

[37]  Ajay N. Jain,et al.  Genomic and transcriptional aberrations linked to breast cancer pathophysiologies. , 2006, Cancer cell.

[38]  Jianmin Zhang,et al.  p16INK4a modulates p53 in primary human mammary epithelial cells. , 2006, Cancer research.

[39]  J. Barrett,et al.  Differential cis-regulation of human versus mouse TERT gene expression in vivo: identification of a human-specific repressive element. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Egozcue,et al.  Telomere dysfunction drives chromosomal instability in human mammary epithelial cells , 2005, Genes, chromosomes & cancer.

[41]  K. Chin,et al.  In situ analyses of genome instability in breast cancer , 2004, Nature Genetics.

[42]  P. Argani,et al.  Telomere Shortening Occurs Early During Breast Tumorigenesis: A Cause of Chromosome Destabilization Underlying Malignant Transformation? , 2004, Journal of Mammary Gland Biology and Neoplasia.

[43]  D. Pellman,et al.  From polyploidy to aneuploidy, genome instability and cancer , 2004, Nature Reviews Molecular Cell Biology.

[44]  P. Yaswen,et al.  Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines , 2003, Oncogene.

[45]  S. Lowe,et al.  Rb-Mediated Heterochromatin Formation and Silencing of E2F Target Genes during Cellular Senescence , 2003, Cell.

[46]  R. Salunga,et al.  Gene expression profiles of human breast cancer progression , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Hicks,et al.  Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. , 2002, Cancer research.

[48]  L. Chin,et al.  Telomere dysfunction provokes regional amplification and deletion in cancer genomes. , 2002, Cancer cell.

[49]  M. Blasco.  Telomerase beyond telomeres , 2002, Nature Reviews Cancer.

[50]  W. Hahn,et al.  A Two-Stage, p16INK4A- and p53-Dependent Keratinocyte Senescence Mechanism That Limits Replicative Potential Independent of Telomere Status , 2002, Molecular and Cellular Biology.

[51]  P. Yaswen,et al.  Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion , 2002, Oncogene.

[52]  L. Holmberg,et al.  Tumour markers in breast carcinoma correlate with grade rather than with invasiveness , 2001, British Journal of Cancer.

[53]  P. Yaswen,et al.  Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[54]  J W Gray,et al.  The ZNF217 gene amplified in breast cancers promotes immortalization of human mammary epithelial cells. , 2001, Cancer research.

[55]  Thea D. Tlsty,et al.  Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes , 2001, Nature.

[56]  P. Yaswen,et al.  Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells , 1999, Oncogene.

[57]  W. Hahn,et al.  Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[58]  T. Kiyono,et al.  Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells , 1998, Nature.

[59]  A. Brenner,et al.  Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation , 1998, Oncogene.

[60]  S. Sukumar,et al.  Careful histological confirmation and microdissection reveal telomerase activity in otherwise telomerase-negative breast cancers. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[61]  A. Maitra,et al.  Telomerase enzyme activity and RNA expression during the multistage pathogenesis of breast carcinoma. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[62]  P. Yaswen,et al.  Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells. , 1997, Molecular biology of the cell.

[63]  A. Brenner,et al.  Chromosome 9p allelic loss and p16/CDKN2 in breast cancer and evidence of p16 inactivation in immortal breast epithelial cells. , 1995, Cancer research.

[64]  C. Greider.  Telomeres, telomerase and senescence , 1990, BioEssays : news and reviews in molecular, cellular and developmental biology.

[65]  Didier Picard,et al.  Chimaeras of Myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells , 1989, Nature.

[66]  M. Stampfer,et al.  Human mammary epithelial cells in culture: differentiation and transformation. , 1988, Cancer treatment and research.

[67]  M. Stampfer,et al.  Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[68]  S. L. Hammond,et al.  Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[69]  Alexandra Eitel.  Senescence , 2014, British medical journal.