EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells
Akihiko Miyanaga,
Masaru Matsumoto,
Jessica A. Beck,
Izumi Horikawa,
Takahiro Oike,
Hirokazu Okayama,
Hiromi Tanaka,
Sandra S. Burkett,
Ana I. Robles,
Mohammed Khan,
Delphine Lissa,
Masahiro Seike,
Akihiko Gemma,
Hiroyuki Mano,
Curtis C. Harris
Affiliations
Akihiko Miyanaga
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Masaru Matsumoto
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Jessica A. Beck
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Izumi Horikawa
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Takahiro Oike
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Hirokazu Okayama
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Hiromi Tanaka
Medical and Molecular Genetics, Indiana University School of Medicine
Sandra S. Burkett
Molecular Cytogenetic Core Facility, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Ana I. Robles
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Mohammed Khan
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Delphine Lissa
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Masahiro Seike
Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School
Akihiko Gemma
Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School
Hiroyuki Mano
Division of Cellular Signaling, National Cancer Center Research Institute
Curtis C. Harris
Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Abstract Background Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. Methods Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. Results The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. Conclusions Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.
