Abstract
Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G(1) phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) and proceeded independently of p53, p14(Arf), and p16(Ink4a). Loss of p21(Cip1/Waf1) or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21(Cip1/Waf1) and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21(Cip1/Waf1).
Highlights
Loss of heterozygosity of chromosomal region 3p21.3 is an early and common event in the development of lung cancer and other human cancers
RASSF1 is one of the genes encoded at this locus, and the expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is frequently lost in cancers of the lung and other organs [1]
To study the functional consequences of RASSF1A expression in human cells, we made use of a tetracycline-inducible expression system [32] and the A549 lung cancer cell line, which is deficient in endogenous RASSF1A, p14Arf, and p16Ink4a expression
Summary
Loss of heterozygosity of chromosomal region 3p21.3 is an early and common event in the development of lung cancer and other human cancers. These findings indicate that RASSF1A exerts its tumor-suppressive activity in the present cancer model mainly by induction of cell cycle arrest and senescence, but not through apoptosis. RASSF1A still up-regulated p21Cip1/Waf1 in A549 cells that display reduced p53 levels through expressing the HPV16 oncogene E6, which destabilizes p53, as well as in NCI-H1299 lung cancer cells, which are homozygously deleted for p53 (Fig. 3B).
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