RASSF1A Mediates p21^Cip1/Waf1-Dependent Cell Cycle Arrest and Senescence through Modulation of the Raf-MEK-ERK Pathway and Inhibition of Akt

¹Center for Biomedicine and Medical Technology, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany; ²Department of Medicine (Cancer Research), West German Cancer Center, University Duisburg-Essen, Essen, Germany; ³Department of Pathology, Johannes Gutenberg University, Mainz, Germany; and ⁴Institute for Genetics, Justus Liebig University, Giessen, Germany

^* To whom correspondence should be addressed. E-mail: martin.schuler{at}uk-essen.de.

	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 G₁ 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. [Cancer Res 2009;69(5):1748–57]

Key Words: p21^Cip1/Waf1, RASSF1A, MAPK, Akt, Senescence

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