Abstract #LB-110: RASSF1A mediates p21Cip1/Waf1-dependent cell cycle arrest and senescence through modulation of the Raf-MEK-ERK pathway and inhibition of Akt in vitro and in a double-conditional xenograft model

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 impacts on 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 tetracycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G1 phase and senescence. RASSF1A-mediated growth inhibition was accompanied by upregulation of the Cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 and proceeded independently of p53, p14Arf and p16Ink4a. Loss of p21Cip1/Waf1 or co-expression of the human papilloma virus (HPV) 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A impacted on mitogen-activated protein kinase (MAPK) and protein kinase B/Akt (Akt) signaling to upregulate p21Cip1/Waf1 and to facilitate its nuclear localization. To further investigate the inhibitory effect RASSF1A on Akt-signaling, we generated double-conditional A549 cells co-expressing tetracycline-inducible RASSF1A and tamoxifen-activatible membrane-targeted Akt (myr-Akt-ERtam). Using this cellular model we observed that RASSF1A leads to inhibition of tumor cell growth even in the presence of activated myr-Akt-ERtam. To further investigate this effect in vivo, A549 cells double-conditional for RASSF1A and myr-Akt-ERtam were injected intravenously into NOD/SCID mice. We observed that tumors of double-conditional A549 cells formed in the lungs of mice treated with doxycycline and tamoxifen display less Ser473-phosphorylated Akt than tumors in the lungs from mice only treated with tamoxifen. Furthermore double-immunofluorescence staining of paraffin-sections for detection of RASSF1A and phospho-AktSer473 in xenografts tumors from doxycycline and tamoxifen treated mice reveal reciprocal staining patterns of RASSF1A and phospho-AktSer473 confirming a function of RASSF1A as Akt inhibitor. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21Cip1/Waf1 caused by modulation of the Raf-MEK-ERK pathway and inhibition of Akt.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-110.