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Various P53 Mutant Proteins Differently Regulate the Ras Circuit to Induce A 3144 Research Article Various p53 mutant proteins differently regulate the Ras circuit to induce a cancer-related gene signature Hilla Solomon1,*, Yosef Buganim1,*, Ira Kogan-Sakin1, Leslie Pomeraniec1, Yael Assia1, Shalom Madar1, Ido Goldstein1, Ran Brosh1, Eyal Kalo1, Tsevi Beatus2, Naomi Goldfinger1 and Varda Rotter1,` 1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel 2Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel *These authors contributed equally to this work `Author for correspondence ([email protected]) Accepted 13 February 2012 Journal of Cell Science 125, 3144–3152 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.099663 Summary Concomitant expression of mutant p53 and oncogenic Ras, leading to cellular transformation, is well documented. However, the mechanisms by which the various mutant p53 categories cooperate with Ras remain largely obscure. From this study we suggest that different mutant p53 categories cooperate with H-Ras in different ways to induce a unique expression pattern of a cancer-related gene signature (CGS). The DNA- contact p53 mutants (p53R248Q and p53R273H) exhibited the highest level of CGS expression by cooperating with NFkB. Furthermore, the Zn+2 region conformational p53 mutants (p53R175H and p53H179R) induced the CGS by elevating H-Ras activity. This elevation in H-Ras activity stemmed from a perturbed function of the p53 transcription target gene, BTG2. By contrast, the L3 loop region conformational mutant (p53G245S) did not affect CGS expression. Our findings were further corroborated in human tumor-derived cell lines expressing Ras and the aforementioned mutated p53 proteins. These data might assist in future tailor-made therapy targeting the mutant p53–Ras axis in cancer. Key words: Ras, Gain of function, Mutant p53 Introduction function, such as transcriptional activation (e.g. MYC, MDR1, The development of tumors is characterized by the accumulation NFkB2), transcriptional repression (e.g. ATF3, CD-95 and of mutations in tumor-suppressor genes and in proto-oncogenes. MST1), unique interaction with specific DNA motives (MAR), Two of the most frequently mutated genes in human cancer are epigenetic modification and interactions with other proteins (e.g. Journal of Cell Science the gene encoding the tumor-suppressor p53 (TP53) and the p63, p73, NFY) (Sigal and Rotter, 2000; Li and Prives, 2007; proto-oncogene Ras (Bos, 1989; Hollstein et al., 1991). Lozano, 2007; Weisz et al., 2007a; Brosh and Rotter, 2009; p53 is a transcription factor that accumulates and is activated in Buganim and Rotter, 2009). response to stress signals. Following its activation, p53 induces Another protein frequently manipulated by cancer cells is the processes such as cell cycle arrest, programmed cell death and Ras proto-oncogene, which becomes hyperactive because of a DNA repair, thereby, guarding the cell from malignancy (Levine, point mutation during tumor development. There are three pivotal 1997). In most cancer cells the p53 pathway is defective, usually Ras genes in the human genome: HRAS, KRAS and NRAS.In because of genetic mutations within the p53 sequence (Guimaraes spite of their high similarity in sequence and function, H-Ras was and Hainaut, 2002). Most of the mutations in p53 are missense found to be a more potent mediator of cellular transformation in mutations that reside within the p53 DNA binding domain (DBD), fibroblast cells, whereas K-Ras and N-Ras are more active in causing an impaired binding of p53 to the DNA. The missense epithelial cells and hematopoetic cells, respectively (Maher et al., mutations can be divided into two rudimentary sub-groups 1995). Ras activation depends on its binding to GDP (the non- according to their impact on the DBD folding. The first group active state) or GTP, which enables its active conformation consists of DNA-contact mutations (e.g. R248, R273), which (Malumbres and Barbacid, 2003). Switching to the active state is affect amino acids that directly interact with DNA while retaining controlled by the guanine nucleotide exchange factor (RasGEF) wild-type p53 conformation. The second group includes p53 enzyme family, which facilitates the replacement of GDP by conformational mutations (e.g. R175, H179), which alter the GTP. Shifting to the non-active state is regulated by the GTPase scaffold that orients the structure of the DNA-binding interface activating protein (RasGAP) enzyme family, which facilitates (Cho et al., 1994). The observation that mutant p53 is accumulated GTP hydrolysis. When mutated, Ras is less sensitive to regulation in tumors, raised the possibility that mutant p53 plays an active by RasGAPs and therefore remains constitutively active. Over- role in the process of malignant transformation. Indeed, it is now activated Ras causes uncontrolled proliferation, survival and accepted that mutant p53 exerts its activity in either a dominant- tumor aggressiveness by induction of signaling pathways such as negative manner, in which it inhibits the normal activity of the MAPK, PI3K and RALGDS (Downward, 2003). wild-type p53, or by a gain-of-function mechanism, in which Early studies in cancer research revealed that mutant p53 mutant p53 undertakes new oncogenic activities. Several and Ras oncogene cooperate to induce cellular transformation mechanisms were suggested to account for mutant p53 gain of (Eliyahu et al., 1984; Parada et al., 1984). Later studies reported Various roles of p53 mutants in cancer 3145 several processes regulated by both p53 and Ras, such as cell sequence], an shCon control vector (vector expressing an shRNA motility, proliferation and survival (Boiko et al., 2006; Song et al., against the mouse Noxa coding sequence) or with a vector 2007; Xia and Land, 2007; Meylan et al., 2009). However, expressing one of the five most frequent p53 mutant forms hitherto, a mechanism underlying the cooperation between (p53R175H, p53H179R, p53G245S, p53R248Q and p53R273H; Fig. 1A). the common forms of mutant p53 and activated Ras, has not These p53 mutations represent three principal categories: been addressed. Because p53 is commonly mutated during conformational mutations within the Zn2+ region (p53R175H, carcinogenesis, and mutated forms of p53 further facilitate p53H179R), conformational mutation within the L3 loop region transformation using a wide range of mechanisms (Buganim and (p53G245S) and DNA-contact mutations (p53R248Q, p53R273H). Rotter, 2009), we decided to focus on the role of the different Next, we compared the expression levels of the CGS in the mutant p53 categories in the crosstalk with Ras. established cell lines using three representative genes (CXCL1, Thus, the main motivation of this work was to identify the IL1B, MMP3). In agreement with our previous data, we found specific molecular details that underlie the crosstalk between Ras that cells expressing the H-RasV12 oncogene along with p53 and the various mutant p53 categories. knock down (hereafter referred to as Ras/shp53 cells) upregulated Recently, we established an in vitro transformation model expression of the CGS both at the mRNA and protein levels and performed a wide genomic profiling to identify clusters of compared with their control counterparts (Con/shCon, Ras/ genes that are related to the different steps of transformation shCon, Con/shp53; Fig. 1B,C). Interestingly, the different p53 (Milyavsky et al., 2005; Buganim et al., 2010). In this analysis we mutant forms had distinct expression patterns. Whereas the Zn2+ identified the cancer-related gene signature (CGS) that was of region conformational mutant (p53R175H, p53H179R) cells particular interest because it was synergistically upregulated upregulated the CGS to a similar level as observed in cells in when H-RasV12 and WTp53 inactivating peptide (GSE56) were which p53 was knocked down, the DNA-contact mutant concomitantly expressed in the cells (Milyavsky et al., 2005; (p53R248Q and p53R273H) cells upregulated the CGS to a much Buganim et al., 2010). The CGS mainly consists of secreted higher extent. Interestingly, the L3 loop region conformational molecules that were shown to have pro-cancerous functions, and mutant (p53G245S) cells exhibited only a minor effect on the CGS at least parts of them were shown to be induced by activated H- expression (Fig. 1B,C). Ras (Sternlicht et al., 1999; Dhawan and Richmond, 2002; To further strengthen this observation, we examined the Mendes et al., 2005; Minn et al., 2005; Wang et al., 2006; mutant-p53-dependent expression of the CGS in six human- Apte and Voronov, 2008). The CGS consists of chemokines tumor-derived cell lines that endogenously express different [chemokine (C-X-C motif) ligand 1, 2 and 3 (CXCL1, CXCL2 mutant p53 types. Accordingly, following mutant p53 knock and CXCL3)], interleukins (IL-1b, IL-6 and CSF2) and extra- down, the representative CGS genes (CXCL1, IL1B, IL8) were cellular matrix (ECM)-related proteins [matrix metallopeptidase downregulated only in cells expressing the p53 DNA-contact 3 (MMP-3), CLECSF2 and TREM-1]. For further investigation, mutations (SW-620, SW-480, NCI-H322), whereas p53 knock we decided to focus on representative genes of this signature, down hardly affected the CGS expression in cells expressing the mainly CXCL1, IL1B and MMP3, that represent the different sub- p53 conformational mutants (NCI-H23, Hs-578-T, SKBR-3; groups of the CGS. In this study we show that the different p53 supplementary
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