Identification of a Novel Germ Line Variant Hotspot Mutant p53-R175L in Pediatric Adrenal Cortical Carcinoma Alina Nico West, 1,3 Raul C. Ribeiro, 4,7 Jesse Jenkins, 5 Carlos Rodriguez-Galindo, 4,7 Bonald C. Figueiredo, 8 Richard Kriwacki, 2,6 and Gerard P. Zambetti 2,3 Departments of 1 Interdisciplinary Sciences and 2 Molecular Sciences, University of Tennessee Health Science Center; Departments of 3 Biochemistry, 4 Hematology and Oncology, 5 Pathology, and 6 Structural Biology, and the 7 International Outreach Program, St. Jude Children’s Research Hospital, Memphis, Tennessee; and 8 Department of Pediatrics, Center for Molecular Genetics and Cancer Research in Children CEGEMPAC, Federal University of Parana ´, Curitiba PR, Brazil Abstract Hotspot mutations in the p53 tumor suppressor gene result in the disruption of DNA contact points or alter the overall structure of the protein to prevent DNA binding. When inherited, hotspot mutants are associated with Li-Fraumeni syndrome (LFS), a familial cancer predisposition. One of the most common hotspot mutations occurs at codon 175, resulting in an arginine to histidine substitution. We have identified a novel germ line variant of the 175 mutant (Arg to Leu; R175L) in a pediatric patient who developed adrenal cortical carcinoma. Surprisingly, the family is not tumor prone or associated with LFS. In vitro , the R175L mutant displayed an attenuated tumor suppressor activity in the regulation of transcription,colonyformation,andapoptosiswhencompared with wild-type p53 and the R175H mutant. These findings suggest that p53-R175L retains sufficient activity to suppress LFS, but not adrenal cortical carcinoma. Therefore, not all hotspot mutants are functionally equivalent and the biochem- ical nature of the mutant may significantly influence clinical outcome. The implications of these results for genetic counseling are discussed. (Cancer Res 2006; 66(10): 5056-62) Introduction Thep53tumorsuppressorplaysaprominentroleintheprevention of cancer by functioning as a transcription factor that induces downstreamtargetswhichnegativelycontrolcellgrowth(e.g.,p21 Cip1 ) and viability (e.g., Puma and Noxa; refs. 1–3). It is, therefore, not surprising that half of all human cancers have sustained inactivating somatic mutations in p53. Several residues are more frequently targeted than others and these are referred to as hotspot mutations (for review, see ref. 4). Interestingly, hotspot mutations disrupt criti- cal DNA contact points or alter the structure in such a way that the protein no longer binds DNA in a sequence-specific manner (5). In either case, prototypical hotspot mutants are unable to efficiently induce target gene expression, cell cycle arrest, and cell death. Germ line p53 hotspot mutations strongly predispose carriers to cancer as children or young adults. Indeed, epidemiology studies estimate that f70% of males and 100% of females who inherit a p53 mutation will develop cancer, including tumors of the breast, brain, soft tissue, bone, blood, and adrenal cortex (6). The extraordinarily high occurrence of tumors associated with germ line p53 mutations is referred to as Li-Fraumeni syndrome (LFS; ref. 7). LFS is formally defined by a proband with a sarcoma before the age of 45 years and a first-degree relative with any cancer under 45 years of age, and an additional first-degree or second-degree family member with any cancer before 45 years or sarcoma at any age (8). Subsequent studies identified a variation of LFS, referred to as Li-Fraumeni-like (LFLS) syndrome, which includes a proband with any childhood cancer or a sarcoma, brain tumor, or adrenal cortical tumor before 45 years of age, and a first-degree or second- degree relative with a LFS-type tumor and an additional first- degree or second-degree relative with any cancer before 60 years of age (9). A telling feature of LFS and LFLS is a child who has developed an adrenal cortical tumor, although other mechanisms (e.g., Beckwith-Wiedemann syndrome) are also recognized to promote childhood adrenal cortical tumorigenesis. Pediatric adrenal cortical tumors are extremely rare, with an annual worldwide incidence of 0.3 to 0.4 per million children under the age of 15 (10). Because childhood adrenal cortical tumors often arise within LFS and LFLS families, they are usually associated with a germ line p53 mutation (11). In many of these cases, the proband has inherited a hotspot p53 mutation. However, childhood adrenal cortical tumors can also occur outside the context of LFS/LFLS and be associated with a constitutional mutant p53 allele (12). For example, recent studies identified a group of pediatric adrenal cortical tumor patients from southern Brazil who inherited a mutation in exon 10, corresponding to an arginine to histidine substitution at amino acid 337 (R337H; ref. 13). Interestingly, the mutation occurs within the COOH-terminal oligomerization domain and not the DNA-binding region. Although the mutation could be tracked through multiple generations within the adrenal cortical tumor families, there were no reports of sarcoma or indications of an increased susceptibility to cancer in general. The R337H mutation strongly predisposes carriers to adrenal cortical tumors, but not to LFS or any other tumors (14). Interestingly, the histidine substitution at this site within the oligomerization domain alters the stability of the protein structure in a pH- dependent manner, which presumably forms the basis for the tumor specificity of this particular mutant (15). The overall structure of p53 relies on arginine 175 (Arg 175 ), which is located in the L2 loop of the DNA binding domain (5). Arg 175 mediates the interaction between the L2 and L3 loops to maintain structural stability. Substitution of histidine for arginine at amino acid 175 (R175H) is one of the most common somatic mutations of p53 detected in human cancers (16, 17). The mutant R175H DNA binding domain is unable to bind specifically to the gadd45 promoter, is significantly less thermodynamically stable than the wild-type domain, and is predicted to be completely denatured at 37jC. Furthermore, full-length mutant p53 R175H Requests for reprints: Gerard P. Zambetti, Department of Biochemistry, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: 901-495-3429; Fax: 901-525-8025; E-mail: gerard.zambetti@stjude.org. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-4580 Cancer Res 2006; 66: (10). May 15, 2006 5056 www.aacrjournals.org Research Article Research. on December 27, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from