Genetic variants of Tgfb1 act as context-dependent modifiers of mouse skin tumor susceptibility Jian-Hua Mao*, Elise F. Saunier*, John P. de Koning*†, Margaret M. McKinnon*, Mamie Nakijama Higgins*, Kathy Nicklas*, Hai-Tao Yang*, Allan Balmain*‡§, and Rosemary J. Akhurst*§¶ʈ *Cancer Research Institute, Comprehensive Cancer Center, Departments of ‡Biochemistry and ¶Anatomy, and §Program in Human Genetics, University of California, San Francisco, CA 94143-0875 Communicated by James E. Cleaver, University of California, San Francisco, CA, April 4, 2006 (received for review November 28, 2005) The human TGFB1 gene is polymorphic, and genetic variants are number of controls (17). Dunning et al. (17) demonstrated that associated with altered cancer risk. However, human genetic homozygosity for the high expressing TGF〉1Pro-10 allele was association studies have had variable outcomes because TGF␤1 associated with an increased invasive cancer risk (odds ratio action is context-dependent. We used the murine skin model of 1.25), which would support a positive role of TGF〉1 in tumor chemical carcinogenesis in genetic linkage analysis of three inde- progression. Conversely, in a cohort study of Ͼ3,000 women Ͼ65 pendent Mus musculus NIH͞Ola ؋ (Mus spretus ؋ M. musculus years old at recruitment, of which 146 developed breast cancer NIH͞Ola)F1 backcrosses, to identify a skin tumor susceptibility over the following 9 years, it was found that women homozygous locus, Skts14, on proximal chromosome 7. Tgfb1 maps at the peak for the high-expressing TGFB1Pro-10 allele were at a reduced risk of linkage. The mouse Tgfb1 gene is polymorphic, resulting in of developing breast cancer, suggesting that TGF␤1 has breast cis-regulated differential allelic mRNA expression between M. tumor-suppressing activity in this cohort (19). One explanation spretus and M. musculus in F1 mouse skin. This phenomenon is for these seemingly discrepant findings is ascertainment bias in reflected in differential phospho-SMAD2 levels, downstream of selecting only young women with invasive breast cancer (17) vs. TGF␤ signaling, between these two mouse species. In normal F1 women who had reached 65 years of age cancer-free (19). Taken mouse skin, the Tgfb1SPR allele is expressed at higher levels than together, these studies support the model of the dual role of the Tgfb1NIH allele, and this differential is accentuated by phorbol TGF␤ in tumorigenesis (4). They illustrate that the prediction of 12-myristate 13-acetate treatment. In benign F1 papillomas, this cancer risk associated with a particular TGF〉1 allele depends on imbalance is reversed, possibly by selection against expression of interacting genetic and environmental factors such that the a hyperactive Tgfb1SPR allele in TGF␤ growth-responsive tumors. hyperactive TGF〉1 allele may confer either cancer protection or We demonstrate that skin tumor susceptibility is altered by Tgfb1 increased risk. This thesis was recently substantiated by the gene dosage, but that manifestation of Tgfb1-linked skin tumor studies of Shin et al. (24) which emphasize the importance of susceptibility in M. musculus NIH͞Ola ؋ (M. spretus ؋ M. musculus understanding the context-dependent action of TGF␤1 and hint 〉 NIH͞Ola)F1 backcross mice depends on interactions with another at the importance of genes that interact with TGF 1 in deter- unlinked tumor modifying locus, Skts15, that overlaps Tgfbm3 on mining disease risk. Similar conclusions were recently made for chromosome 12. These findings illustrate the power of complex risk of invasive prostate cancer (18). genetic interactions in determining disease outcome and have In the current study, we demonstrate the existence of another major implications to the assessment of disease risk in individuals skin tumor susceptibility locus, Skts14, containing the Tgfb1 gene harboring variant TGFB1 alleles. on proximal chromosome 7. We show that cis-acting regulatory elements of the mouse Tgfb1 gene are polymorphic, leading to carcinoma ͉ chemical skin carcinogenesis ͉ genetic interaction ͉ TGF␤ allelic mRNA expression, which could account for altered tumor susceptibility between mouse strains. Importantly, we demon- GENETICS GF␤ acts as a negative growth regulator of normal and strate that the outcome of Tgfb1 allelic variation depends on Tbenign proliferative epithelial cells (1–3), but stimulates genetic context, particularly with respect to another modifier tumor progression once accumulation of oncogenic mutations locus, Skts15, on chromosome 12 with which Tgfb1 interacts. ␤ dampens the tumor’s negative growth response to TGF (4–6). Results Additionally, TGF␤ overproduced by both the malignant cell and tumor stroma may act on the tumor microenvironment to Skts14, a Skin Papilloma Susceptibility Locus on Proximal Mouse Chromosome 7. Previous studies demonstrated the existence of a indirectly stimulate tumor progression (7, 8). TGF␤ alters stro- skin tumor susceptibility locus, Skts1, on proximal chromosome mal cell characteristics, such as extracellular matrix deposition, 7 that controls papilloma development (25). This linkage was secretion of proteases and other cytokines that enhance angio- originally mapped at a low resolution with a peak located at Ϸ50 genesis, and tumor growth and plasticity (9, 10). Moreover, it can Mb. In the current study, a panel of markers mapping at a higher act as a very potent local and systemic immunosuppressor resolution was used to regenotype the chromosome 7-linkage (11–13). region in 306 (NIH͞Ola ϫ outbred Mus spretus)F ϫ NIH͞Ola Genes encoding components of the TGF␤1-signaling path- 1 (NSP) backcross animals, revealing two linkage peaks and way, including TGF〉1 (14) and TGFBRI (15, 16), have been suggesting two distinct loci, with a more proximal locus, Skts14, shown to be functionally polymorphic in humans, and genetic mapping at Ϸ14 Mb on chromosome 7 (Fig. 1). associations have been found between carriers of specific TGFB1 and TGFBR1 polymorphic variants and cancer susceptibility (15–20). The TGFB1 gene harbors polymorphisms in its pro- Conflict of interest statement: No conflicts declared. moter, plus amino acid polymorphisms in its signal peptide that Abbreviations: NSP, (NIH͞Ola ϫ outbred M. spretus) ϫ NIH͞Ola backcross; PMA, phorbol influence protein secretion and levels of freely circulating 12-myristate 13-acetate; LOD, logarithm of the odds; ⌬CT, differential between threshold TGF␤1 (14, 21–23). Several independent groups have demon- cycles. strated a genetic association between variant TGFB1 alleles and †Present address: Semaia Pharmaceuticals, Buntlaan 44, 3971 JD, Driebergen, The Nether- altered risk for breast cancer (17, 19, 20, 24). The most extensive lands. report was that of a case-control study of Ͼ3,900 early-onset ʈTo whom correspondence should be addressed. E-mail: [email protected]. (median age 50) invasive breast cancer patients and a similar © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602581103 PNAS ͉ May 23, 2006 ͉ vol. 103 ͉ no. 21 ͉ 8125–8130 Downloaded by guest on September 27, 2021 Fig. 1. Skts14, a skin tumor susceptibility locus on proximal mouse chromo- some 7. LOD scores for genetic linkage on chromosome 7 in the F1 NSP backcross (n ϭ 206; ᭜), and in that subset of the NSP backcross sharing Fig. 2. Haploinsufficiency for Tgfb1 increases papilloma incidence. Twenty- Haplotype 4 (n ϭ 44; E). Also shown is a linkage analysis in the N4 backcross three WT and 30 Tgfb1ϩ͞Ϫ age-matched adult female mice were subjected to (n ϭ 76; ‚). a standard chemical carcinogenesis protocol, and papilloma numbers were counted weekly. Tgfb1ϩ͞Ϫ developed significantly more papillomas than Tgfb1ϩ͞ϩ mice (P ϭ 0.025 at 20 weeks; Student’s t test). Information on shared haplotypes in outbred populations can be used to refine the locations of potential disease susceptibility genes (26). A haplotype map was previously constructed for Table 1 shows that homozygosity for NIH alleles at Skts14 and chromosome 7 by using variation in microsatellite lengths be- Skts15 confers higher tumor susceptibility in two independent M. tween the M. spretus alleles in the outbred colony (26). Inter- spretus ϫ NIH backcrosses (Table 1). The presence of a single M. estingly, when the linkage data were stratified according to M. spretus allele at either of these interacting loci results in relative spretus haplotype, it was found that mice from one haplotype, resistance to papilloma development, with additional M. spretus namely ‘‘Haplotype 4,’’ showed linkage only to the Skts14 locus, alleles (Tgfb1Spr/NIH;D12Nds11Spr/NIH) having little additional ef- in the absence of linkage to the original, more distal, Skts1 locus, fect (Table 1). The Skts1͞Skts5 interaction also shows this effect, thus demonstrating that the two chromosome 7 loci act inde- but only in the NSP cross (Table 1), emphasizing the indepen- pendently (Fig. 1). Skts14 was further validated by linkage dence of the two interactions and the importance of the Skts14͞ analysis on N4 backcross mice that had been backcrossed four Skts15 interaction. more generations to the NIH strain while selecting for those with papilloma resistance at each generation (N4 mice). The Haploinsufficiency for Tgfb1, a Candidate Gene at Skts14, Increases LODMAX at Skts1 was less than LODMAX Ϫ 1atSkts14, again Papilloma Incidence. Tgfb1 is an excellent candidate as a tumor suggesting independence of Skts14 from Skts1 (Fig. 1). susceptibility gene at Skts14, especially in light of the evidence that