Allele-Specific Deletions in Mouse Tumors Identify Fbxw7 as Germline Modifier of Tumor Susceptibility

Genome-wide association studies (GWAS) have been successful in finding associations between specific genetic variants and cancer susceptibility in human populations. These studies have identified a range of highly statistically significant associations between single nucleotide polymorphisms (SNPs) and susceptibility to development of a range of human tumors. However, the effect of each SNP in isolation is very small, and all of the SNPs combined only account for a relatively minor proportion of the total genetic risk (5–10%). There is therefore a major requirement for alternative routes to the discovery of genetic risk factors for cancer. We have previously shown using mouse models that chromosomal regions harboring susceptibility genes identified by linkage analysis frequently exhibit allele-specific genetic alterations in tumors. We demonstrate here that the Fbxw7 gene, a commonly mutated gene in a wide range of mouse and human cancers, shows allele-specific deletions in mouse lymphomas and skin tumors. Lymphomas from three different F1 hybrids show 100% allele-specificity in the patterns of allelic loss. Parental alleles from 129/Sv or Spretus/Gla mice are lost in tumors from F1 hybrids with C57BL/6 animals, due to the presence of a specific non-synonymous coding sequence polymorphism at the N-terminal portion of the gene. A specific genetic test of association between this SNP and lymphoma susceptibility in interspecific backcross mice showed a significant linkage (p = 0.001), but only in animals with a functional p53 gene. These data therefore identify Fbxw7 as a p53-dependent tumor susceptibility gene. Increased p53-dependent tumor susceptibility and allele-specific losses were also seen in a mouse skin model of skin tumor development. We propose that analysis of preferential allelic imbalances in tumors may provide an efficient means of uncovering genetic variants that affect mouse and human tumor susceptibility

Pten regulates Aurora-A and cooperates with Fbxw7 in modulating radiation-induced tumor development

PMCID: PMC3388112.-- et al.The Aurora-A kinase gene is frequently amplified and/or overexpressed in a variety of human cancers, leading to major efforts to develop therapeutic agents targeting this pathway. Here, we show that Aurora-A is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3β. Using a series of truncated Aurora-A proteins and site-directed mutagenesis, we identified distinct FBXW7 and GSK3β-binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation of Aurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3β pathway. Moreover, radiation-induced tumor latency is significantly shortened in Fbxw7 +/-Pten +/- mice as compared with either Fbxw7 +/- or Pten +/- mice, indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novel posttranslational regulatory network in which the Pten and Fbxw7 pathways appear to converge on the regulation of Aurora-A level. ©2012 AACR.J.-H. Mao was supported by the NIH, National Cancer Institute grant R01 CA116481, the Low Dose Scientific Focus Area, Office of Biological and Environmental Research, of the U.S. Department of Energy under contract no. DEAC02- 05CH11231, and Laboratory Directed Research and Development Program (LDRD). A. Balmain was supported by the Department of Energy Low Dose Radiation Research Program DESC0003679 and the National Cancer Institute grant U01 CA141455. J. Losada-Losada was partially supported by FEDER, MICINN (PLE2009-119), FIS (PI10/00328), and "Fundación Eugenio Rodríguez- Pascual."Peer Reviewe

K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling

SummaryK-Ras and H-Ras share identical effectors and have similar properties; however, the high degree of tumor-type specificity associated with K-Ras and H-Ras mutations suggests that they have unique roles in oncogenesis. Here, we report that oncogenic K-Ras, but not H-Ras, suppresses non-canonical Wnt/Ca2+ signaling, an effect that contributes strongly to its tumorigenic properties. K-Ras does this by binding to calmodulin and so reducing CaMKii activity and expression of Fzd8. Restoring Fzd8 in K-Ras mutant pancreatic cells suppresses malignancy, whereas depletion of Fzd8 in H-RasV12-transformed cells enhances their tumor initiating capacity. Interrupting K-Ras-calmodulin binding using genetic means or by treatment with an orally active protein kinase C (PKC)-activator, prostratin, represses tumorigenesis in K-Ras mutant pancreatic cancer cells. These findings provide an alternative way to selectively target this “undruggable” protein

Skin cancer induction by DMBA-TPA protocol in <i>Fbxw7</i> deficient mice.

<p>(A) Loss of a single copy of <i>Fbxw7</i> does not significantly affect papilloma development: Average of papillomas in <i>Fbxw7+/−</i> and wild type mice. (B) Loss of <i>Fbxw7</i> increases susceptibility to papilloma development in a <i>p53</i>-heterozygous background. There were significant differences in the average of papilloma development between <i>p53+/−Fbxw7+/+</i> and <i>p53+/−Fbxw7+/−</i> mice (Mann-Whitney test).</p

Allele-specific change and linkage analysis of Fbxw7 locus in skin tumors.

<p>(A) Analysis of allele-specific <i>Fbxw7</i> gene copy number by QPCR in primary skin tumors. The <i>Fbxw7</i> allele from the <i>Mus spretus</i> parent was deleted in early stages of skin cancer development. Samples 1, 6 and 7 are papillomas; samples 13 and 14 are spindle carcinomas; all the rest are squamous carcinomas. ΔΔCT less than −0.5 indicates loss of the <i>Mus Spretus</i> allele. (B) LOD scores for number of papillomas on Chromosome 3 were generated by R/QTL.</p

Distribution of <i>Fbxw7</i> alleles among different mouse strains by sequencing analysis.

<p>Distribution of <i>Fbxw7</i> alleles among different mouse strains by sequencing analysis.</p

Detection of allele-specific loss of <i>Fbxw7</i> gene in radiation-induced thymic lymphomas.

<p>(A) PCR analysis at the <i>Fbxw7</i> locus: Tumors derived from F1 hybrids between <i>C57BL/6</i> and <i>129/Sv</i>, or <i>C57BL/6</i> and <i>Mus spretus</i>, showed consistent loss of the <i>129/Sv</i> or <i>Mus spretus</i> alleles, respectively; whereas tumors derived from <i>129/Sv</i> and <i>Mus spretus</i> hybrids showed loss of either <i>129/Sv</i> or <i>Spretus</i> alleles at the same frequency. (B) Sequence analysis showing the polymorphism in <i>Fbxw7</i> between <i>C57BL/6</i> (B6) and <i>Mus spretus</i> (Spr). (C) Comparison of the amino acid sequences at the <i>Fbxw7α</i> polymorphic region in <i>Mus spretus</i>, <i>129/Sv</i>, <i>C57BL/6</i>, <i>FVB/N</i>, <i>NIH/Ola</i> strain by sequencing.</p

Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers

Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies