The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germline mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that female mice heterozygous for the "scurfin" mutation of the Foxp3 gene (Foxp3(sf/+)) developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and HER-2/ErbB2 was overexpressed. Foxp3 bound and repressed the HER-2/ErbB2 promoter. Deletion, functionally significant somatic mutations, and downregulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2/ErbB2 overexpression, regardless of the status of HER-2 amplification. Our data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.

A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex.Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2. IntroductionCancer pathogenesis involves both the inactivation of tumor suppressor genes and the activation of oncogenes (1, 2). One of the most fascinating aspects of cancer biology is the interaction between cancer suppressor genes and oncogenes. Most of these interactions are at posttranslational levels. For instance, proteins encoded by tumor suppressor genes can inactivate oncogenes. One of the most clearly studied cases is tumor suppressor Rb, which inhibits the E2F family members of oncogenes (1,2). Conversely, oncogenes can overcome the tumor suppressor proteins. For example, S-phase kinase-associated protein 2 (SKP2) causes the degradation of tumor suppressor FOXO (3) as well as CDK inhibitors, such as p27 (4, 5). It is less clear whether such antagonism exists at the transcriptional level. However, a recent study suggests that FOXO may repress the expression of cyclin D (6), a well-known oncogene. Likewise, we have recently demonstrated that forkhead box P3 (FOXP3), an X-linked tumor suppressor, represses transcription of ERBB2/HER2 oncogene (7).The high-level expression of SKP2 was reported in a significant proportion of cancers (5). SKP2 is a component of the E3 ubiquitin ligase SCF with specificity for CDK inhibitor p27. However, under physiological conditions, Skp2 expression is maximal at the S and G2 phases and appears to primarily mediate p27 degradation at the G2 (8, 9) but not G0 and G1 phases (10). As such, Skp2 was found to be essential for progression into mitosis in cell cycles (4, 9, 11). Targeted mutation of Skp2 causes delayed animal growth and cellular polyplo...