Yuichi Wakabayashi

Bcl11b is required for differentiation and survival of αβ T lymphocytes

The gene Bcl11b, which encodes zinc finger proteins, and its paralog, Bcl11a, are associated with immune-system malignancies. We have generated Bcl11b-deficient mice that show a block at the CD4−CD8− double-negative stage of thymocyte development without any impairment in cells of B- or γδ T cell lineages. The Bcl11b−/− thymocytes showed unsuccessful recombination of Vβ to Dβ and lacked the pre–T cell receptor (TCR) complex on the cell surface, owing to the absence of Tcrb mRNA expression. In addition, we saw profound apoptosis in the thymus of neonatal Bcl11b−/− mice. These results suggest that Bcl11b is a key regulator of both differentiation and survival during thymocyte development.

Promotion of Hras-induced squamous carcinomas by a polymorphic variant of the Patched gene in FVB mice

Mice of the C57BL/6 strain are resistant to the development of skin squamous carcinomas (SCCs) induced by an activated Ras oncogene, whereas FVB/N mice are highly susceptible. The genetic basis of this difference in phenotype is unknown. Here we show that susceptibility to SCC is under the control of a carboxy-terminal polymorphism in the mouse Ptch gene. F1 hybrids between C57BL/6 and FVB/N strains ((B6FVB)F1) are resistant to Ras-induced SCCs, but resistance can be overcome either by elimination of the C57BL/6 Ptch allele (PtchB6) or by overexpression of the FVB/N Ptch allele (PtchFVB) in the epidermis of K5Hras-transgenic (B6FVB)F1 hybrid mice. The human Patched (PTCH) gene is a classical tumour suppressor gene for basal cell carcinomas and medulloblastomas, the loss of which causes increased signalling through the Sonic Hedgehog (SHH) pathway. SCCs that develop in PtchB6+/- mice do not lose the wild-type Ptch gene or show evidence of increased SHH signalling. Although PtchFVB overexpression can promote SCC formation, continued expression is not required for tumour maintenance, suggesting a role at an early stage of tumour cell lineage commitment. The Ptch polymorphism affects Hras-induced apoptosis, and binding to Tid1, the mouse homologue of the Drosophila l(2)tid tumour suppressor gene. We propose that Ptch occupies a critical niche in determining basal or squamous cell lineage, and that both tumour types can arise from the same target cell depending on carcinogen exposure and host genetic background.

Homozygous deletions and point mutations of the Rit1/Bcl11b gene in γ-ray induced mouse thymic lymphomas

Allelic loss (LOH) mapping and sequence analysis were conducted for gamma-ray induced mouse thymic lymphomas and a novel tumor suppressor gene, Rit1/Bcl11b, on chromosome 12 was isolated. Bi-allelic changes were found in 17 of the 66 p53-proficient lymphomas with Rit1 LOH but in only 2 of the 54 p53-deficient lymphomas. This suggests an association between the presence of functional p53 and inactivation of the Rit1 gene in the lymphoma development. Introduction of Rit1 into HeLa cells lacking Rit1 expression suppressed cell growth. These results indicate that loss-of-function mutations of Rit1 contribute to mouse lymphomagenesis and possibly to human cancer development.

Allelic loss analysis of gamma-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16.

A total of 429 γ-ray-induced thymic lymphomas were obtained from F1 and backcross mice between BALB/c and MSM strains, about a half of which carried a p53-deficient allele. A genome-wide allelic loss analysis has revealed two loci exhibiting frequent allelic losses but no allelic preference, one is localized within a 2.9 cM region between D12Mit53 and D12Mit279 loci on chromosome 12, and the other is near the D16Mit122/D16Mit162 loci on chromosome 16. The frequency of allelic loss in the D12Mit279 region is 62% and does not differ in tumors between the presence and absence of the p53-deficient allele. In contrast, the loss frequency of D16Mit122 is raised by the existence of p53-deficient allele: 62% for p63(−/+) and 13% for p53(+/+), suggesting cooperative function of the two losses. The D12Mit279 and D16Mit122 regions probably harbor different types of tumor suppressor gene that play key roles in lymphoma development.

Genetic loci controlling susceptibility to γ-ray-induced thymic lymphoma

BALB/c is a susceptible strain for the development of γ-ray induced mouse thymic lymphoma whereas MSM shows resistance. Association analysis of 220 backcross mice between the two strains using 67 markers was carried out to identify loci involved in the control of susceptibility. The genotype of mice with lymphoma showed excess heterozygosity relative to MSM homozygosity at D2Mit15 and D4Mit12 and was skewed toward MSM-derived alleles at D5Mit5. The P values in Mantel-Cox test were 0.0048 (D2Mit15), 0.0034 (D4Mit12) and 0.0048 (D5Mit5), suggesting association at the three loci in the susceptibility. Cooperative effect on lymphomagenesis was also observed among the three loci. To obtain independent evidence for linkage at D4Mit12, we made partially congenic mice in which a D4Mit12 region in BALB/c was replaced by MSM-derived homolog. Examination for the lymphoma susceptibility in 78 progeny of the congenic mice confirmed the effect of the locus near D4Mit12 (P=0.0037). The result, together with the linkage analysis, shows that the locus near D4Mit12 is regarded as a confirmed linkage but the other two loci as marginally suggestive.

Ptch1 overexpression drives skin carcinogenesis and developmental defects in K14PtchFVB mice

Ptch1 is a key regulator of embryonic development, acting through the sonic hedgehog (SHH) signaling pathway. Ptch1 is best known as a tumor suppressor, since germline or somatic mutations in Ptch1 lead to the formation of skin basal cell carcinomas (BCCs). Here, we show that Ptch1 also acts as a lineage-dependent oncogene, as overexpression of Ptch1 in adult skin in K14PtchFVB transgenic mice synergizes with chemically induced Hras mutations to promote squamous carcinoma development. These effects were not due to aberrant activation of SHH signaling by the K14PtchFVB transgene, as developmental defects in the highest expressing transgenic lines were consistent with inhibition of this pathway. Carcinomas from K14PtchFVB transgenic mice had only a small number of non-proliferative Ptch1 transgene positive cells, suggesting that the Ptch1 transgene is not required for tumor maintenance, but may play a critical role in cell fate determination at the initiation stage.

Meis1 Regulates Epidermal Stem Cells and Is Required for Skin Tumorigenesis

Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1.

Multiple self-healing squamous epithelioma (MSSE): rare variants in an adjacent region of chromosome 9q22.3 to known TGFBR1 mutations suggest a digenic or multilocus etiology.

Multiple self-healing squamous epithelioma (MSSE, OMIM132800) also known as Ferguson-Smith disease (FSD) is a rare inherited skin cancer syndrome characterized by multiple invasive keratoacanthoma-like skin tumors that regress spontaneously leaving pitted scars (Ferguson-Smith, 1934; Goudie et al., 1993). The majority of the first-identified affected families have a shared Scottish ancestry, but MSSE has also been described in families of non-Scottish origin (D’Alessandro et al., 2007). Recently, the causative gene for MSSE was identified as TGFBR1, encoding a transmembrane serine/threonine kinase receptor involved in TGF-β signaling (Goudie et al., 2011). TGFBR1 mutations in MSSE are functionally null implying a tumor suppressor action in this disease, whereas mis sense mutations in the same gene can lead to Marfan syndrome-related disorders (Goudie et al., 2011; Loeys et al., 2005). TGFBR1 had previously been excluded as a MSSE candidate because the gene lies outside the original shared at-risk haplotype (SRH) in the Scottish families (Bose et al., 2006). However using large scale sequencing technology, Goudie et al. (2011) identified TGFBR1 germline mutations in a total of 18 MSSE families including 12 Scottish families. Of 9 Scottish families with the SRH, 7 families shared the same TGFBR1 mutation (p.G52R), importantly, however 2 families had different causative mutations (p.N45S and p.R414X). This suggests that unidentified rare variants associated with MSSE pathogenesis may exist in the non-TGFBR1 SRH region that has been conserved in 9 families. The fact that this relatively large conserved haplotype had not undergone recombination over many generations in these 9 affected Scottish MSSE families suggested that manifestation of the disease may require both the causative TGFBR1 mutation and additional variant(s) located within the SRH region. To search for these SRH variants we used the Agilent SureSelect Target Enrichment system followed by sequencing on the Illumina GAII platform to sequence all of the target genes between the markers D9S197 and D9S1809 on 9q22 31 (D’Alessandro et al., 2007). 13 families including 7 Scottish families with the SRH were available (Table S1). Family numbers hereafter correspond to the study by Goudie et al. 2011. We performed targeted sequencing in 5 families with the SRH and 5 families without the SRH, along with 6 CEPH controls. Coding regions of the 15 then-known genes were sequenced previously (D’Alessandro et al., 2007), but based on NCBI36 (hg18), there are now 29 known genes in the targeted region (Table S5), encompassing ~2.2-Mb in total, and this entire genomic region of these genes was sequenced. After filtering out common variants included in dbSNP and shared with CEPH controls, we identified 9 non-coding variants shared by all 5 families with the SRH. None of the 9 rare variants were detected in MSSE families without the SRH. These 9 variants are located in intronic regions of 5 different genes: FAM120A, PHF2, C9orf3, FANCC, and PTCH1 (Table 1). Sanger dideoxy sequencing confirmed these 9 variants in all 5 families plus 2 additional families (Table S1, Figure S1). Thus, all 9 variants were conserved over this ~2.2Mb region in 7 Scottish families with the SRH, although TGFBR1 mutations were different in families 2 and 18 (Figure 1a). In 231 unrelated healthy controls, including 118 Scottish individuals, the minor allele frequency (MAF) of the 9 variants was rare, ranging from 0 to 0.022 and the association of these variants with the MSSE phenotype was highly significant (Table 1). This suggests that these 9 variants are MSSE-associated and segregated in individuals with this rare skin malignancy condition. Interestingly, these variants are located at either end of the ~2.2-Mb target region leaving a ~1.4-Mb central region where 24 genes are densely located (Figure 1a). Further analysis of MSSE families lacking the SRH identified distinct MSSE-associated rare variants in two Scottish families (Table S2-3). In family 17, we identified 8 distinct variants that were detected in 4 affected family members (Table S3). Interestingly, these 8 variants were all clustered in a ~1.4-Mb central region of the SRH that excluded the 9 MSSE associated variants discussed above (Figure 1a). This family harbored the most complex TGFBR1 mutation (c.1059_1062delACTGinsCAATAA) that was not observed in other families. All of these variants are non-coding and not frequently found in 162 healthy Scottish controls tested. Figure 1 Schematic summary of the 9 rare variants identified in this study and functional effect (a) 7 Scottish MSSE families shared all the 9 non-coding non-TGFBR1 variants identified in this study, but the TGFBR1 mutations are different amongst these families ... Table 1 9 rare non-coding variants identified in 7 Scottish MSSE families with the SRH proximal to the causative TGFBR1 locus. Three of the 9 variants found were located in intronic regions of the PTCH1 gene, of which germline loss of function mutations are responsible for nevoid basal cell carcinoma syndrome (NBCCS) (Hahn et al., 1996; Johnson et al., 1996). Our previous study using mouse models suggested that a gain of function polymorphic variant in the mouse Ptch1 gene conferred susceptibility to squamous cell carcinoma (SCC) development (Wakabayashi et al., 2007). We considered the possibility that these rare PTCH1 variants may play a role in predisposition to SCC development in MSSE patients. We carried out a computational analysis of the possible functional significance of all variants using the program AliBaba2.1. This analysis identified the 97309311G>C PTCH1 variant as having possible functional significance. An electromobility shift assay (EMSA) was performed on the major 97309311G>C variant, which is reported to bind to multiple transcription factors including SP1 and PU.1 (http://genome.ucsc.edu/ENCODE/). While the major allele (97309311G) could bind to SP1 and PU.1 transcription factors in nuclear extracts from HaCaT immortalized human keratinocyte cells, the minor allele (97309311C) showed complete disruption of this binding, indicating that the MSSE-associated rare variant perturbs normal interaction between this binding site and transcription factors (Figure 1b). In our study, we identified a rare MSSE-associated haplotype comprising 9 variants spanning a 2.2Mb region lying 1.5Mb proximal to the causative TGFBR1 gene. Several of the genes in this region have been implicated in cancer development, and regulatory polymorphisms may affect tumor susceptibility (Sinha et al., 2008). The co inheritance of these variants in the majority of Scottish families with MSSE suggests that these variants, or other as yet unknown linked variants (or their combinations), may affect the expression of the skin cancer phenotype induced by the mutations in TGFBR1. Their locations at opposite ends of the conserved haplotype may suggest that they modify chromatin loop structure to influence expression of genes within the region. Alternatively, the variants may directly affect expression of relevant genes, particularly the PTCH1 gene, which is implicated in susceptibility to different forms of skin cancer and carries three intronic variants, one of which affects transcription factor binding. Functional analysis of the possible roles of these and other variants in determining the MSSE phenotype, for example by gene expression or ChIP-seq analysis to assess differential transcription factor binding, will require further studies using keratinocytes or cultured primary tumor cells from MSSE affected patients. Finally, it is possible that since loss of function germline mutations in TGFBR1 are very rare, the conserved haplotype may act as a modifier to increase survival of individuals carrying strong mutations in this potent developmental regulator. This mechanism is supported by the identification of unlinked variant genetic modifiers that are preferentially inherited in mice haploinsufficient for Tgfb1 (Benzinou et al. 2012).

Mapping of genetic modifiers of thymic lymphoma development in p53-knockout mice.

The strain dependency of the spectrum and latency of tumors has been reported in p53-deficient (KO) mice, suggesting the presence of modifiers for the outcome of the p53 deficiency. The modifiers provide clues to the oncogenic pathway in cells lacking p53, the most frequently mutated gene in a wide variety of human cancers. To search the modifiers, we induced 160 lymphomas and 69 skin tumors by γ-irradiation of p53(KO/+) backcross mice between BALB/c and MSM strains and performed genome scan. BALB/c-derived alleles at three loci on chromosome 19, Mp53D1 (modifier of p53-deficiency) at D19Mit5, Mp53D2 at D19Mit90 and Mp53D3 at D19Mit123, extended the latency of thymic lymphoma development (P values in Mantel–Cox test were 0.0007, 0.0007 and 0.0003, respectively). Mp53D3 also increased the latency of skin tumors (P value, 0.0008). The linkage of Mp53D2 was confirmed by the experiment using 94 p53-KO mice consomic for chromosome 19, providing a significant linkage. However, the linkage was not confirmed for Mp53D1 or Mp53D3, suggesting epistasis of genes involved in the tumorigenesis.

Independent genetic control of early and late stages of chemically induced skin tumors in a cross of a Japanese wild-derived inbred mouse strain, MSM/Ms

MSM/Ms is an inbred mouse strain derived from a Japanese wild mouse, Mus musculus molossinus. In this study, we showed that MSM/Ms mice exhibit dominant resistance when crossed with susceptible FVB/N mice and subjected to the two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA)/ 12-O-tetradecanoylphorbol-13-acetate (TPA). A series of F1 backcross mice were generated by crossing p53(+/+) or p53(+/-) F1 (FVB/N × MSM/Ms) males with FVB/N female mice. These generated 228 backcross animals, approximately half of which were p53(+/-), enabling us to search for p53-dependent skin tumor modifier genes. Highly significant linkage for papilloma multiplicity was found on chromosomes 6 and 7 and suggestive linkage was found on chromosomes 3, 5 and 12. Furthermore, in order to identify stage-dependent linkage loci we classified tumors into three categories (<2mm, 2-6mm and >6mm), and did linkage analysis. The same locus on chromosome 7 showed strong linkage in groups with <2mm or 2-6mm papillomas. No linkage was detected on chromosome 7 to papillomas >6mm, but a different locus on chromosome 4 showed strong linkage both to papillomas >6mm and to carcinomas. This locus, which maps near the Cdkn2a/p19(Arf) gene, was entirely p53-dependent, and was not seen in p53 (+/-) backcross animals. Suggestive linkage conferring susceptibility to carcinoma was also found on chromosome 5. These results clearly suggest distinct loci regulate each stage of tumorigenesis, some of which are p53-dependent.