Rb inactivation accelerates neoplastic growth and substitutes for recurrent amplification of cIAP1, cIAP2 and Yap1 in sporadic mammary carcinoma associated with p53 deficiency.

Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis. We used newly generated MMTV-Cre105Ayn mice to inactivate p53 and/or Rb strictly in the mammary epithelium, and to determine recurrent genomic changes associated with deficiencies of these genes. p53 inactivation led to formation of estrogen receptor-positive raloxifene-responsive mammary carcinomas with features of luminal subtype B. Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation. Genome-wide analysis of mammary carcinomas identified a recurrent amplification at chromosome band 9A1, a locus orthologous to human 11q22, which contains protooncogenes cIAP1 (Birc2), cIAP2 (Birc3) and Yap1. It is interesting that this amplicon was preferentially detected in carcinomas carrying wild-type Rb. However, all three genes were overexpressed in carcinomas with p53 and Rb inactivation, likely due to E2F-mediated transactivation, and cooperated in carcinogenesis according to gene knockdown experiments. These findings establish a model of luminal subtype B mammary carcinoma, identify critical role of cIAP1, cIAP2 and Yap1 co-expression in mammary carcinogenesis and provide an explanation for the lack of recurrent amplifications of cIAP1, cIAP2 and Yap1 in some tumors with frequent Rb deficiency, such as mammary carcinoma.

The retinoblastoma gene regulates somatic growth during mouse development.

Overexpression of the retinoblastoma gene (Rb) in mice leads to the dwarf phenotype. To explore the potential mechanism of Rb effects on the somatic growth, bitransgenic mice with tetracycline-regulated Rb expression were generated, and their phenotypes were compared with those of previously established Rb mouse models. By gestational day 12.5, embryos lacking Rb and those expressing twice the regular amount of Rb are 15% larger and 10-30% smaller, respectively, compared with their wild-type littermates. The dwarf phenotype is associated with increased plasma levels of insulin-like growth factor-I (IGF-I) but not with growth hormone and glucose concentrations. Down-regulation of the Rb transgene expression results in a reduction of the IGF-I plasma concentrations to normalcy and an increase of somatic growth prenatally and postnatally. Consistent with the in vivo results, cells overexpressing Rb require higher thresholds of IGF-I to stimulate proliferation. Thus, Rb plays an integral role for mouse somatic growth and maintenance during ontogenesis, and IGF-I pathway is likely to be a target for such regulation.

Sequence-specific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1.

BRCA1 has been implicated in the transcriptional regulation of DNA damage-inducible genes that function in cell cycle arrest. To explore the mechanistic basis for this regulation, a novel human gene, ZBRK1, which encodes a 60 kDa protein with an N-terminal KRAB domain and eight central zinc fingers, was identified by virtue of its interaction with BRCA1 in vitro and in vivo. ZBRK1 binds to a specific sequence, GGGxxx CAGxxxTTT, within GADD45 intron 3 that supports the assembly of a nuclear complex minimally containing both ZBRK1 and BRCA1. ZBRK1 represses transcription through this recognition sequence in a BRCA1-dependent manner. These results thus reveal a novel corepressor function for BRCA1 and provide a mechanistic basis for the biological activity of BRCA1 through sequence-specific transcriptional regulation.

Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development.

BRCA1 is proposed to be a tumor suppressor gene. To explore the biological function of BRCA1, a partial deletion (amino acids 300-361) of mouse Brca1 exon 11 was introduced into the genome of embryonic stem (ES) cells by homologous recombination. Mice carrying one mutated allele of Brca1 appear normal and are fertile up to 10 months of age without any sign of illness. However, no viable progeny homozygous for the Brca1 mutant allele were obtained. Detailed analysis of large numbers of embryos at different stages of development indicated that the homozygous mutant concepti are severely retarded in growth as early as embryonic day 4.5 (E4.5) and are resorbed completely by E8.5. Although the homozygotes at E5.5-E6.5 are able to synthesize DNA and display distinguishable embryonic and extraembryonic structures, they fail to differentiate and form egg cylinders. Consequently, they were unable to form primitive streaks and undergo gastrulation. Consistent with these in vivo results, blastocysts homozygous for mutated Brca1 alleles are at a considerable disadvantage when grown in vitro. These observations suggest that Brca1 has an important role in the early development of mouse embryos.

Wild type neu transgene counteracts mutant homologue in malignant transformation of rat Schwann cells.

Mutational activation of the neu (erbB-2) receptor protein tyrosine kinase gene appears to be the triggering event in the process of oncogenesis induced by N-ethyl-N-nitrosourea (EtNU) in immature Schwann cells of the rat peripheral nervous system. Subsequent loss of the wild-type neu allele may represent a critical secondary step towards malignancy. Developmentally-regulated expression of a wild-type rat neu transgene (neu cDNA under the control of the rat Po promoter) in the Schwann cells of transgenic BDIX and Sprague-Dawley rats exposed to EtNU on postnatal day 1 results in a lower incidence of early atypical proliferates in the trigeminal nerve. Furthermore, re-introduction of the wild-type neu gene into homozygous neu mutant schwannoma cells counteracts the expression of the tumorigenic phenotype. The suppressive action of the wild-type gene over its mutationally activated oncogenic homologue underlines the critical function of the neu gene in the control of differentiation in the Schwann cell lineage, and provides evidence for the responsiveness of cellular phenotypes towards quantitative shifts in the dosage of wild-type vs mutant signal transducing molecules.