Int6 Expression Predicts Survival in Early Stage Non-Small Cell Lung Cancer Patients. The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus (MMTV) in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a polyhedric protein involved in the regulation of protein translation and degradation through binding with three cellular complexes: the eukaryotic translation initiation factor 3 (eIF3), the proteasome regulatory lid, and the COP9 signalosome. In the present study, we have determined the steady state levels of Int6 RNA by quantitative real-time RT-PCR in 101 human lung tumors and matched normal lung tissues obtained from stage I non-small cell lung cancers (NSCLC) patients with long-term follow-up. In 27% of the tumors Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 RNA levels the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P<0.000001). Low levels of Int6 RNA were found to be a significant predictor of overall and disease-free survival (p= 0.0004 and p= 0.0020, respectively). A multivariate analysis confirmed that low Int6 RNA level was the only independent and significant factor to predict poor prognosis, for both overall (P = 0.0006) and disease-free (P = 0.024) survival. Overexpression of human Cripto-1 in transgenic mice impairs mammary gland development and differentiation and induces mammary tumorigenesis. Cripto-1, a member of the EGF-CFC gene family, performs an important role in vertebrate development. Overexpression of Cripto-1 has been reported in several types of cancer in mice and humans, including human breast cancer. To investigate the role of human Cripto-1 in mammary gland development and carcinogenesis, we have developed transgenic mice expressing of the human Cripto-1 gene under the regulation of the whey acidic protein (WAP) promoter in FVB/N mice. We found that the Cripto-1 transgene begins to be expressed during late-pregnancy and that b-casein expression was suppressed in the pregnant mammary glands of WAP-Cripto-1 transgenic mice compared to FVB/N mice. During early lactation alveolar structures in the mammary glands of WAP-Cripto-1 expressing mice were less differentiated and delayed in their development compared to FVB/N mice. As a result, WAP-Cripto-1 females have impaired lactation as compared to FVB/N mice. Involution is enhanced in the mammary glands of WAP-Cripto-1 mice compared to FVB/N mice. These results indicate that Cripto-1 inhibits mammary gland development / differentiation and enhanced apoptosis. In addition, 15 out of 30 Wap-Cripto-1 multiparous females developed mammary tumors. These tumors exhibit a phenotype that is similar to mammary tumors that are induced by Wnt-1. The role of cKit in """"""""activated"""""""" Notch4/Int3 induced mammary tumors. Differential display analysis of RNA from mammary tissue of pregnant normal FVB and WAP-Notch4/Int3 ICD mice and a mammary tumor from a WAP-Notch4/Int3 ICD female showed that high levels of cKit were detectable in the tumor compared normal pregnant FVB mammary tissue. Intermediate levels of cKit RNA was present in mammary tissue from pregnant WAP-Notch4/Int3 ICD females. In fact all mammary tumors from WAP-Notch4/Int3 ICD, MMTV LTR-Notch4/Int3 ICD, and WAP-Notch4/Int3sh transgenic mice express high levels of cKit. The drug Gleevec has been shown to specifically inhibit the activity of cAbl, cKit, PDGFR tyrosine kinases. We have treated WAP-Notch4/Int3 ICD tumor bearing mice with increasing amounts of Gleevec. In these mice the tumors either regresses or remains stationary in size. This suggests that in the context of """"""""activated"""""""" Notch4/Int3 cKit activity is both necessary and sufficient for mammary tumor growth.
