Non-small cell lung cancers (NSCLCs) harbor thousands of passenger events that hide genetic drivers. Even highly recurrent events in NSCLC, such as mutations in PTEN, EGFR, KRAS, and ALK, are only detected in, at most, 30% of patients. Thus, many unidentified low-penetrant events are causing a significant portion of lung cancers. To detect low-penetrance drivers of NSCLC a forward genetic screen was performed in mice using the Sleeping Beauty (SB) DNA transposon as a random mutagen to generate lung tumors in a Pten deficient background. SB mutations coupled with Pten deficiency were sufficient to produce lung tumors in 29% of mice. Pten deficiency alone, without SB mutations, resulted in lung tumors in 11% of mice, while the rate in control mice was ~3%. In addition, thyroid cancer and other carcinomas as well as the presence of bronchiolar and alveolar epithelialization in mice deficient for Pten were also identified. Analysis of common transposon insertion sites identified 76 candidate cancer driver genes. These genes are frequently dysregulated in human lung cancers and implicate several signaling pathways. Cullin3 (Cul3), a member of an ubiquitin ligase complex that plays a role in the oxidative stress response pathway, was identified in the screen and evidence demonstrates that Cul3 functions as a tumor suppressor.

Bone morphogenetic proteins (BMPs) are involved in embryonic mammary gland (MG) development and can be dysregulated in breast cancer. However, the role BMPs play in the postnatal MG remains virtually unknown. BMPs are potent morphogens that are involved in cell fate determination, proliferation, apoptosis and adult tissue homeostasis. Twisted gastrulation (TWSG1) is a secreted BMP binding protein that modulates BMP ligand availability in the extracellular space. Here we investigate the consequences of TWSG1 deletion on development of the postnatal MG. At puberty, Twsg1 is expressed in the myoepithelium and in a subset of body cells of the terminal end buds. In the mature duct, Twsg1 expression is primarily restricted to the myoepithelial layer. Global deletion of Twsg1 leads to a delay in ductal elongation, reduced secondary branching, enlarged terminal end buds, and occluded lumens. This is associated with an increase in luminal epithelial cell number and a decrease in apoptosis. In the MG, pSMAD1/5/8 level and the expression of BMP target genes are reduced, consistent with a decrease in BMP signaling. GATA-3, which is required for luminal identity, is reduced in Twsg1−/− MGs, which may explain why K14 positive cells, which are normally restricted to the myoepithelial layer, are found within the luminal compartment and shed into the lumen. In summary, regulation of BMP signaling by TWSG1 is required for normal ductal elongation, branching of the ductal tree, lumen formation, and myoepithelial compartmentalization in the postnatal MG.

The severity of numerous developmental abnormalities can vary widely despite shared genetic causes. Mice deficient in Twisted gastrulation (Twsg1−/−) display such phenotypic variation, developing a wide range of craniofacial malformations on an isogenic C57BL/6 strain background. To examine the molecular basis for this reduced penetrance and variable expressivity, we used exon microarrays to analyze gene expression in mandibular arches from several distinct, morphologically defined classes of Twsg1−/− and wild type (WT) embryos. Hierarchical clustering analysis of transcript levels identified numerous differentially expressed genes, clearly distinguishing severely affected and unaffected Twsg1−/− mutants from wild type (WT) embryos. Several genes that play well-known roles in craniofacial development were upregulated in unaffected Twsg1−/− mutant embryos, suggesting that they may compensate for the loss of TWSG1. Imprinted genes were overrepresented among genes that were differentially expressed particularly between affected and unaffected mutants. The most severely affected embryos demonstrated increased p53 signaling and increased expression of its target, Trp53inp1. The frequency of craniofacial defects significantly decreased with a reduction of p53 gene dosage from 44% in Twsg1−/−p53+/+ pups (N=675) to 30% in Twsg1−/−p53+/− (N=47, p=0.04) and 15% in Twsg1−/−p53−/− littermates (N=39, p=0.001). In summary, these results demonstrate that phenotypic variability in Twsg1−/− mice is associated with differential expression of certain developmentally regulated genes, and that craniofacial defects can be partially rescued by reduced p53 levels. We postulate that variable responses to stress may contribute to variable craniofacial phenotypes by triggering differential expression of genes and variable cellular apoptosis.