Before metastasis, certain organs have already been influenced by primary tumors. However, the exact alterations and regulatory mechanisms of the premetastatic organs remain poorly understood. Here, we report that, in the premetastatic stage, angiopoietin 2 (Angpt2), matrix metalloproteinase (MMP) 3, and MMP10 are up-regulated in the lung by primary B16/F10 tumor, which leads to the increased permeability of pulmonary vasculatures and extravasation of circulating tumor cells. Subsequent studies show that Angpt2, MMP3, and MMP10 have a synergistic effect on disrupting vascular integrity in both in vitro and in vivo models. Lentivirus-based in vivo RNA interference of Angpt2, MMP3, and MMP10 attenuates the pulmonary vascular permeability and suppresses the infiltration of myeloid cells in the premetastatic lung. Moreover, knocking down these factors significantly inhibits the spontaneous lung metastasis in the model by orthotopic implantation of MDA-MB-231-Luc-D3H1 cells in nude mice. Further investigations reveal that the malignancy of tumor cells is positively correlated with their capabilities to induce the expression of Angpt2, MMP3, and MMP10. Luciferase reporter assay and chromatin immunoprecipitation assay also suggest that transforming growth factor-B1 and tumor necrosis factor-A signaling are involved in the regulation of these premetastatic factors. Our study shows that pulmonary vascular destabilization in the premetastatic phase promotes the extravasation of tumor cells and facilitates lung metastasis, which may provide potential targets for clinical prevention of metastasis.

In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2—the strongest drivers of growth in a KRAS-driven model—reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment. Significance: By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease. This article is highlighted in the In This Issue feature, p. 1601

To identify the genetic defect associated with autosomal dominant congenital nuclear cataract in a Chinese family, molecular genetic investigation via haplotype analysis and direct sequencing were performed Sequencing of the CRYGD gene revealed a c.127T>C transition, which resulted in a substitution of a highly conserved tryptophan with arginine at codon 43 (p.Trp43Arg). This mutation co-segregated with all affected individuals and was not observed in either unaffected family members or in 200 normal unrelated individuals. Biophysical studies indicated that the p.Trp43Arg mutation resulted in significant tertiary structural changes. The mutant protein was much less stable than the wild-type protein, and was more prone to aggregate when subjected to environmental stresses such as heat and UV irradiation. © 2010 Wiley-Liss, Inc.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS–driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. Significance: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS–driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models. This article is highlighted in the In This Issue feature, p. 1601

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher-order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

Indian Hedgehog (Ihh) regulates chondrocyte and osteoblast differentiation through the Glioma-associated oncogene homolog (Gli) transcription factors. Previous in vitro studies suggested that Speckletype POZ protein (Spop), part of the Cullin-3 (Cul3) ubiquitin ligase complex, targets Gli2 and Gli3 for degradation and negatively regulates Hedgehog (Hh) signaling. In this study, we found defects in chondrocyte and osteoblast differentiation in Spop-null mutant mice. Strikingly, both the full-length and repressor forms of Gli3, but not Gli2, were up-regulated in Spop mutants, and Ihh target genes Patched 1 (Ptch1) and parathyroid hormone-like peptide (Pthlh) were down-regulated, indicating compromised Hh signaling. Consistent with this finding, reducing Gli3 dosage greatly rescued the Spop mutant skeletal defects. We further show that Spop directly targets the Gli3 repressor for ubiquitination and degradation. Finally, we demonstrate in a conditional mutant that loss of Spop results in brachydactyly and osteopenia, which can be rescued by reducing the dosage of Gli3. In summary, Spop is an important positive regulator of Ihh signaling and skeletal development.T he human skeleton provides essential mechanical support, mobility, and mineral storage critical for health (1). In development, most bones form through endochondral ossification in which chondrocytes in the growth plate proliferate, undergo hypertrophic differentiation, and secrete calcium-containing extracellular matrix (2). The osteoblasts in the perichondrium, a thin layer of tissue surrounding the cartilage, replace the dying chondrocytes and secrete more bone matrix. On the other hand, osteoclasts, derived from white blood cells, invade and digest bone matrix. The balance between the osteoblast and osteoclast activities allows calcium homeostatic control and bone health. In osteopenia and osteoporosis, conditions afflicting more than 10 million Americans, an abnormal decrease in osteoblast activity or increase in osteoclast activity results in the loss of bone mass (1, 3). Unfortunately, our understanding of these bone diseases has been hindered by incomplete knowledge in the molecular mechanisms underlying endochondral bone development and remodeling.Indian Hedgehog (Ihh), a member of the Hedgehog (Hh) family of signaling proteins, is essential for endochondral bone development (4). Ihh regulates gene expression through the Gli family of transcription factors, which act as both transcriptional activators and repressors (5). In the absence of Hh, efficient proteolytic processing turns Gli3 into a transcriptional repressor (Gli3R) whereas processing of Gli2 is rather inefficient (6-8). Hh inhibits Gli processing and converts the full-length Gli proteins into transcriptional activators. Both Gli2 and Gli3 play important roles in the regulation of bone formation downstream of Ihh (9-12).Ihh maintains the expression of parathyroid hormone-like peptide (Pthlh) (Mouse Genome Informatics) in the periarticular perichondrium (13), which then stimulates the prol...

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. Significance: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G