Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

In contrast to the aberrant control of proliferation, apoptosis, angiogenesis and lifespan, the cellular mechanisms that cause local invasion and metastasis of tumour cells are still poorly understood. New experimental approaches have identified different types of epithelial-plasticity changes in tumour cells towards fibroblastoid phenotypes as crucial events that occur during metastasis, and many molecules and signalling pathways cooperate to trigger these processes.

Extensive prior research has focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearranging cis-regulatory elements remains unclear. Here, we present a framework for inferring cancer-related gene overexpression resulting from cis-regulatory element reorganization (e.g., enhancer hijacking), by integrating SCNAs, gene expression data, and information on chromatin interaction domains. Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer associates with recurrent deletions in cis, and present evidence supporting a tumor-promoting role. We additionally pursued cancer type-specific analyses, uncovering IGF2 as a target for enhancer hijacking in colorectal cancer. IGF2-containing tandem duplications result in the de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, which mediates high-level gene activation. Our framework enables systematic inference of cis-regulatory element rearrangements mediating dysregulation in cancer.

No abstract

Erk/MAPK and TGFbeta signaling cause epithelial to mesenchymal transition (EMT) and metastasis in mouse mammary epithelial cells (EpH4) transformed with oncogenic Ras (EpRas). In trials to unravel underlying mechanisms, expression profiling for EMT-specific genes identified a secreted interleukin-related protein (ILEI), upregulated exclusively at the translational level. Stable overexpression of ILEI in EpH4 and EpRas cells caused EMT, tumor growth, and metastasis, independent of TGFbeta-R signaling and enhanced by Bcl2. RNAi-mediated knockdown of ILEI in EpRas cells before and after EMT (EpRasXT) prevented and reverted TGFbeta-dependent EMT, also abrogating metastasis formation. ILEI is overexpressed and/or altered in intracellular localization in multiple human tumors, an event strongly correlated to invasion/EMT, metastasis formation, and survival in human colon and breast cancer.

In cerebellum, GABA A receptors containing ␣ 6 subunits are expressed exclusively in granule cells. The number of ␣ 6 receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on ␣ 6 subunit-specific antibodies indicated that 45% of GABA A receptors in cerebellar extracts contained ␣ 6 subunits. Western blot analysis demonstrated that ␣ 1 , ␤ 1 , ␤ 2 , ␤ 3 , ␥ 2 , and ␦ subunits co-purified with ␣ 6 subunits, suggesting the existence of multiple ␣ 6 receptor subtypes. These subtypes were identified using a new method based on the one-by-one immunochromatographic elimination of receptors containing the copurifying subunits in parallel or subsequent experiments. By quantification and Western blot analysis of ␣ 6 receptors remaining in the extract, the proportion of ␣ 6 receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that ␣ 6 receptors in cerebellum are composed predominantly ofOther experiments indicated that 10%, 51%, or 21% of ␣ 6 receptors contained homogeneous ␤ 1 , ␤ 2 , or ␤ 3 subunits, respectively, whereas two different ␤ subunits were present in 18% of all ␣ 6 receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABA A receptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors. (Sieghart, 1995). So far six ␣, four ␤, three ␥, one ␦, one ⑀, and three subunits have been cloned and sequenced from mammalian brain (Sieghart, 1995;Ogurusu and Shingai, 1996;Davies et al., 1997), and it is assumed that five subunits assemble to form f unctional GABA A receptors (Nayeem et al., 1994;Tretter et al., 1997). E xpression studies have indicated that ␣, ␤, and ␥ subunits have to combine to form receptors closely resembling native receptors. Depending on the type of ␣, ␤, and ␥ subunits used for transfection of cells, however, recombinant receptors with different pharmacological properties do arise (Sieghart, 1995). The distinct but overlapping regional and cellular expression of the individual subunits (Persohn et al., 1992;Wisden et al., 1992) raises the possibility of the existence of an extremely large variety of GABA A receptor subtypes in the brain. So far the actual extent of GABA A receptor heterogeneity is not known.GABA A receptors containing ␣ 6 subunits are expressed in cerebellar granule cells and in the embryologically related granule cells of the cochlear nucleus only Persohn et al., 1992;Wisden et al., 1992;Varecka et al., 1994;Jones et al., 1997). Thus, all ␣ 6 receptors from cerebellum are expressed in the same cell type. In addition, receptors consisting of ␣ 6 ␤ x ␥ 2 subunits have special properties because they exhibit a high affinity for the inverse benzodiazepine agonist Ro 15-4513 but no affinity for the benzodiazepine agonist diazepam (Sieghart, 1995).Several studies have investigated the subunit compo...

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and transdifferentiation to mesenchymal-like cells with invasive behavior.

The acquisition of metastatic ability by tumor cells is considered a late event in the evolution of malignant tumors. We report that untransformed mouse mammary cells that have been engineered to express the inducible oncogenic transgenes MYC and Kras D12 , or polyoma middle T, and introduced into the systemic circulation of a mouse can bypass transformation at the primary site and develop into metastatic pulmonary lesions upon immediate or delayed oncogene induction. Therefore, previously untransformed mammary cells may establish residence in the lung once they have entered the bloodstream and may assume malignant growth upon oncogene activation. Mammary cells lacking oncogenic transgenes displayed a similar capacity for long-term residence in the lungs but did not form ectopic tumors.Metastatic dissemination of cancer cells, the major cause of cancer mortality, is traditionally viewed as a late-stage event (1), although mammary epithelial cells have been shown to disseminate systemically from early neoplastic lesions in transgenic mice and from ductal carcinoma in situ in women (2). There is ample evidence that the ability of fully transformed tumor cells to metastasize depends on the regulation of developmental programs and external environmental cues (3-11), but to what extent the seeding or growth of tumor cells at the ectopic site is dependent on the initiating transforming event(s) is a subject of debate (12). We have developed a system that separates the process of seeding cells in the lung from the process of malignant growth at an ectopic site by using animals engineered to express potent oncogenes in a doxycycline-dependent mammary-specific manner. After intravenous (IV) injection of marked mammary cells that have different genetic potentials [no oncogenes, or oncogenes that will be expressed only after cells have taken up residence in an ectopic site (the lungs)], normal mammary cells can lodge in the lungs, grow slowly, and become frank metastatic malignancies once potent oncogenes are turned on.We recently described tri-transgenic TetO-MYC;TetO-Kras D12 ;MMTV-rtTA (TOM;TOR; MTB) mice that coordinately express MYC and mutant Kras oncogenes in mammary epithelial cells when fed doxycycline (13). Doxycycline-naïve animals do not express the transgenic oncogenes and have morphologically and functionally normal mammary glands, but they develop diffuse autochthonous tumors within 3 to 4 weeks after doxycycline exposure. Tumors that form because of the expression of these oncogenes display malignant characteristics, such as transplantability and metastasis ( fig. S1) To investigate whether mammary cells from these mice can be induced to form metastasis in the absence of transformation at the primary site, we modified the traditional experimental metastasis assay (14). In the modified approach, instead of IV delivery of tumor cells from doxycycline-treated animals into new recipients, we injected dissociated morphologically normal mammary cells from mature TOM;TOR;MTB animals never exposed to doxycy...