Somatic mutations affect key pathways in lung adenocarcinoma.

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Recurring mutations found by sequencing an acute myeloid leukemia genome.

BACKGROUND: The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known. METHODS: We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome. RESULTS: We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis. CONCLUSIONS: By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.

Akt-dependent cell size regulation by the adhesion molecule on glia occurs independently of phosphatidylinositol 3-kinase and Rheb signaling.

The role of cell adhesion molecules in mediating interactions with neighboring cells and the extracellular matrix has long been appreciated. More recently, these molecules have been shown to modulate intracellular signal transduction cascades critical for cell growth and proliferation. Expression of adhesion molecule on glia (AMOG) is downregulated in human and mouse gliomas, suggesting that AMOG may be important for growth regulation in the brain. In this report, we examined the role of AMOG expression on cell growth and intracellular signal transduction. We show that AMOG does not negatively regulate cell growth in vitro or in vivo. Instead, expression of AMOG in AMOG-deficient cells results in a dramatic increase in cell size associated with protein kinase B/Akt hyperactivation, which occurs independent of phosphatidylinositol 3-kinase activation. AMOG-mediated Akt phosphorylation specifically activates the mTOR/p70S6 kinase pathway previously implicated in cell size regulation, but it does not depend on tuberous sclerosis complex/Ras homolog enriched in brain (Rheb) signaling. These data support a novel role for a glial adhesion molecule in cell size regulation through selective activation of the Akt/mTOR/S6K signal transduction pathway.

Effect of merlin phosphorylation on neurofibromatosis 2 (NF2) gene function.

The neurofibromatosis 2 (NF2) tumor suppressor gene product, merlin, belongs to the ezrin-radixin-moesin (ERM) subgroup of the Protein 4.1 family, which links cell surface glycoproteins to the actin cytoskeleton. Previous studies have suggested that phosphorylation of merlin, similar to other ERM proteins, may regulate its function. To determine whether merlin phosphorylation has functional consequences for merlin suppression of cell growth and motility, we generated doxycycline-regulatable RT4 schwannoma cell lines that inducibly express full-length merlin with mutations at two potential phosphorylation sites (amino-acid residues S518 and T576). Whereas a mutation at S518 that mimics constitutive phosphorylation (S518D) abrogates the ability of merlin to suppress cell growth and motility, the S518A merlin mutant, which mimics nonphosphorylated merlin, functions equivalently to wild-type merlin. Similar mutations involving T576, the analogous phosphorylation site in ERM proteins important for regulating their function, had no effect. In contrast to other functionally inactive missense merlin mutants, the regulated overexpression of S518D merlin resulted in dramatic changes in cell shape and the elaboration of filopodial extensions. These results provide the first direct demonstration that the S518D merlin mutation, which mimics merlin phosphorylation, impairs not only merlin growth and motility suppression but also leads to an acquisition of a novel phenotype previously ascribed to ERM proteins.

Serine 518 phosphorylation modulates merlin intramolecular association and binding to critical effectors important for NF2 growth suppression.

The neurofibromatosis 2 (NF2) tumor suppressor protein, merlin, functions as a negative growth regulator; however, the molecular mechanisms that underlie merlin regulation remain elusive. Recent studies have implicated merlin phosphorylation in regulating merlin subcellular localization and growth suppression. P21-activated kinase (PAK), a downstream target of Rac1/Cdc42, directly phosphorylates merlin at Serine 518. In this report, we show that PAK2 directly phosphorylates wild-type merlin, whereas merlin truncation mutants with impaired GST-amino-terminal domain (N-term or NTD)/GST-carboxy-terminal domain (C-term or CTD) intramolecular association exhibit impaired S518 phosphorylation. We directly demonstrate that PAK2 phosphorylation impairs merlin N-term/C-term binding in vitro and in vivo. Lastly, we show that PAK2 phosphorylation impairs the ability of merlin to bind to two interacting proteins, CD44 and hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), both critical for merlin growth suppression. These observations suggest that merlin S518 phosphorylation directly modulates merlin intramolecular and intermolecular associations important for the ability of merlin to function as a tumor suppressor.

Functional significance of S6K overexpression in meningioma progression.

One common genetic change in anaplastic meningiomas is amplification of chromosome 17q23 containing the S6 kinase (S6K) gene. We show, for the first time to our knowledge, increased S6K mRNA expression in anaplastic meningiomas compared with benign tumors. To evaluate S6K as a candidate meningioma progression gene, we generated IOMM-Lee human meningioma cell lines overexpressing S6K. Whereas no effect of S6K overexpression on meningioma cell growth, motility, or adhesion was observed in vitro, S6K overexpression resulted in increased tumor size in vivo. Collectively, these results suggest that S6K is functionally important for meningioma progression and may represent a target for future meningioma therapy.

Functional analysis of the relationship between the neurofibromatosis 2 tumor suppressor and its binding partner, hepatocyte growth factor-regulated tyrosine kinase substrate.

Individuals with the neurofibromatosis 2 (NF2) inherited tumor predisposition syndrome are prone to the development of nervous system tumors, including schwannomas and meningiomas. The NF2 tumor suppressor protein, merlin or schwannomin, inhibits cell growth and motility as well as affects actin cytoskeleton-mediated processes. Merlin interacts with several proteins that might mediate merlin growth suppression, including hepatocyte growth factor-regulated tyrosine kinase substrate (HRS or HGS). Previously, we demonstrated that regulated overexpression of HRS in RT4 rat schwannoma cells had the same functional consequences as regulated overexpression of merlin. To determine the functional significance of this interaction, we generated a series of HRS truncation mutants and defined the regions of HRS required for merlin binding and HRS growth suppression. The HRS domain required for merlin binding was narrowed to a region (residues 470-497) containing the predicted coiled-coil domain whereas the major domain responsible for HRS growth suppression was distinct (residues 498-550). To determine whether merlin growth suppression required HRS, we demonstrated that merlin inhibited growth in HRS (+/+), but not HRS( -/-) mouse embryonic fibroblast cells. In contrast, HRS could suppress cell growth in the absence of Nf2 expression. These results suggest that merlin growth suppression requires HRS expression and that the binding of merlin to HRS may facilitate its ability to function as a tumor suppressor.

Genetic heterogeneity of stably transfected cell lines revealed by expression profiling with oligonucleotide microarrays.

Large-scale gene expression measurements with oligonucleotide microarrays have contributed tremendously to biological research. However, to distinguish between relevant expression changes and falsely identified positives, the source and magnitude of errors must be understood. Here, we report a source of biological variability in microarray experiments with stably transfected cell lines. Mouse embryonic fibroblast (MEF/3T3) and rat schwannoma (RT4) cell lines were generated to provide regulatable schwannomin expression. The expression levels of 29 samples from five different mouse embryonic fibroblast clonal cell lines and 18 samples from 3 RT4 cell lines were monitored with oligonucleotide microarrays. Using hierarchical clustering, we determined that the changes in gene expression induced by schwannomin overexpression were subtle when compared with those detected as a consequence of clonal selection during generation of the cell lines. The hierarchical clustering implies that significant alterations of gene expression were introduced during the transfection and selection processes. A total of 28 genes were identified by Kruskal-Wallis rank test that showed significant variation between clonal lines. Most of them were related to cytoskeletal function and signaling pathways. Based on these analyses, we recommend that replications of experiments with several selected cell lines are necessary to assess biological effects of induced gene expression.