5q- myelodysplastic syndromes: chromosome 5q genes direct a tumor-suppression network sensing actin dynamics.

Complete loss or interstitial deletions of chromosome 5 are the most common karyotypic abnormality in myelodysplastic syndromes (MDSs). Isolated del(5q)/5q- MDS patients have a more favorable prognosis than those with additional karyotypic defects, who tend to develop myeloproliferative neoplasms (MPNs) and acute myeloid leukemia. The frequency of unbalanced chromosome 5 deletions has led to the idea that 5q harbors one or more tumor-suppressor genes that have fundamental roles in the growth control of hematopoietic stem/progenitor cells (HSCs/HPCs). Cytogenetic mapping of commonly deleted regions (CDRs) centered on 5q31 and 5q32 identified candidate tumor-suppressor genes, including the ribosomal subunit RPS14, the transcription factor Egr1/Krox20 and the cytoskeletal remodeling protein, alpha-catenin. Although each acts as a tumor suppressor, alone or in combination, no molecular mechanism accounts for how defects in individual 5q candidates may act as a lesion driving MDS or contributing to malignant progression in MPN. One candidate gene that resides between the conventional del(5q)/5q- MDS-associated CDRs is DIAPH1 (5q31.3). DIAPH1 encodes the mammalian Diaphanous-related formin, mDia1. mDia1 has critical roles in actin remodeling in cell division and in response to adhesive and migratory stimuli. This review examines evidence, with a focus on mouse gene-targeting experiments, that mDia1 acts as a node in a tumor-suppressor network that involves multiple 5q gene products. The network has the potential to sense dynamic changes in actin assembly. At the root of the network is a transcriptional response mechanism mediated by the MADS-box transcription factor, serum response factor (SRF), its actin-binding myocardin family coactivator, MAL, and the SRF-target 5q gene, EGR1, which regulate the expression of PTEN and p53-family tumor-suppressor proteins. We hypothesize that the network provides a homeostatic mechanism balancing HPC/HSC growth control and differentiation decisions in response to microenvironment and other external stimuli.

Identification of deregulated oncogenic pathways in renal cell carcinoma: an integrated oncogenomic approach based on gene expression profiling.

In this age of targeted therapy, identification of molecular pathways that are deregulated in cancer will not only elucidate underlying tumorigenic mechanisms, but may also help to determine the classes of drugs that are used for treatment. In kidney cancer, a spectrum of histological subtypes exists that are characterized both by distinct molecular signatures and increasingly by distinct molecular pathways that are deregulated in each subtype. For example, the VHL/hypoxia pathway is well-known to be deregulated in clear cell renal cell carcinoma (RCC) whereas in papillary RCC activation of the HGF/Met pathway has been implicated. Additional molecular pathways, many not yet identified, may also be involved in the development of the different histologic subtypes. Moreover, differences in pathway activation may reflect differences in tumor progression and response to treatment. In this article, we describe an oncogenomic approach, based on integrative analysis of gene expression profiling data. In this approach, gene expression data is used to identify both cytogenetic abnormalities and molecular pathways that are deregulated in RCC. Ideally, predicted pathway abnormalities can be linked to predicted cytogenetic abnormalities to identify likely candidate genes. Although further cellular and functional studies are warranted to validate the computational models, development of such models in RCC have the potential to open up new avenues of molecular research and may have significant diagnostic and therapeutic implications.

Microarray profiling of the effects of histone deacetylase inhibitors on gene expression in cancer cell lines.

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase.

Mutations in the Ras family of GTP binding proteins represent one of the most frequently observed genetic alterations in human cancers. We and others have recently demonstrated that expression of Met, the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), is significantly up-regulated in Ras-transformed cells. Because HGF/SF-Met signaling is proposed to play a prominent role in tumor development and progression, we assessed the possible requirement for Met during Ras-mediated tumor growth and metastasis. To disrupt endogenous Met signaling, we constructed dominant-negative mutants of both human and murine Met and showed that these can inhibit HGF/SF-mediated Met signaling and cell invasion of ras-transformed cells in vitro. Moreover, ectopic expression of dominant-negative Met mutants reduced the s.c. tumor growth of ras-transformed cells and dramatically suppressed their ability to form lung metastases in vivo. Our data demonstrate that Met plays a prominent role during Ras-mediated tumor growth and metastasis, and further suggest that agents that inhibit HGF/SF-Met signaling may represent an important therapeutic avenue for the treatment of a variety of malignant tumors.

Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification.

To better understand the molecular mechanisms that underlie the tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC), we studied the gene expression profiles of 29 ccRCC tumors obtained from patients with diverse clinical outcomes by using 21,632 cDNA microarrays. We identified gene expression alterations that were both common to most of the ccRCC studied and unique to clinical subsets. There was a significant distinction in gene expression profile between patients with a relatively nonaggressive form of the disease [100% survival after 5 years with the majority (15/17 or 88%) having no clinical evidence of metastasis] versus patients with a relatively aggressive form of the disease (average survival time 25.4 months with a 0% 5-year survival rate). Approximately 40 genes most accurately make this distinction, some of which have previously been implicated in tumorigenesis and metastasis. To test the robustness and potential clinical usefulness of this molecular distinction, we simulated its use as a prognostic tool in the clinical setting. In 96% of the ccRCC cases tested, the prediction was compatible with the clinical outcome, exceeding the accuracy of prediction by staging. These results suggest that two molecularly distinct forms of ccRCC exist and that the integration of expression profile data with clinical parameters could serve to enhance the diagnosis and prognosis of ccRCC. Moreover, the identified genes provide insight into the molecular mechanisms of aggressive ccRCC and suggest intervention strategies.

Met receptor tyrosine kinase: enhanced signaling through adapter proteins.

The Met receptor tyrosine kinase is the prototypic member of a small subfamily of growth factor receptors that when activated induce mitogenic, motogenic, and morphogenic cellular responses. The ligand for Met is hepatocyte growth factor/scatter factor (HGF/SF) and while normal HGF/SF-Met signaling is required for embryonic development, abnormal Met signaling has been strongly implicated in tumorigenesis, particularly in the development of invasive and metastatic phenotypes. Following ligand binding and autophosphorylation, Met transmits intercellular signals using a unique multisubstrate docking site present within the C-terminal end of the receptor. The multisubstrate docking site mediates the binding of several adapter proteins such as Grb2, SHC, Crk/CRKL, and the large adapter protein Gab1. These adapter proteins in turn recruit several signal transducing proteins to form an intricate signaling complex. Analysis of how these adapter proteins bind to the Met receptor and what signal transducers they recruit have led to more substantial models of HGF/SF-Met signal transduction and have uncovered new potential pathways that may be involved into Met mediated tumor cell invasion and metastasis.

Loss of Rhb1, a Rheb-related GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that caused by nitrogen starvation.

The Rheb GTPase is most similar in primary sequence to the Ras, Rap, R-Ras, and Ral GTPases, which regulate cell growth and differentiation in many cell types. A likely fission yeast homologue of mammalian Rheb, which we designated Rhb1, was identified by genome sequencing. Our investigation of rhb1 showed that rhb1(-) cells arrested cell growth and division with a terminal phenotype similar to that of nitrogen-starved cells. In particular, cells depleted of Rhb1 arrested as small, round cells with 1N DNA content, arrested more quickly in low-nitrogen medium, and induced expression of fnx1 and mei2 mRNA, two mRNAs that were normally induced by nitrogen starvation. Since mammalian Rheb binds and may regulate Raf-1, a Ras effector, we tested for functional overlap between Ras1 and Rhb1 in fission yeast. This analysis showed that Ras1 overexpression did not suppress rhb1(-) mutant phenotypes, Rhb1 overexpression did not suppress ras1(-) mutant phenotypes, and ras1(-) rhb1(-) double mutants had phenotypes equal to the sum of the corresponding single-mutant phenotypes. Hence, there is no evidence for overlapping functions between Ras1 and Rhb1. On the basis of this study, we hypothesize that Rhb1 negatively regulates entry into stationary phase when extracellular nitrogen levels are adequate for growth. If this hypothesis is correct, then Rhb1 and Ras1 regulate alternative responses to limiting nutrients.

Byr4 localizes to spindle-pole bodies in a cell cycle-regulated manner to control Cdc7 localization and septation in fission yeast.

Cytokinesis and septation in the fission yeast Schizosaccharomyces pombe are studied as a model for mammalian cell division. In fission yeast, septation is positively regulated by Spg1, a Ras family GTPase that localizes to spindle-pole bodies (SPBs) throughout the cell cycle. As cells enter mitosis, Spg1 accumulates in an active, GTP-bound form and binds the Cdc7 protein kinase to cause Cdc7 translocation to SPBs. Cdc7 disappears from one SPB in mid-anaphase and from the second SPB in late mitosis. Byr4 plus Cdc16 negatively regulate septation by forming a two-component GTPase-activating protein for Spg1. These results led us to hypothesize that Byr4 localization to SPBs regulated the nucleotide state of Spg1, due to its ability to form Spg1GAP activity with Cdc16 and thus the binding of Cdc7 to Spg1 at SPBs. To test this hypothesis, Byr4 localization was determined using indirect immunofluorescence. This analysis revealed that Byr4 was localized to SPBs that did not contain Cdc7. In byr4(-) mutants, Cdc7 localized to interphase SPBs and only symmetrically localized to mitotic SPBs. In contrast, Byr4 overexpression prevented Spg1 and Cdc7 localization to SPBs. These results suggest that Byr4 localization to SPBs maintains Spg1 in an inactive form, presumably by stimulating Spg1 GTPase activity with Cdc16, and that loss of Byr4 from mitotic SPBs increases the active fraction of Spg1 and thereby increases Spg1-Cdc7 binding. Byr4 localization to SPBs was decreased in spg1, cdc16, sid4, and cdc11 mutants as well as in several mutants that affect medial F-actin structures, suggesting that multiple pathways regulate Byr4 localization to SPBs.

Regions of Byr4, a regulator of septation in fission yeast, that bind Spg1 or Cdc16 and form a two-component GTPase-activating protein with Cdc16.

In the fission yeast Schizosaccharomyces pombe, septation and constriction of the actomyosin ring for cell division are positively regulated by the Spg1 GTPase, a member of the Ras superfamily. Spg1 is negatively regulated by Byr4 and Cdc16, which together form a two-component GTPase-activating protein for the Spg1 GTPase. To better understand how Byr4 regulates septation, Byr4 mutants were tested for in vitro functions. This analysis revealed that Byr4 contained one Cdc16-binding site and four Spg1-binding sites (SBS), designated SBS1-SBS4. Although mutants with a single SBS bound Spg1 and inhibited GTP dissociation, the equilibrium binding affinity of these mutants was 28-280-fold weaker than Byr4. Because some Byr4 mutants with multiple SBSs bound Spg1 tighter than the corresponding mutants with a single SBS, multiple SBSs probably interact to cause the high affinity binding of Byr4 to Spg1. A region of Byr4 that bound Spg1, SBS4, and the region that bound Cdc16, Cdc16-binding site, was necessary and sufficient to form Cdc16-dependent Spg1GAP activity that was similar to that of wild-type Byr4 with Cdc16.

Byr4 and Cdc16 form a two-component GTPase-activating protein for the Spg1 GTPase that controls septation in fission yeast.

BACKGROUND: Spatial and temporal control of cytokinesis ensures the accurate transmission of genetic material and the correct development of multicellular organisms. An excellent model system in which to study cytokinesis is Schizosaccharomyces pombe because there are similarities between cytokinesis in S. pombe and mammals and because genes involved in S. pombe cytokinesis have been characterized. In particular, formation of the septum is positively regulated by the Spg1 GTPase and its effector, the Cdc7 kinase. Septation is negatively regulated by Cdc16, a protein similar to GTPase-activating proteins (GAPs) for Ypt GTPases, and by Byr4, a protein of unknown biochemical function. This study investigates the relationship between Byr4, Cdc16, and Spg1. RESULTS: Genetic interactions were observed between byr4, cdc16, and spg1 mutants. Byr4 bound to Cdc16 and Spg1 in yeast two-hybrid assays and in coprecipitations in vitro and in yeast. Byr4 inhibited the dissociation and hydrolysis of GTP bound to Spg1, but when Byr4 and Cdc16 were combined together they displayed Spg1GAP activity in vitro; Cdc16 alone had no detectable GAP activity. The binding of Byr4 to Spg1 and the Byr4-Cdc16 Spg1GAP activity were specific because Byr4 and Cdc16 did not bind to or affect the GTPase activities of the seven known S pombe Ypt family GTPase. CONCLUSIONS: Byr4 and Cdc16 form a two-component GAP for the Spg1 GTPase. Byr4 and Cdc16 appear to negatively regulate septation in S. pombe by modulating the nucleotide state of Spg1 possibly in a spatially or temporally controlled manner.