High-resolution ROMA CGH and FISH analysis of aneuploid and diploid breast tumors.

Combining representational oligonucleotide microarray analysis (ROMA) of tumor DNA with fluorescence in situ hybridization (FISH) of individual tumor cells provides the opportunity to detect and validate a wide range of amplifications, deletions, and rearrangements directly in frozen tumor samples. We have used these combined techniques to examine 101 aneuploid and diploid breast tumors for which long-term follow-up and detailed clinical information were available. We have determined that ROMA provides accurate and sensitive detection of duplications, amplifications, and deletions and yields defined boundaries for these events with a resolution of <50 kbp in most cases. We find that diploid tumors exhibit fewer rearrangements on average than aneuploids, but rearrangements occur at the same locations in both types. Diploid tumors reflect at least three consistent patterns of rearrangement. The reproducibility and frequency of these events, especially in very early stage tumors, provide insight into the earliest chromosomal events in breast cancer. We have also identified correlations between certain sets of rearrangement events and clinically relevant parameters such as long-term survival. These correlations may enable novel prognostic indicators for breast and other cancers as more samples are analyzed.

The Ras-Byr2RBD complex: structural basis for Ras effector recognition in yeast.

BACKGROUND: The small GTP binding protein Ras has important roles in cellular growth and differentiation. Mutant Ras is permanently active and contributes to cancer development. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase. The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms. RESULTS: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components. The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization. A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region. CONCLUSIONS: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.

Design of a retroviral-mediated ecdysone-inducible system and its application to the expression profiling of the PTEN tumor suppressor.

We have engineered the ecdysone-inducible mammalian expression system for general retroviral delivery to cultured mammalian cells. We inducibly expressed PTEN in the glioblastoma cell line, U87MG, lacking this gene. Because nearly all cells are recruited on induction, we find both up- and down-regulated genes by cDNA microarray analysis. The changes we see are similar to those observed after treatment with LY294002, an inhibitor of phosphatidylinositol 3-OH kinase, fully consistent with the model that PTEN antagonizes phosphatidylinositol 3-OH kinase. Both treatments result in suppressed expression of the transforming growth factor (TGF)-beta gene and the genes of the cholesterol biosynthesis pathway. Our results illustrate the power of using a fully inducible expression system in conjunction with cDNA microarray analysis for exploring gene function.

PTEN controls tumor-induced angiogenesis.

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

Detecting gene copy number fluctuations in tumor cells by microarray analysis of genomic representations.

In this work, we explore the use of representations in conjunction with DNA microarray technology to measure gene copy number changes in cancer. We demonstrate that arrays of DNA probes derived from low-complexity representations can be used to detect amplifications, deletions, and polymorphic differences when hybridized to representations of genomic DNA. The method is both reproducible and verifiable, and is applicable even to microscopic amounts of primary tumors. We also present a mathematical model for array performance that is useful for designing and understanding DNA microarray hybridization protocols. The future applications and challenges of this approach are discussed.

Molecular forceps from combinatorial libraries prevent the farnesylation of Ras by binding to its carboxyl terminus.

INTRODUCTION: Ras is one of the major oncogenes. In order to function properly it has to undergo post-translational processing at its carboxyl terminus. It has been shown that inhibitors of farnesyl transferase, the first enzyme in the processing chain, can suppress the transforming activity of oncogenic Ras. RESULTS: We have identified molecular forceps, branched peptidic molecules, from combinatorial libraries that bind to the carboxyl terminus of Ras and interfere with its farnesylation without inhibiting the farnesyl transferase. The active molecules were selected by a screening against the carboxy-terminal octapeptide of Ras. CONCLUSIONS: The implications of our findings are twofold. First, we demonstrate that it is possible to prevent enzymatic transformations by blocking the enzyme's access to its substrate using a synthetic small molecule to mask the substrate. Second, we show that it is feasible to derive molecules from combinatorial libraries that bind a specific epitope on a protein by selecting these molecules with the isolated peptide epitope.

The lipid phosphatase activity of PTEN is critical for its tumor supressor function.

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.

Signaling pathways in Ras-mediated tumorigenicity and metastasis.

The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes in athymic nude mice when expressed in NIH 3T3 fibroblasts. All mutants displayed comparable tumorigenic properties, but only the mutant that activates the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) 1/2 pathway, V12S35 Ras, induced tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK1/2 pathway in NIH 3T3 cells by mos or a constitutively active form of MEK was sufficient to induce metastasis whereas R-Ras, which fails to activate the ERK1/2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells expressed higher levels of activated ERK1/2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK1/2 occurred during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors did not show changes in the level of ERK1/2 activation when compared with the parental cells. When tumor-explanted cell lines derived from each of the effector domain mutants were passaged one additional time in vivo, all mediated rapid tumor growth, but, again, only cells derived from V12S35 Ras-tumors formed numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK1/2 activation, experimental metastasis appears to require constitutive activation of the ERK1/2 pathway.

Rapid isolation of cDNA by hybridization.

The isolation of genes from a given genomic region can be a rate-limiting step in the discovery of disease genes. We describe an approach to the isolation of cDNAs that have sequences in common with large genomic clones such as bacterial artificial chromosomes. We applied this method to loci both amplified and deleted in cancer, illustrating its usage in the identification of both oncogenes and tumor suppressor genes, respectively. The method, called rapid isolation of cDNAs by hybridization (RICH), depends on solution hybridization, enzymatic modification, and amplification/selection of sequences present in both cDNA populations and the genomic clones. The method should facilitate the development of transcription maps for large genomic clones, possibly even yeast artificial chromosomes.

Genetic analysis using genomic representations.

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible "representations" of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase.

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan-Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.

PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.

Mapping of homozygous deletions on human chromosome 10q23 has led to the isolation of a candidate tumor suppressor gene, PTEN, that appears to be mutated at considerable frequency in human cancers. In preliminary screens, mutations of PTEN were detected in 31% (13/42) of glioblastoma cell lines and xenografts, 100% (4/4) of prostate cancer cell lines, 6% (4/65) of breast cancer cell lines and xenografts, and 17% (3/18) of primary glioblastomas. The predicted PTEN product has a protein tyrosine phosphatase domain and extensive homology to tensin, a protein that interacts with actin filaments at focal adhesions. These homologies suggest that PTEN may suppress tumor cell growth by antagonizing protein tyrosine kinases and may regulate tumor cell invasion and metastasis through interactions at focal adhesions.

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.

Evaluation of the FHIT gene in colorectal cancers.

A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.

A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation.

Oncogenic Ras transforms cells through the activation of multiple downstream pathways mediated by separate effector molecules, one of which is Raf. Here we report the identification of a second ras-binding protein that can induce cellular transformation in parallel with activation of the Raf/mitogen-activated protein kinase cascade. The Ral guanine nucleotide dissociation stimulator (RalGDS) was isolated from a screen for Ras-binding proteins that specifically interact with a Ras effector-loop mutant, ras(12V,37G), that uncouples Ras from activation of Raf1. RalGDS, like ras(12V, 37G), cooperates synergistically with mutationally activated Raf to induce foci of growth and morphologically transformed NIH 3T3 cells. RalGDS does not significantly enhance MAP kinase activation by activated Raf, suggesting that the cooperativity in focus formation is due to a distinct pathway acting downstream of Ras and parallel to Raf.

Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS.

The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS.

Multiple Ras functions can contribute to mammalian cell transformation.

We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

Comparative genomic analysis of tumors: detection of DNA losses and amplification.

We demonstrate the use of representational difference analysis for cloning probes that detect DNA loss and amplification in tumors. Using DNA isolated from human tumor cell lines to drive hybridization against matched normal DNA, we were able to identify six genomic regions that are homozygously deleted in cultured cancer cells. When this method was applied in the reverse way, using normal DNA to drive hybridization against tumor cell DNA, we readily isolated probes detecting amplification. Representational difference analysis was also performed on DNAs derived from tumor biopsies, and we thereby discovered a probe detecting very frequent homozygous loss in colon cancer cell lines and located on chromosome 3p.

Cooperative interaction of S. pombe proteins required for mating and morphogenesis.

We isolated two S. pombe genes, scd1 and scd2, that are required for normal morphology and mating. scd1 and scd2 are homologous to CDC24 and BEM1, respectively, of S. cerevisiae. Epistasis analyses indicate that scd2 and ras1 converge upon scd1, which, in turn, interacts with cdc42sp, a RHO-like GTPase. Studies with the yeast two-hybrid system indicate that scd2 forms complexes with both scd1 and cdc42sp. Furthermore, biochemical studies indicate that the interaction between scd1 and scd2 is direct. The yeast two-hybrid data further suggest that scd1, scd2, cdc42sp, and ras1, in its GTP-bound state, act cooperatively to form a protein complex.