Preclinical evaluation of the combination of mTOR and proteasome inhibitors with radiotherapy in malignant peripheral nerve sheath tumors.

About one half of malignant peripheral nerve sheath tumors (MPNST) have Neurofibromin 1 (NF1) mutations. NF1 is a tumor suppressor gene essential for negative regulation of RAS signaling. Survival for MPNST patients is poor and we sought to identify an effective combination therapy. Starting with the mTOR inhibitors rapamycin and everolimus, we screened for synergy in 542 FDA approved compounds using MPNST cells with a native NF1 loss in both alleles. We further analyzed the cell cycle and signal transduction. In vivo growth effects of the drug combination with local radiation therapy (RT) were assessed in MPNST xenografts. The synergistic combination of mTOR inhibitors with bortezomib yielded a reduction in MPNST cell proliferation. The combination of mTOR inhibitors and bortezomib also enhanced the anti-proliferative effect of radiation in vitro. In vivo, the combination of mTOR inhibitor (everolimus) and bortezomib with RT decreased tumor growth and proliferation, and augmented apoptosis. The combination of approved mTOR and proteasome inhibitors with radiation showed a significant reduction of tumor growth in an animal model and should be investigated and optimized further for MPNST therapy.

Human genes containing polymorphic trinucleotide repeats.

Expansions of trinucleotide repeats within gene transcripts are responsible for fragile X syndrome, myotonic dystrophy and spinal and bulbar muscular atrophy. To identify other human genes with similar features as candidates for triplet repeat expansion mutations, we screened human cDNA libraries with repeat probes and searched databases for transcribed genes with repeats. From both strategies, 40 genes were identified and 14 characterized. Five were found to contain repeats which are highly polymorphic including the N-cadherin, BCR, glutathione-S-transferase and Na+/K+ ATPase (beta-subunit) genes. These data demonstrate the occurrence of other human loci which may undergo this novel mechanism of mutagenesis giving rise to genetic disease.

Mad-related genes in the human.

Resistance to the growth inhibitory effects of TGF-beta is common in human cancers. However, the mechanism(s) by which tumour cells become resistant to TGF-beta are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-beta family members. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-beta-like ligands.

Genes expressed in human tumor endothelium.

To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.

Investigation of chromosome 1q reveals differential expression of members of the S100 family in clinical subgroups of intracranial paediatric ependymoma.

Gain of 1q is one of the most common alterations in cancer and has been associated with adverse clinical behaviour in ependymoma. The aim of this study was to investigate this region to gain insight into the role of 1q genes in intracranial paediatric ependymoma. To address this issue we generated profiles of eleven ependymoma, including two relapse pairs and seven primary tumours, using comparative genome hybridisation and serial analysis of gene expression. Analysis of 656 SAGE tags mapping to 1q identified CHI3L1 and S100A10 as the most upregulated genes in the relapse pair with de novo 1q gain upon recurrence. Moreover, three more members of the S100 family had distinct gene expression profiles in ependymoma. Candidates (CHI3L1, S100A10, S100A4, S100A6 and S100A2) were validated using immunohistochemistry on a tissue microarray of 74 paediatric ependymoma. In necrotic cases, CHI3L1 demonstrated a distinct staining pattern in tumour cells adjacent to the areas of necrosis. S100A6 significantly correlated with supratentorial tumours (P<0.001) and S100A4 with patients under the age of 3 years at diagnosis (P=0.038). In conclusion, this study provides evidence that S100A6 and S100A4 are differentially expressed in clinically relevant subgroups, and also demonstrates a link between CHI3L1 protein expression and necrosis in intracranial paediatric ependymoma.

Transcriptional response to hypoxia in human tumors.

BACKGROUND: The presence of hypoxic regions within solid tumors is associated with a more malignant tumor phenotype and worse prognosis. To obtain a blood supply and protect against cellular damage and death, oxygen-deprived cells in tumors alter gene expression, resulting in resistance to therapy. To investigate the mechanisms by which cancer cells adapt to hypoxia, we looked for novel hypoxia-induced genes. METHODS: The transcriptional response to hypoxia in human glioblastoma cells was quantified with the use of serial analysis of gene expression. The time course of gene expression in response to hypoxia in a panel of various human tumor cell lines was measured by real-time polymerase chain reaction. Hypoxic regions of human carcinomas were chemically marked with pimonidazole. Immunohistochemistry and in situ hybridization were used to examine gene expression in the tumor's hypoxic regions. RESULTS: From the 24 504 unique transcripts expressed, 10 new hypoxia-regulated genes were detected-all induced, to a greater extent than vascular endothelial growth factor, a hypoxia-induced mitogen that promotes blood vessel growth. These genes also responded to hypoxia in breast and colon cancer cells and were activated by hypoxia-inducible factor 1, a key regulator of hypoxic responses. In tumors, gene expression was limited to hypoxic regions. Induced genes included hexabrachion (an extracellular matrix glycoprotein), stanniocalcin 1 (a calcium homeostasis protein), and an angiopoietin-related gene. CONCLUSIONS: We have identified the genes that are transcriptionally activated within hypoxic malignant cells, a crucial first step in understanding the complex interactions driving hypoxia response. Within our catalogue of hypoxia-responsive genes are novel candidates for hypoxia-driven angiogenesis.

Frequency of Smad gene mutations in human cancers.

Much excitement has recently been generated by the discovery of the Smad genes, encoding proteins that transduce signals from the transforming growth factor beta family of cytokines. Here, we report the completion of cloning of the six known human Smads, providing novel sequences for Smad5 and Smad6. Previously, Smad4 and Smad2 were shown to be mutated in human cancers. However, analysis of the other four Smad genes revealed no mutations in a total of 167 tumors, including those from colon, breast, lung, and pancreas. These results suggest that the various Smad genes have different functions and demonstrate that mutations in these four genes do not, in general, account for the widespread resistance to transforming growth factor beta that is found in human tumors.

Absence of secretory phospholipase A2 gene alterations in human colorectal cancer.

A potent modifying locus of intestinal tumorigenesis in the mouse was recently identified as secretory phospholipase A2 (sPLA2). The human homologue of sPLA2 maps to chromosome 1p35, a region frequently lost in human tumors. To evaluate the possibility that sPLA2 was a tumor suppressor gene that was the target of the 1p loss events, we identified polymorphisms within the human sPLA2 gene. Using these polymorphisms, 31% of 16 colorectal carcinomas were found to lose a sPLA2 allele. However, sequence analysis of the complete coding region of sPLA2 revealed no somatic mutations in the remaining allele of those tumors with allelic loss, nor in 18 additional colorectal cancers. Thus, sPLA2 is within the chromosomal region often lost during colorectal tumorigenesis, but mutations of this gene do not appear to play a major role in colorectal cancer development, and sPLA2 is unlikely to be the 1p35 tumor suppressor.

Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer.

Difficulties in the detection, diagnosis, and treatment of ovarian cancer result in an overall low survival rate of women with this disease. A better understanding of the pathways involved in ovarian tumorigenesis will likely provide new targets for early and effective intervention. Here, we have used serial analysis of gene expression (SAGE) to generate global gene expression profiles from various ovarian cell lines and tissues, including primary cancers, ovarian surface epithelia cells, and cystadenoma cells. The profiles were used to compare overall patterns of gene expression and to identify differentially expressed genes. We have sequenced a total of 385,000 tags, yielding >56,000 genes expressed in 10 different libraries derived from ovarian tissues. In general, ovarian cancer cell lines showed relatively high levels of similarity to libraries from other cancer cell lines, regardless of the tissue of origin (ovarian or colon), indicating that these lines had lost many of their tissue-specific expression patterns. In contrast, immortalized ovarian surface epithelia and ovarian cystadenoma cells showed much higher similarity to primary ovarian carcinomas than to primary colon carcinomas. Primary tissue specimens therefore appeared to be a better model for gene expression analyses. Using the expression profiles described above and stringent selection criteria, we have identified a number of genes highly differentially expressed between nontransformed ovarian epithelia and ovarian carcinomas. Some of the genes identified are already known to be overexpressed in ovarian cancer, but several represent novel candidates. Many of the genes up-regulated in ovarian cancer represent surface or secreted proteins such as claudin-3 and -4, HE4, mucin-1, epithelial cellular adhesion molecule, and mesothelin. Interestingly, both apolipoprotein E (ApoE) and ApoJ, two proteins involved in lipid homeostasis, are among the genes highly up-regulated in ovarian cancer. Selected serial analysis of gene expression results were further validated through immunohistochemical analysis of ApoJ, claudin-3, claudin-4, and epithelial cellular adhesion molecule in archival material. These experiments provided additional evidence of the relevance of our findings in vivo. The publicly available expression data reported here should stimulate and aid further research in the field of ovarian cancer.

A public database for gene expression in human cancers.

A public database, SAGEmap, was created as a component of the Cancer Genome Anatomy Project to provide a central location for depositing, retrieving, and analyzing human gene expression data. This database uses serial analysis of gene expression to quantify transcript levels in both malignant and normal human tissues. By accessing SAGEmap (http://www.ncbi.nlm.nih.gov/SAGE) the user can compare transcript populations between any of the posted libraries. As an initial demonstration of the database's utility, gene expression in human glioblastomas was compared with that of normal brain white matter. Of the 47,174 unique transcripts expressed in these two tissues, 471 (1.0%) were differentially expressed by more than 5-fold (P<0.001). Classification of these genes revealed functions consistent with the biological properties of glioblastomas, in particular: angiogenesis, transcription, and cell cycle related genes.

Gene discovery using the serial analysis of gene expression technique: implications for cancer research.

Cancer is a genetic disease. As such, our understanding of the pathobiology of tumors derives from analyses of the genes whose mutations are responsible for those tumors. The cancer phenotype, however, likely reflects the changes in the expression patterns of hundreds or even thousands of genes that occur as a consequence of the primary mutation of an oncogene or a tumor suppressor gene. Recently developed functional genomic approaches, such as DNA microarrays and serial analysis of gene expression (SAGE), have enabled researchers to determine the expression level of every gene in a given cell population, which represents that cell population's entire transcriptome. The most attractive feature of SAGE is its ability to evaluate the expression pattern of thousands of genes in a quantitative manner without prior sequence information. This feature has been exploited in three extremely powerful applications of the technology: the definition of transcriptomes, the analysis of differences between the gene expression patterns of cancer cells and their normal counterparts, and the identification of downstream targets of oncogenes and tumor suppressor genes. Comprehensive analyses of gene expression not only will further understanding of growth regulatory pathways and the processes of tumorigenesis but also may identify new diagnostic and prognostic markers as well as potential targets for therapeutic intervention.

Characterization of a highly polymorphic dinucleotide repeat 150 KB proximal to the fragile X site.

Fragile X [fra (X)] syndrome is a frequently encountered form of mental retardation and is inherited as an X-linked semi-dominant trait with reduced penetrance. We report here the characterization of a highly polymorphic dinucleotide repeat, DXS 548, which is approximately 150 kb proximal to the fra(X) site and the associated FMR-1 gene. DXS 548 is tightly linked to the fra (X) syndrome locus (FRAXA) without recombination (LOD = 9.07 with q of 0) in selected families with crossovers between FRAXA and very closely linked flanking markers. This dinucleotide repeat could be useful in determining the parental origin of a new fra (X) mutations and evaluating the role of FMR-1 in X-linked non-specific mental retardation.

Using Serial Analysis of Gene Expression to identify tumor markers and antigens.

Tumor markers and antigens are normally highly expressed in malignant tissue, but not in the surrounding normal tissue. Serial Analysis of Gene Expression (SAGE) is a technology that counts mRNA transcripts and can be used to find those genes most highly induced in malignant tissues. SAGE produces a comprehensive profile of gene expression and can be used to search for tumor biomarkers in a limited number of samples. Public sources of SAGE data, in particular through the Cancer Genome Anatomy Project, increase the value of this technology by making a large source of information on many tumors and normal tissues available for comparison. Although the perfect tumor-specific gene does not exist, the differences in gene expression between tumor and normal can be exploited for therapeutic or diagnostic purposes.

Ham56-immunoreactive macrophages in untreated infiltrating gliomas.

CONTEXT: Classic diagnostic neuropathologic teachings have cautioned against making the diagnosis of neoplasia in the presence of a macrophage population. The knowledge of macrophage distribution should prove useful when confronted with an infiltrating glioma containing macrophages. OBJECTIVE: To identify macrophages in untreated, infiltrating gliomas using the monoclonal antibody HAM56, and to confirm their presence in an untreated glioblastoma multiforme (GBM) with the serial analysis of gene expression (SAGE) method. METHODS: We evaluated the presence of macrophages in 16 cases of untreated, supratentorial infiltrating gliomas with the macrophage monoclonal antibody HAM56. We performed SAGE for one case of GBM and for normal brain tissue. RESULTS: In World Health Organization (WHO) grade II well-differentiated astrocytoma and oligodendroglioma, HAM56 reactivity was noted only in endothelial cells, and unequivocal macrophages were not identified. In WHO grade III anaplastic astrocytoma and anaplastic oligodendroglioma, rare HAM56-positive macrophages were noted in solid areas of tumor. In WHO grade IV GBM, HAM56-positive macrophages were identified in areas of solid tumor (mean labeling index, 8.6%). In all cases of GBM, nonquantitated HAM56-positive macrophages were identified in foci of pseudopalisading cells abutting necrosis and in foci of microvascular proliferations. In none of the cases were granulomas or microglial nodules found, and there was no prior history of surgical intervention, radiation therapy, chemotherapy, or head trauma in these cases. By SAGE, the macrophage-related proteins osteopontin and macrophage-capping protein were overexpressed 12-fold and eightfold, respectively, in one untreated GBM compared with normal brain tissue. In this case, numerous HAM56-positive macrophages (labeling index, 24.5%) were present in the solid portion of tumor, and abundant nonquantified macrophages were identified in foci of pseudopalisading cells abutting necrosis and in foci of microvascular proliferations. CONCLUSIONS: This study confirms the utility of the monoclonal antibody HAM56 in identifying macrophages within untreated infiltrating gliomas. The overexpression of macrophage-related proteins in one case of GBM as detected by SAGE signifies that macrophages may be present in untreated GBMs.

What can digital transcript profiling reveal about human cancers?

Important biological and clinical features of malignancy are reflected in its transcript pattern. Recent advances in gene expression technology and informatics have provided a powerful new means to obtain and interpret these expression patterns. A comprehensive approach to expression profiling is serial analysis of gene expression (SAGE), which provides digital information on transcript levels. SAGE works by counting transcripts and storing these digital values electronically, providing absolute gene expression levels that make historical comparisons possible. SAGE produces a comprehensive profile of gene expression and can be used to search for candidate tumor markers or antigens in a limited number of samples. The Cancer Genome Anatomy Project has created a SAGE database of human gene expression levels for many different tumors and normal reference tissues and provides online tools for viewing, comparing, and downloading expression profiles. Digital expression profiling using SAGE and informatics have been useful for identifying genes that have a role in tumor invasion and other aspects of tumor progression.

Genome and genetic resources from the Cancer Genome Anatomy Project.

The Cancer Genome Anatomy Project (CGAP) is a collaborative network of cancer researchers with a common goal: to decipher the genetic changes that occur during cancer formation and progression. The project brings together several recent technologies capable of high-throughput analysis to help achieve this goal. Automated sequencing of cDNA libraries is a primary focus and is geared towards providing a comprehensive and annotated set of human and mouse transcribed sequences. This effort includes full-length transcript sequence generated by CGAP's new Mammalian Gene Collection initiative. Single nucleotide polymorphisms (SNPs) within human gene sequences (Genetic Annotation Initiative) and chromosomal rearrangements within cancer cells (Cancer Chromosome Aberration Project) are also being cataloged as part of CGAP. Finally, to help determine gene expression patterns related to cancer, CGAP provides a quantitative catalog of data through its SAGEmap initiative. The genome and genetic analysis tools listed in this review are all freely distributed by CGAP (http://cgap.nci.nih.gov/) without restriction.

Serial analysis of gene expression: probing transcriptomes for molecular targets.

Serial analysis of gene expression (SAGE) is a method to rapidly count expressed RNA transcripts in a population of cells. The basic approach is to isolate a small transcript tag, clone multiple tags into a sequencing vector and efficiently count the tags by automated sequencing. The result is the fractional representation of nearly every transcript (the transcriptome), in a digital format. These tag counts can be compared to other SAGE libraries yielding differentially expressed genes. Analysis of the differentially expressed genes has been used to determine which genes are involved in a disease process. The promise of this technology is that by understanding when genes are pathologically altered in expression, therapies can be formulated to target the appropriate gene or pathway.

A database for regional gene expression in the human brain.

Alterations in gene expression levels have been widely studied for various neurological diseases, but few studies have sought to characterize genome-wide gene expression patterns from various regions of the normal brain. A sensitive method for quantifying transcript levels, Serial Analysis of Gene Expression (SAGE), was used to assay expression levels in white matter, thalamus, and cerebellum from the same normal brain, obtained by rapid autopsy. The complete dataset for these SAGE libraries are posted on the Cancer Genome Anatomy Project sponsored SAGEmap website where library comparisons can be made, or the data downloaded for local analysis. The expression of several region-specific genes--neurogenic differentiation 1 (cerebellum), cocaine- and amphetamine-regulated transcript (thalamus), and neurogranin (white matter)--was confirmed using quantitative fluorescent real-time RT-PCR. Further informatics analysis of the data yielded a list of brain-specific genes. The database formed by this analysis provides a means to investigate the expression status of genes involved in the region specific functions of the normal brain. These normal brain gene expression levels also are useful for comparison to pathological expression levels and a sensitive means to determine gene expression in a normal adult brain prior to formulating therapeutic strategies.

CGG-repeat polymorphism of the BCR gene rules out predisposing alleles leading to the Philadelphia chromosome.

The Philadelphia chromosome (Ph) is associated with leukemia, most frequently of the chronic myelogenous variety. The Ph chromosome is a translocation chromosome which gains oncogenic potential through the fusion of the ABL oncogene of chromosome 9 with the BCR gene of chromosome 22. The Ph is believed to arise from random chromosome rearrangement with a subsequent selective advantage of the malignant cell line. However, alleles may be present in the population which predispose toward this specific rearrangement. We used a highly polymorphic CGG-repeat polymorphism within the first exon of the BCR gene to determine BCR allele frequencies among 26 leukemia patients with the Ph chromosome and 63 control individuals. Eight BCR alleles of variable CGG-repeat length were present in both groups at statistically similar frequencies and in Hardy-Weinberg equilibrium. We therefore concluded that there are no alleles of the BCR gene that have a major predisposing influence on the development of the Ph chromosome and subsequent leukemia.