Human DNA replication depends on the activation of thousands of origins distributed within the genome. The actual distribution of origins is not known, nor whether this distribution is unique to a cell type, or if it changes with the proliferative state of the cell. In this study we have employed a real time PCR-based nascent strand DNA abundance assay, to determine the location of origins along a 78 kb region on Ch2q34. Preliminary studies using nascent DNA strands isolated from either HeLa and normal skin fibroblast cells showed that in both cell lines peaks of high origin activity mapped in similar locations. However, the overall origin profile in HeLa cells corresponded to broad origin activation zones, whereas in fibroblasts a more punctuated profile of origin activation was observed. To investigate the relevance of this differential origin profile, we compared the origin distribution profiles in breast cancer cell lines MDA-MB-231, BT-474, and MCF-7, to their normal counterpart MCF-10A. In addition, the CRL7250 cell line was also used as a normal control. Our results validated our earlier observation and showed that the origin profile in normal cell lines exhibited a punctuated pattern, in contrast to broader zone profiles observed in the cancer cell lines. A quantitative analysis of origin peaks revealed that the number of activated origins in cancer cells is statistically larger than that obtained in normal cells, suggesting that the flexibility of origin usage is significantly increased in cancer cells compared to their normal counterparts.

Diagnostic biopsy is not routinely performed for children with diffuse intrinsic pontine glioma (DIPG). Consequently, our understanding of DIPG biology is hindered by limited tissue availability. We performed comparative genomic hybridization (CGH) on autopsy specimens to examine the feasibility of determining DNA genomic copy number aberrations on formalin-fixed, paraffin-embedded (FFPE) blocks. Histology on FFPE blocks obtained from autopsy of pediatric patients with DIPG was reviewed. Regions were marked for processing, and DNA was extracted from the tissue core, labeled by chemical coupling with Cy5, and hybridized to 105K oligonucleotide CGH arrays. After hybridization and washing, arrays were scanned, and data segmented and processed with Nexus software. Twenty-two samples from 13 subjects were obtained. Histologic variability was noted. CGH was successfully performed on 18 of 22 samples, representing 11 of 13 subjects. All demonstrated DNA copy number abnormalities. High copy number amplification of known oncogenes and homozygous deletions of known tumor suppressor genes were observed. Additional regions of high copy number amplification and homozygous deletion and geographical variations in the CGH patterns were found. CGH performed on FFPE tissue obtained from autopsy yields satisfactory results. This sample set from patients with DIPG was highly informative, with the majority of specimens showing ≥1 abnormality related to a known cancer gene. Aberrations in candidate drug targets were observed. This study establishes the feasibility of genomic DNA analysis from DIPG autopsy material, identifies several targets for which molecular targeted therapy exists, and suggests significant heterogeneity among patients with DIPG.

Background

The Sequence Read Archive (SRA) is the largest public repository of sequencing data from the next generation of sequencing platforms including Illumina (Genome Analyzer, HiSeq, MiSeq, .etc), Roche 454 GS System, Applied Biosystems SOLiD System, Helicos Heliscope, PacBio RS, and others.

Results

SRAdb is an attempt to make queries of the metadata associated with SRA submission, study, sample, experiment and run more robust and precise, and make access to sequencing data in the SRA easier. We have parsed all the SRA metadata into a SQLite database that is routinely updated and can be easily distributed. The SRAdb R/Bioconductor package then utilizes this SQLite database for querying and accessing metadata. Full text search functionality makes querying metadata very flexible and powerful. Fastq files associated with query results can be downloaded easily for local analysis. The package also includes an interface from R to a popular genome browser, the Integrated Genomics Viewer.

Conclusions

SRAdb Bioconductor package provides a convenient and integrated framework to query and access SRA metadata quickly and powerfully from within R.

Purposes

Pancreatic cancer is the 4th-leading cause of cancer-related death, and studies on the clinical relevance of its genomic imbalances are warranted.

Experimental Design

Recurrent copy number alterations of cytobands and genes were analyzed by array comparative genomic hybridization (aCGH) in 44 resected pancreatic cancer specimens. Prognostic markers identified by aCGH were validated by PCR gene copy number assay in an independent validation cohort of 61 resected pancreatic cancers. The functions of gene identified were evaluated by proliferation, cell cycle and migration assays in pancreatic cancer cells.

Results

We demonstrated recurrent copy number gains and losses in the first cohort. Loss of 18q22.3 was significantly associated with short-term overall survival in the first cohort (p=0.019). This cytoband includes the Carboxypeptidase of glutamate-like (CPGL) gene. CPGL gene deletion was associated with shorter overall survival in the validation cohort (p=0.003). CPGL deletion and mutations of TP53 or Kras appear to be independent events. A Cox model analysis of the two cohorts combined showed that loss of 18q22.3/deletion of the CPGL gene was an independent poor prognostic factor for overall survival (hazard ratio=2.72, p=0.0007). Reconstitution of CPGL or its splicing-variant CPGL-B into CPGL-negative pancreatic cancer cells attenuated cell growth, migration, and induced G1-accumulation.

Conclusion

Loss of 18q22.3/deletion of the CPGL gene is a poor prognostic marker in resected pancreatic cancer, and functional studies suggest the CPGL gene as growth suppressor gene in pancreatic cancer.

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X4-N1-30-X4) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif (C/GCCGGAAGCGGAA) and the ETS⇔CRE motif (C/GCGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif.

Background

Metastasis is characterized by spreading of neoplastic cells to an organ other than where they originated and is the predominant cause of death among cancer patients. This holds true for melanoma, whose incidence is increasing more rapidly than any other cancer and once disseminated has few therapeutic options. Here we performed whole exome sequencing of two sets of matched normal and metastatic tumor DNAs.

Results

Using stringent criteria, we evaluated the similarities and differences between the lesions. We find that in both cases, 96% of the single nucleotide variants are shared between the two metastases indicating that clonal populations gave rise to the distant metastases. Analysis of copy number variation patterns of both metastatic sets revealed a trend similar to that seen with our single nucleotide variants. Analysis of pathway enrichment on tumor sets shows commonly mutated pathways enriched between individual sets of metastases and all metastases combined.

Conclusions

These data provide a proof-of-concept suggesting that individual metastases may have sufficient similarity for successful targeting of driver mutations.

Dysregulation of mechanisms that govern the control of epithelial cell polarity, morphology and plasticity are emerging as key processes in tumor progression. In this study we report amplification and overexpression of PAR6B, an essential component in epithelial cell tight junction (TJ) formation and maintenance of apico-basal polarity, in breast cancer cell lines. Analysis of chromosome 20q13.13 in 11 breast cancer cell lines by fluorescence in situ hybridization (FISH) identified a novel small amplicon centered at PARD6B in 5 cell lines, with copy number ranging from 7 to 27. The presence of the PARD6B amplicon correlated with PARD6B transcript and PAR6B protein abundance. Expression of related isoforms PARD6A and PARD6G were detectable at significantly lower levels. PARD6B overexpression correlated with TJ network formation in cultured cell monolayers. SiRNA-mediated inhibition of PAR6B in MCF7 resulted in loss of TJ assembly and membrane localization of atypical PKCζ (aPKC), but did not affect adherens junction formation. SiRNA-mediated inhibition of CDC42 in MCF7 also resulted in loss of TJ networks, confirming the requirement of a complete PAR6-aPKC-CDC42-PAR3 complex to activate and stabilize TJs. Immunohistochemical analysis of PAR6B expression on breast tumor microarrays indicated exquisite epithelial cell-specificity. Few quantitative differences in staining were observed between normal epithelium and adjacent tumor margins. However staining appeared reduced and cytoplasmic in more poorly differentiated tumors. We propose that quantitative imbalances in the components of pathways governing normal epithelial cell polarity arising from gain or loss of function may radically alter epithelial cell architecture and contribute to tumor progression.

AIM: To explore the association between methylation in leukocyte DNA and colorectal cancer (CRC) risk in male smokers using the α-tocopherol, β-carotene cancer prevention study.

METHODS: About 221 incident CRC cases, and 219 controls, frequency-matched on age and smoking intensity were included. DNA methylation of 1505 CpG sites selected from 807 genes were evaluated using Illumina GoldenGate Methylation Cancer Panel I in pre-diagnostic blood leukocytes of study subjects. Tertiles of methylation level classified according to the distribution in controls for each CpG site were used to analyze the association between methylation level and CRC risk with logistic regression. The time between blood draw to cancer diagnosis (classifying cases according to latency) was incorporated in further analyses using proportional odds regression.

RESULTS: We found that methylation changes of 31 CpG sites were associated with CRC risk at P < 0.01 level. Though none of these 31 sites remained statistically significant after Bonferroni correction, the most statistically significant CpG site associated with CRC risk achieved a P value of 1.0 × 10-4. The CpG site is located in DSP gene, and the risk estimate was 1.52 (95% CI: 0.91-2.53) and 2.62 (95% CI: 1.65-4.17) for the second and third tertile comparing with the lowest tertile respectively. Taking the latency information into account strengthened some associations, suggesting that the methylation levels of corresponding sites might change over time with tumor progression.

CONCLUSION: The results suggest that the methylation level of some genes were associated with cancer susceptibility and some were related to tumor development over time. Further studies are warranted to confirm and refine our results.

Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.

Author Summary

Here we investigate the relationship between somatic variants and non-coding regulatory regions. To do this, we develop a new algorithm for identifying single nucleotide somatic variants in whole-genome sequencing data and apply it to a metastatic melanoma sample and a cell culture derived from this sample. Our results show that the two genomes are similar at the level of single nucleotide changes and more variable at larger copy number changes. We further observe that patterns of somatic mutation accumulation in non-coding regulatory regions suggests that the metastatic melanoma cells de-differentiated into a more basal regulatory state. That is, by simply looking at mutation accumulation across cell-type-specific non-coding functional regions, one can clearly see patterns that are indicative of cell state de-differentiation. Results from genome-wide functional regulatory region experimental mapping support this observation.

SUMMARY

Embryonal rhabdomyosarcoma (eRMS) shows the most myodifferentiation amongst sarcomas, yet the precise cell of origin remains undefined. Using Ptch1, p53 and/or Rb1 conditional mouse models and controlling prenatal or postnatal myogenic cell of origin, we demonstrate that eRMS and undifferentiated pleomorphic sarcoma (UPS) lie in a continuum, with satellite cells predisposed to giving rise to UPS. Conversely, p53 loss in maturing myoblasts gives rise to eRMS, which have the highest myodifferentiation potential. Irrespective of origin, Rb1 loss modifies tumor phenotype to mimic UPS. In human sarcomas that lack pathognomic chromosomal translocations, p53 loss of function is prevalent whereas Shh or Rb1 alterations likely act primarily as modifiers. Thus, sarcoma phenotype is strongly influenced by cell of origin and mutational profile.

The PAX3-FKHR fusion protein is present in the majority of alveolar rhabdomyosarcoma (ARMS) associated with increased aggressiveness and poor prognosis. To better understand the molecular pathogenesis of PAX3-FKHR, we carried out the first and unbiased genome-wide identification of PAX3-FKHR binding sites and associated target genes in ARMS. The data shows that PAX3-FKHR binds to the same sites as PAX3 at both MYF5 and MYOD enhancers. The genome-wide analysis reveals that the PAX3-FKHR sites are: 1) mostly distal to transcription start sites; 2) conserved; 3) enriched for PAX3 motifs; and 4) strongly associated with genes over-expressed in PAX3-FKHR positive RMS cells and tumors. There is little evidence in our dataset for PAX3-FKHR binding at the promoters. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common co-regulation for many target genes. We further provide the first direct evidence that FGFR4 and IGF1R are the targets for PAX3-FKHR. The map of PAX3-FKHR binding sites provides the frame work for the understanding of PAX3-FKHR pathogenic roles and molecular targets to allow a systematic evaluation of agents against this aggressive RMS.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, whose incidence continues to rise. Epidemiological studies reveal that the major etiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor (HGF/SF) transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and etiologic criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4. However, mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, and suggest that IFN-γ-R signaling represents a novel therapeutic melanoma target.

Hypoxia is an important condition in the tumor cell microenvironment and approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Since, by immunohistochemistry, HIF-1α expression was readily detectable in 18/28 primary Ewing's sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in-vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is up-regulated by hypoxia in a HIF-1α-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis related genes, hypoxia stimulated the invasiveness and soft-agar colony formation of ESFT cells in-vitro. Our data represents the first transcriptome analysis of hypoxic ESFT cells and identifies hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT.

Background

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.

Methodology/Principal Findings

We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.

Conclusions/Significance

Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.

Metastasis is the major cause of thyroid cancer-related death. However, little is known about the genes involved in the metastatic spread of thyroid carcinomas. We have created a mouse that spontaneously develops metastatic follicular thyroid carcinoma (FTC). This mouse harbors a targeted mutation (denoted TRβPV) in the thyroid hormone receptor β gene (ThrbPV/PV mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in ThrbPV/PV mice promotes proliferation of thyroid tumor cells, but requires the collaboration of the oncogenic action of TRβPV to empower the tumor cells to undergo distant metastasis. To uncover genes destined to drive the metastatic process, we used cDNA microarrays to compare the genomic expression profile of laser capture microdissected thyroid tumor lesions of ThrbPV/PV mice with that of hyperplastic thyroid cells of wild-type mice having elevated TSH induced by treatment with the anti-thyroid drug propylthiouracil (WT-PTU mice). Analyses of microarray data indicated that the expressions of 150 genes were significantly altered between ThrbPV/PV and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in ThrbPV/PV mice than in WT-PTU mice). Thirty-six percent of genes with altered expression function as key regulators in metastasis. The remaining genes were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix signaling. The present studies have uncovered novel genes responsible for the metastatic spread of FTC and, furthermore, have shown that the metastatic process of thyroid cancer requires effective collaboration among genes with diverse cellular functions. Importantly, the present studies indicate that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Thus, our study has provided new insights into the understanding of the metastatic spread of human thyroid cancer.

Thyroid hormone receptors (TRs) are critical in regulating gene expression in normal physiological processes. Decreased expression and/or somatic mutations of TRs have been shown to be associated several types of human cancers including liver, breast, lung, and thyroid. To understand the molecular mechanisms by which mutated TRs promote carcinogenesis, an animal model of follicular thyroid carcinoma (FTC) (ThrbPV/PV mice) was used in the present study. The ThrbPV/PV mouse harbors a knockin dominant negative PV mutation, identified in a patient with resistance to thyroid hormone. To understand whether oncogenic actions of PV involve not only the loss of normal TR functions but also gain-of-function activities, we compared the gene expression profiles of thyroid lesions in ThrbPV/PV mice and Thra1−/−Thrb−/− mice that also spontaneously develop FTC, but with less severe malignancy. Analysis of the cDNA microarray data derived from microdissected thyroid tumor cells of these two mice showed contrasting global gene expression profiles. With stringent selection using 2.5-fold change (p<0.01) in cDNA microarray analysis, 241 genes with altered gene expression were identified. Nearly half of the genes (n=103: 42.7% of total) with altered gene expression in thyroid tumor cells of ThrbPV/PV mice were associated with tumorigenesis and metastasis; some of these genes function as oncogenes in human thyroid cancers. The remaining genes were found to function in transcriptional regulation, RNA processing, cell proliferation, apoptosis, angiogenesis, and cytoskeleton modification. These results indicate that the more aggressive thyroid tumor progression in ThrbPV/PV mice was not due simply to the loss of tumor suppressor functions of TR via mutation but also, importantly, to gain-of-function in the oncogenic activities of PV to drive thyroid carcinogenesis. Thus, the present study identifies a novel mechanism by which a mutated TRβ evolves with an oncogenic advantage to promote thyroid carcinogenesis.

The CRTC1-MAML2 fusion oncogene underlies the etiology of mucoepidermoid salivary gland carcinoma (MEC) where it confers a favorable survival outcome as compared with fusion-negative MEC. While these analyses suggested that detection of CRTC1-MAML2 serves as a useful prognostic biomarker, we recently identified outlier cases of fusion-positive MEC associated with advanced-staged lethal disease. To identify additional genetic alterations that might cooperate with CRTC1-MAML2 to promote disease progression, we performed a pilot high-resolution oligonucleotide array CGH (aCGH) and PCR-based genotyping study on 23 MEC samples including14 fusion-positive samples for which we had clinical outcome information. Unbiased aCGH analysis identified inactivating deletions within CDKN2A as a candidate poor prognostic marker which was confirmed by PCR-based analysis (CDKN2A deletions in 5/5 unfavorable fusion-positive cases and 0/9 favorable fusion-positive cases). We did not detect either activating EGFR mutations, nor copy number gains at the EGFR or ERBB2 loci as poor prognostic features for fusion-positive MEC in any of the tumor specimens. Prospective studies with larger case series will be needed to confirm that combined CRTC1-MAML2 and CDKN2A genotyping will optimally stage this disease.

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Thyroid hormone receptors (TRs) are critical in regulating gene expression in normal physiological processes. Decreased expression and/or somatic mutations of TRs have been shown to be associated several types of human cancers including liver, breast, lung, and thyroid. To understand the molecular mechanisms by which mutated TRs promote carcinogenesis, an animal model of follicular thyroid carcinoma (FTC) (Thrbpv/pv mice) was used in the present study. The Thrbpv/pv mouse harbors a knockin dominant negative PV mutation, identified in a patient with resistance to thyroid hormone. To understand whether oncogenic actions of PV involve not only the loss of normal TR functions but also gain-of-function activities, we compared the gene expression profiles of thyroid lesions in Thrbpv/pv mice and Thra1-/- Thrb-/- mice that also spontaneously develop FTC, but with less severe malignancy. Analysis of the cDNA microarray data derived from microdissected thyroid tumor cells of these two mice showed contrasting global gene expression profiles. With stringent selection using 2.5-fold change (p<0.01) in cDNA microarray analysis, 241 genes with altered gene expression were identified. Nearly half of the genes (n=103: 42.7% of total) with altered gene expression in thyroid tumor cells of Thrbpv/pv mice were associated with tumorigenesis and metastasis; some of these genes function as oncogenes in human thyroid cancers. The remaining genes were found to function in transcriptional regulation, RNA processing, cell proliferation, apoptosis, angiogenesis, and cytoskeleton modification. These results indicate that the more aggressive thyroid tumor progression in Thrbpv/pv mice was not due simply to the loss of tumor suppressor functions of TR via mutation but also, importantly, to gain-of-function in the oncogenic activities of PV to drive thyroid carcinogenesis. Thus, the present study identifies a novel mechanism by which a mutated TRβ evolves with an oncogenic advantage to promote thyroid carcinogenesis.

A major challenge in studies of etiologic heterogeneity in breast cancer has been the limited throughput, accuracy and reproducibility of measuring tissue markers. Computerized image analysis systems may help address these concerns but published reports of their use are limited. We assessed agreement between automated and pathologist scores of a diverse set of immunohistochemical (IHC) assays performed on breast cancer TMAs.

TMAs of 440 breast cancers previously stained for ER-α, PR, HER-2, ER-β and aromatase were independently scored by two pathologists and three automated systems (TMALabII, TMAx, Ariol). Agreement between automated and pathologist scores of negative/positive was measured using the area under the receiver operator characteristics curve (AUC) and weighted kappa statistics (κ) for categorical scores. We also investigated the correlation between IHC scores and mRNA expression levels.

Agreement between pathologist and automated negative/positive and categorical scores was excellent for ER-α and PR (AUC range =0.98-0.99; κ range =0.86-0.91). Lower levels of agreement were seen for ER-β categorical scores (AUC=0.99-1.0; κ=0.80-0.86) and both negative/positive and categorical scores for aromatase (AUC=0.85-0.96; κ=0.41-0.67) and HER2 (AUC=0.94-0.97; κ=0.53-0.72). For ER-α and PR, there was strong correlation between mRNA levels and automated (ρ=0.67-0.74) and pathologist IHC scores (ρ=0.67-0.77). HER2 mRNA levels were more strongly correlated with pathologist (ρ=0.63) than automated IHC scores (ρ=0.41-0.49).

Automated analysis of IHC markers is a promising approach for scoring large numbers of breast cancer tissues in epidemiologic investigations. This would facilitate studies of etiologic heterogeneity which ultimately may allow improved risk prediction and better prevention approaches.

A major challenge in studies of etiologic heterogeneity in breast cancer has been the limited throughput, accuracy and reproducibility of measuring tissue markers. Computerized image analysis systems may help address these concerns but published reports of their use are limited. We assessed agreement between automated and pathologist scores of a diverse set of immunohistochemical (IHC) assays performed on breast cancer TMAs.

TMAs of 440 breast cancers previously stained for ER-α, PR, HER-2, ER-β and aromatase were independently scored by two pathologists and three automated systems (TMALabII, TMAx, Ariol). Agreement between automated and pathologist scores of negative/positive was measured using the area under the receiver operator characteristics curve (AUC) and weighted kappa statistics (κ) for categorical scores. We also investigated the correlation between IHC scores and mRNA expression levels.

Agreement between pathologist and automated negative/positive and categorical scores was excellent for ER-α and PR (AUC range =0.98-0.99; κ range =0.86-0.91). Lower levels of agreement were seen for ER-β categorical scores (AUC=0.99-1.0; κ=0.80-0.86) and both negative/positive and categorical scores for aromatase (AUC=0.85-0.96; κ=0.41-0.67) and HER2 (AUC=0.94-0.97; κ=0.53-0.72). For ER-α and PR, there was strong correlation between mRNA levels and automated (ρ=0.67-0.74) and pathologist IHC scores (ρ=0.67-0.77). HER2 mRNA levels were more strongly correlated with pathologist (ρ=0.63) than automated IHC scores (ρ=0.41-0.49).

Automated analysis of IHC markers is a promising approach for scoring large numbers of breast cancer tissues in epidemiologic investigations. This would facilitate studies of etiologic heterogeneity which ultimately may allow improved risk prediction and better prevention approaches.

Metastasis is the major cause of thyroid cancer-related death. However, little is known about the genes involved in the metastatic spread of thyroid carcinomas. We have created a mouse that spontaneously develops metastatic follicular thyroid carcinoma (FTC). This mouse harbors a targeted mutation (denoted TRβPV) in the thyroid hormone receptor β gene (ThrbPV/PV mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in ThrbPV/PV mice promotes proliferation of thyroid tumor cells, but requires the collaboration of the oncogenic action of TRβPV to empower the tumor cells to undergo distant metastasis. To uncover genes destined to drive the metastatic process, we used cDNA microarrays to compare the genomic expression profile of laser capture microdissected thyroid tumor lesions of ThrbPV/PV mice with that of hyperplastic thyroid cells of wild-type mice having elevated TSH induced by treatment with the anti-thyroid drug propylthiouracil (WT-PTU mice). Analyses of microarray data indicated that the expressions of 150 genes were significantly altered between ThrbPV/PV and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in ThrbPV/PV mice than in WT-PTU mice). Thirty-six percent of genes with altered expression function as key regulators in metastasis. The remaining genes were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix signaling. The present studies have uncovered novel genes responsible for the metastatic spread of FTC and, furthermore, have shown that the metastatic process of thyroid cancer requires effective collaboration among genes with diverse cellular functions. Importantly, the present studies indicate that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Thus, our study has provided new insights into the understanding of the metastatic spread of human thyroid cancer.

Brain metastases of breast cancer appear to be increasing in incidence as systemic therapy improves. Metastatic disease in the brain is associated with high morbidity and mortality. We present the first gene expression analysis of laser captured epithelial cells from resected human brain metastases of breast cancer compared to unlinked primary breast tumors. The tumors were matched for histology, TNM stage and hormone receptor status. Most differentially expressed genes were down-regulated in the brain metastases which included, surprisingly, many genes associated with metastasis. Q-PCR analysis confirmed statistically significant differences or strong trends in the expression of six genes: BMP1, PEDF, LAMγ3, SIAH, STHMN3 and TSPD2. Hexokinase 2 (HK2) was also of interest because of its increased expression in brain metastases. HK2 is important in glucose metabolism and apoptosis. In agreement with our microarray results, HK2 levels (both mRNA and protein) were elevated in a brain metastatic derivative (231-BR) of the human breast carcinoma cell line MDA-MB-231 relative to the parental cell line (231-P), in vitro. Knockdown of HK2 expression in 231-BR cells using shRNA reduced cell proliferation when cultures were maintained in glucose limiting conditions. Finally, HK2 expression was analyzed in a cohort of 123 resected brain metastases of breast cancer. High HK2 expression was significantly associated with poor patient survival post-craniotomy (P=0.028). The data suggest that HK2 overexpression is associated with metastasis to the brain in breast cancer and it may be a therapeutic target.

Background

Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease.

Methods

We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase–polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy.

Results

In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor κB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA.

Conclusions

The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa.