Establishing and characterizing patient-derived xenografts using pre-chemotherapy percutaneous biopsy and post-chemotherapy surgical samples from a prospective neoadjuvant breast cancer study.

BACKGROUND: Patient-derived xenografts (PDXs) are increasingly used in cancer research as a tool to inform cancer biology and drug response. Most available breast cancer PDXs have been generated in the metastatic setting. However, in the setting of operable breast cancer, PDX models both sensitive and resistant to chemotherapy are needed for drug development and prospective data are lacking regarding the clinical and molecular characteristics associated with PDX take rate in this setting. METHODS: The Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective neoadjuvant chemotherapy (NAC) trial of stage I-III breast cancer patients treated with neoadjuvant weekly taxane+/-trastuzumab followed by anthracycline-based chemotherapy. Using percutaneous tumor biopsies (PTB), we established and characterized PDXs from both primary (untreated) and residual (treated) tumors. Tumor take rate was defined as percent of patients with the development of at least one stably transplantable (passed at least for four generations) xenograft that was pathologically confirmed as breast cancer. RESULTS: Baseline PTB samples from 113 women were implanted with an overall take rate of 27.4% (31/113). By clinical subtype, the take rate was 51.3% (20/39) in triple negative (TN) breast cancer, 26.5% (9/34) in HER2+, 5.0% (2/40) in luminal B and 0% (0/3) in luminal A. The take rate for those with pCR did not differ from those with residual disease in TN (p = 0.999) and HER2+ (p = 0.2401) tumors. The xenografts from 28 of these 31 patients were such that at least one of the xenografts generated had the same molecular subtype as the patient. Among the 35 patients with residual tumor after NAC adequate for implantation, the take rate was 17.1%. PDX response to paclitaxel mirrored the patients' clinical response in all eight PDX tested. CONCLUSIONS: The generation of PDX models both sensitive and resistant to standard NAC is feasible and these models exhibit similar biological and drug response characteristics as the patients' primary tumors. Taken together, these models may be useful for biomarker discovery and future drug development.

RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing.

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.

Analysis of alternative splicing associated with aging and neurodegeneration in the human brain.

Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.

Human transcriptome array for high-throughput clinical studies.

A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB.

To gain global insights into the role of the well-known repressive splicing regulator PTB, we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB-repressed and PTB-activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTB-activated exon to a PTB-repressed exon. Our results show that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.

Exon-level microarray analyses identify alternative splicing programs in breast cancer.

Protein isoforms produced by alternative splicing (AS) of many genes have been implicated in several aspects of cancer genesis and progression. These observations motivated a genome-wide assessment of AS in breast cancer. We accomplished this by measuring exon level expression in 31 breast cancer and nonmalignant immortalized cell lines representing luminal, basal, and claudin-low breast cancer subtypes using Affymetrix Human Junction Arrays. We analyzed these data using a computational pipeline specifically designed to detect AS with a low false-positive rate. This identified 181 splice events representing 156 genes as candidates for AS. Reverse transcription-PCR validation of a subset of predicted AS events confirmed 90%. Approximately half of the AS events were associated with basal, luminal, or claudin-low breast cancer subtypes. Exons involved in claudin-low subtype-specific AS were significantly associated with the presence of evolutionarily conserved binding motifs for the tissue-specific Fox2 splicing factor. Small interfering RNA knockdown of Fox2 confirmed the involvement of this splicing factor in subtype-specific AS. The subtype-specific AS detected in this study likely reflects the splicing pattern in the breast cancer progenitor cells in which the tumor arose and suggests the utility of assays for Fox-mediated AS in cancer subtype definition and early detection. These data also suggest the possibility of reducing the toxicity of protein-targeted breast cancer treatments by targeting protein isoforms that are not present in limiting normal tissues.

Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis.

Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

Identification of common genetic variants that account for transcript isoform variation between human populations.

In addition to the differences between populations in transcriptional and translational regulation of genes, alternative pre-mRNA splicing (AS) is also likely to play an important role in regulating gene expression and generating variation in mRNA and protein isoforms. Recently, the genetic contribution to transcript isoform variation has been reported in individuals of recent European descent. We report here results of an investigation of the differences in AS patterns between human populations. AS patterns in 176 HapMap lymphoblastoid cell lines derived from individuals of European and African ancestry were evaluated using the Affymetrix GeneChip Human Exon 1.0 ST Array. A variety of biological processes such as response to stimulus and transcription were found to be enriched among the differentially spliced genes. The differentially spliced genes also include some involved in human diseases that have different prevalence or susceptibility between populations. The genetic contribution to the population differences in transcript isoform variation was then evaluated by a genome-wide association using the HapMap genotypic data on single nucleotide polymorphisms (SNPs). The results suggest that local and distant genetic variants account for a substantial fraction of the observed transcript isoform variation between human populations. Our findings provide new insights into the complexity of the human genome as well as the health disparities between the two populations.

HITS-CLIP yields genome-wide insights into brain alternative RNA processing.

Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.

Genetic architecture of transcript-level variation in humans.

We report here the results of testing the pairwise association of 12,747 transcriptional gene-expression values with more than two million single-nucleotide polymorphisms (SNPs) in samples of European (CEPH from Utah; CEU) and African (Yoruba from Ibadan; YRI) ancestry. We found 4,677 and 5,125 significant associations between expression quantitative nucleotides (eQTNs) and transcript clusters in the CEU and the YRI samples, respectively. The physical distance between an eQTN and its associated transcript cluster was referred to as the intrapair distance. An association with 4 Mb or less intrapair distance was defined as local; otherwise, it was defined as distant. The enrichment analysis of functional categories shows that genes harboring the local eQTNs are enriched in the categories related to nucleosome and chromatin assembly; the genes harboring the distant eQTNs are enriched in the categories related to transmembrane signal transduction, suggesting that these biological pathways are likely to play a significant role in regulation of gene expression. We highlight in the EPHX1 gene a deleterious nonsynonymous SNP that is distantly associated with gene expression of ORMDL3, a susceptibility gene for asthma.

Evaluation of genetic variation contributing to differences in gene expression between populations.

Gene expression is a complex quantitative trait partially regulated by genetic variation in DNA sequence. Population differences in gene expression could contribute to some of the observed differences in susceptibility to common diseases and response to drug treatments. We characterized gene expression in the full set of HapMap lymphoblastoid cell lines derived from individuals of European and African ancestry for 9156 transcript clusters (gene-level) evaluated with the Affymetrix GeneChip Human Exon 1.0 ST Array. Gene expression was found to differ significantly between these samples for 383 transcript clusters. Biological processes including ribosome biogenesis and antimicrobial humoral response were found to be enriched in these differential genes, suggesting their possible roles in contributing to the population differences at a higher level than that of mRNA expression and in response to environmental information. Genome-wide association studies for local or distant genetic variants that correlate with the differentially expressed genes enabled identification of significant associations with one or more single-nucleotide polymorphisms (SNPs), consistent with the hypothesis that genetic factors and not simply population identity or other characteristics (age of cell lines, length of culture, etc.) contribute to differences in gene expression in these samples. Our results provide a comprehensive view of the genes differentially expressed between populations and the enriched biological processes involved in these genes. We also provide an evaluation of the contributions of genetic variation and nongenetic factors to the population differences in gene expression.

Identification of genetic variants contributing to cisplatin-induced cytotoxicity by use of a genomewide approach.

Cisplatin, a platinating agent commonly used to treat several cancers, is associated with nephrotoxicity, neurotoxicity, and ototoxicity, which has hindered its utility. To gain a better understanding of the genetic variants associated with cisplatin-induced toxicity, we present a stepwise approach integrating genotypes, gene expression, and sensitivity of HapMap cell lines to cisplatin. Cell lines derived from 30 trios of European descent (CEU) and 30 trios of African descent (YRI) were used to develop a preclinical model to identify genetic variants and gene expression that contribute to cisplatin-induced cytotoxicity in two different populations. Cytotoxicity was determined as cell-growth inhibition at increasing concentrations of cisplatin for 48 h. Gene expression in 176 HapMap cell lines (87 CEU and 89 YRI) was determined using the Affymetrix GeneChip Human Exon 1.0 ST Array. We identified six, two, and nine representative SNPs that contribute to cisplatin-induced cytotoxicity through their effects on 8, 2, and 16 gene expressions in the combined, Centre d'Etude du Polymorphisme Humain (CEPH), and Yoruban populations, respectively. These genetic variants contribute to 27%, 29%, and 45% of the overall variation in cell sensitivity to cisplatin in the combined, CEPH, and Yoruban populations, respectively. Our whole-genome approach can be used to elucidate the expression of quantitative trait loci contributing to a wide range of cellular phenotypes.

Heritability of alternative splicing in the human genome.

Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5' splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled.

A correlation with exon expression approach to identify cis-regulatory elements for tissue-specific alternative splicing.

Correlation of motif occurrences with gene expression intensity is an effective strategy for elucidating transcriptional cis-regulatory logic. Here we demonstrate that this approach can also identify cis-regulatory elements for alternative pre-mRNA splicing. Using data from a human exon microarray, we identified 56 cassette exons that exhibited higher transcript-normalized expression in muscle than in other normal adult tissues. Intron sequences flanking these exons were then analyzed to identify candidate regulatory motifs for muscle-specific alternative splicing. Correlation of motif parameters with gene-normalized exon expression levels was examined using linear regression and linear splines on RNA words and degenerate weight matrices, respectively. Our unbiased analysis uncovered multiple candidate regulatory motifs for muscle-specific splicing, many of which are phylogenetically conserved among vertebrate genomes. The most prominent downstream motifs were binding sites for Fox1- and CELF-related splicing factors, and a branchpoint-like element acuaac; pyrimidine-rich elements resembling PTB-binding sites were most significant in upstream introns. Intriguingly, our systematic study indicates a paucity of novel muscle-specific elements that are dominant in short proximal intronic regions. We propose that Fox and CELF proteins play major roles in enforcing the muscle-specific alternative splicing program, facilitating expression of unique isoforms of cytoskeletal proteins critical to muscle cell function.

A genome-wide approach to identify genetic variants that contribute to etoposide-induced cytotoxicity.

Large interindividual variance has been observed in sensitivity to drugs. To comprehensively decipher the genetic contribution to these variations in drug susceptibility, we present a genome-wide model using human lymphoblastoid cell lines from the International HapMap consortium, of which extensive genotypic information is available, to identify genetic variants that contribute to chemotherapeutic agent-induced cytotoxicity. Our model integrated genotype, gene expression, and sensitivity of HapMap cell lines to drugs. Cell lines derived from 30 trios of European descent (Center d'Etude du Polymorphisme Humain population) and 30 trios of African descent (Yoruban population) were used. Cell growth inhibition at increasing concentrations of etoposide for 72 h was determined by using alamarBlue assay. Gene expression on 176 HapMap cell lines (87 Center d'Etude du Polymorphisme Humain population and 89 Yoruban population) was determined by using the Affymetrix GeneChip Human Exon 1.0ST Array. We evaluated associations between genotype and cytotoxicity, genotype and gene expression and correlated gene expression of the identified candidates with cytotoxicity. The analysis identified 63 genetic variants that contribute to etoposide-induced toxicity through their effect on gene expression. These include genes that may play a role in cancer (AGPAT2, IL1B, and WNT5B) and genes not yet known to be associated with sensitivity to etoposide. This unbiased method can be used to elucidate genetic variants contributing to a wide range of cellular phenotypes induced by chemotherapeutic agents.

Discovery of tissue-specific exons using comprehensive human exon microarrays.

BACKGROUND: Higher eukaryotes express a diverse population of messenger RNAs generated by alternative splicing. Large-scale methods for monitoring gene expression must adapt in order to accurately detect the transcript variation generated by this splicing. RESULTS: We have designed a high-density oligonucleotide microarray with probesets for more than one million annotated and predicted exons in the human genome. Using these arrays and a simple algorithm that normalizes exon signal to signal from the gene as a whole, we have identified tissue-specific exons from a panel of 16 different normal adult tissues. RT-PCR validation confirms approximately 86% of the predicted tissue-enriched probesets. Pair-wise comparisons between the tissues suggest that as many as 73% of detected genes are differentially alternatively spliced. We also demonstrate how an inclusive exon microarray can be used to discover novel alternative splicing events. As examples, 17 new tissue-specific exons from 11 genes were validated by RT-PCR and sequencing. CONCLUSION: In conjunction with a conceptually simple algorithm, comprehensive exon microarrays can detect tissue-specific alternative splicing events. Our data suggest significant expression outside of known exons and well annotated genes and a high frequency of alternative splicing events. In addition, we identified and validated a number of novel exons with tissue-specific splicing patterns. The tissue map data will likely serve as a valuable source of information on the regulation of alternative splicing.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array.

BACKGROUND: Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. RESULTS: We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. CONCLUSION: Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.

Matrix metalloproteinases contribute to insulin insufficiency in Zucker diabetic fatty rats.

To assess the molecular changes associated with pancreatic beta-cell dysfunction occurring during the onset of type 2 diabetes, we profiled pancreatic islet mRNAs from diabetic male and high-fat-fed female Zucker diabetic fatty (ZDF) rats and their nondiabetic lean counterparts on custom islet-specific oligonucleotide arrays. The most prominent changes in both the male and female models of type 2 diabetes were increases in the mRNAs encoding proteases and extracellular matrix components that are associated with tissue remodeling and fibrosis. The mRNAs for metalloproteinase (MMP)-2, -12, and -14 were sharply increased with the onset of islet dysfunction and diabetes. Zymography of islet extracts revealed a concurrent, >10-fold increase in MMP-2 protease activity in islets from 9-week-old male ZDF rats. Treatment of female ZDF rats receiving a diabetogenic diet with PD166793, a broad-spectrum MMP inhibitor, substantially prevented diabetes. The effect of this compound was due in part to marked beta-cell expansion. These studies indicate that MMPs contribute to islet fibrosis and insulin insufficiency in ZDF rats. Class-targeted protease inhibitors should be explored for their potential therapeutic utility in preservation of beta-cell mass in type 2 diabetes.

Overexpression of repressive cAMP response element modulators in high glucose and fatty acid-treated rat islets. A common mechanism for glucose toxicity and lipotoxicity?

The hyperlipidemia and hyperglycemia of the diabetic state accelerate beta-cell dysfunction, yet the mechanisms are not fully defined. We used rat islet-specific oligonucleotide arrays (Metabolex Rat Islet Genechips) to identify genes that are coordinately regulated by high glucose and free fatty acids (FFA). Exposure of rat islets to FFA (125 microM for 2 days) or glucose (27 mM for 4 days) reduced glucose-stimulated insulin secretion by 70 +/- 5 and 40 +/- 4%, respectively, relative to control-cultured islets. These treatments also substantially reduced the insulin content of the islets. Islet Genechips analysis revealed that the mRNA levels of cAMP response element modulator (CREM)-17X and inducible cAMP early repressor were significantly increased in both 27 mM glucose- and FFA-treated islets. Removing FFA or high glucose from the culture medium restored glucose-stimulated insulin secretion and the mRNA levels of the two CREM repressors to normal. Northern blot analysis revealed a 5-fold increase in the abundance of CREM-17X mRNA and a concomitant 50% reduction in the insulin mRNA in FFA-treated islets. Transient transfection of the insulin-secreting beta HC9 cells with CREM-17X suppressed rat insulin promoter activity by nearly 50%. Overexpression of CREM-17X in intact islets via adenovirus infection decreased islet insulin mRNA levels and insulin content and resulted in a significant decrease in glucose- or KCl-induced insulin secretion. Taken together, these data suggest that up-regulation of CREM repressors by either FFA or high glucose exacerbates beta-cell failure in type 2 diabetes by suppressing insulin gene transcription.