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.

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.

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.

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.

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.

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.

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.

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.