Michelle J. Tang

Alternative expression analysis by RNA sequencing

In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription–PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.

SPARC in cancer biology: Its role in cancer progression and potential for therapy

The ability to effectively target a tumor to achieve complete regression and cure is the ultimate goal that drives our need to better understand tumor biology. Recently, SPARC has generated considerable interest as a multi-faceted protein that belongs to a family of matricellular proteins. It functions not only to modulate cell-cell and cell-matrix interactions, but its de-adhesive and growth inhibitory properties in non-transformed cells have led to studies to assess its role in cancer. Its divergent actions reflect the complexity of this protein, because in certain types of cancers, such as melanomas and gliomas, SPARC is associated with a highly aggressive tumor phenotype, while in others, mainly ovarian, neuroblastomas and colorectal cancers, SPARC may function as a tumor suppressor. Recent studies have also demonstrated a role for SPARC in sensitizing therapy-resistant cancers. Here, the role of SPARC in cancer progression and its potential application in cancer therapy is discussed.

A Novel Interaction between Procaspase 8 and SPARC Enhances Apoptosis and Potentiates Chemotherapy Sensitivity in Colorectal Cancers

Chemotherapy resistance accounts for the high mortality rates in patients with advanced cancers. We previously used a genomics approach to determine novel genes associated with this phenomenon and identified secreted protein acidic and rich in cysteine (SPARC) as a chemosensitizer capable of reversing therapy resistance in colorectal cancer cells by enhancing apoptosis in vitro and tumor regression in vivo. Here, we examined the mechanisms by which SPARC enhances apoptosis in the presence of chemotherapy. We show that SPARC potentiates apoptosis by augmenting the signaling cascade in a caspase-8-dependent manner, because apoptosis can be abolished by caspase 8 small interfering RNA in the presence of SPARC. This occurs independently of death receptor activation and leads to downstream involvement of Bid and subsequent apoptosis. Interestingly, this results from an interaction between SPARC and the N terminus of the procaspase-8 DED-containing domain. These exciting findings provide an initial map of the apoptosis signaling events mediated by SPARC and how this can ultimately result in the reversal of chemotherapy resistance and enhanced tumor regression. This signaling cascade can be exploited therapeutically and may have potential clinical implications for patients with advanced and therapy-refractory cancers.

Synergism between vitamin D and secreted protein acidic and rich in cysteine–induced apoptosis and growth inhibition results in increased susceptibility of therapy-resistant colorectal cancer cells to chemotherapy

Patients with advanced colorectal cancer continue to have poor outcomes because of therapy-refractory disease. We previously showed that secreted protein acidic and rich in cysteine (SPARC) gene and protein could function as a chemotherapy sensitizer by enhancing tumor regression in response to radiation and chemotherapy in tumor xenograft models of chemotherapy-resistant tumors. This function of SPARC was gleamed from a microarray analysis that also revealed down-regulation of the vitamin D receptor (VDR) in therapy-refractory colorectal cancer cells. This study examines the potential synergistic effect of SPARC and vitamin D, which up-regulates VDR, in enhancing chemotherapy response in colorectal cancer. Using MIP101 colorectal cancer cell lines and SPARC-overexpressing MIP101 cells, we were able to show that, in the presence of SPARC, exposure to low doses of 1α,25-dihydroxyvitamin D3 significantly reduces cell viability, enhances chemotherapy-induced apoptosis, and inhibits the growth of colorectal cancer cells. Moreover, in tumor xenograft mouse models, up-regulation of VDR was seen in tumors that had the greatest regression following treatment that combined SPARC with chemotherapy. Therefore, our findings reveal a synergistic effect between SPARC and low doses of 1α,25-dihydroxyvitamin D3 that further augments the sensitivity of tumors to chemotherapy. This combination may prove to be a useful adjunct in the treatment of colorectal cancer, especially in those patients with therapy-refractory disease. [Mol Cancer Ther 2007;6(1):309–17]

A Peptide of SPARC Interferes with the Interaction between Caspase8 and Bcl2 to Resensitize Chemoresistant Tumors and Enhance Their Regression In Vivo

SPARC, a matricellular protein with tumor suppressor properties in certain human cancers, was initially identified in a genome-wide analysis of differentially expressed genes in chemotherapy resistance. Its exciting new role as a potential chemosensitizer arises from its ability to augment the apoptotic cascade, although the exact mechanisms are unclear. This study further examines the mechanism by which SPARC may be promoting apoptosis and identifies a smaller peptide analogue with greater chemosensitizing and tumor-regressing properties than the native protein. We examined the possibility that the apoptosis-enhancing activity of SPARC could reside within one of its three biological domains (N-terminus (NT), the follistatin-like (FS), or extracellular (EC) domains), and identified the N-terminus as the region with its chemosensitizing properties. These results were not only confirmed by studies utilizing stable cell lines overexpressing the different domains of SPARC, but as well, with a synthetic 51-aa peptide spanning the NT-domain. It revealed that the NT-domain induced a significantly greater reduction in cell viability than SPARC, and that it enhanced the apoptotic cascade via its activation of caspase 8. Moreover, in chemotherapy resistant human colon, breast and pancreatic cancer cells, its chemosensitizing properties also depended on its ability to dissociate Bcl2 from caspase 8. These observations translated to clinically significant findings in that, in-vivo, mouse tumor xenografts overexpressing the NT-domain of SPARC had significantly greater sensitivity to chemotherapy and tumor regression, even when compared to the highly-sensitive SPARC-overexpressing tumors. Our results identified an interplay between the NT-domain, Bcl2 and caspase 8 that helps augment apoptosis and as a consequence, a tumors response to therapy. This NT-domain of SPARC and its 51-aa peptide are highly efficacious in modulating and enhancing apoptosis, thereby conferring greater chemosensitivity to resistant tumors. Our findings provide additional insight into mechanisms involved in chemotherapy resistance and a potential novel therapeutic that specifically targets this devastating phenomenon.

Identification of COS markers in the Pinaceae.

Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism.

ALEXA: a microarray design platform for alternative expression analysis

To the editor: Eukaryotic genomes are predicted to contain about 7,000–29,000 genes1. Each of these genes may be alternatively processed to produce multiple distinct mRNAs by alternative transcript initiation, splicing and polyadenylation (collectively referred to as alternative expression). Although analysis of available transcript resources indicates that up to ~75% of genes are alternatively processed, most microarray expression platforms cannot detect alternative transcripts2. Proof-of-principle experiments have described the use of oligonucleotide microarrays to profile transcript isoforms generated by alternative expression, but resources to create such arrays are lacking3,4. To address this limitation we created a microarray design platform for alternative expression analysis (ALEXA), which is capable of designing arrays that can detect all of the major categories of alternative expression. The ALEXA platform facilitates selection and annotation of oligonucleotide probes representing alternative expression events for any species in the EnsEMBL database1. For each target gene, probes are selected within every exon, intron, exon junction and exon boundary. This approach allows for the detection of constitutive and alternative exons, canonical exon junctions, junctions of known or new exon-skipping events, alternative exon boundaries and retained introns (Supplementary Fig. 1 online). We designed the platform to be flexible to the user’s experimental interests and preferred array manufacturer. The user may limit probe selection to known alternative expression events or include all possible exon junctions and boundaries to drive the discovery of transcripts. Probes may be designed for an arbitrary subset of genes or for all genes. Most technical parameters of the design can be modified by the user, including: the amount and types of control probes; the use of varying or fixed probe length; and the thresholds for filtering of probe sequences. The probe design process begins with retrieval of genomic sequences from EnsEMBL, removal of pseudogenes, masking of repeat elements and extraction of probe sequences. Random probe sequences are generated to uniformly represent the melting temperature and length of all experimental probes. Extracted and randomly generated probes are scored according to their melting temperature, folding potential, complexity and specificity (Supplementary Methods online). Although several publications have described using microarrays to study alternative expression in model organisms and specific tissues2, to our knowledge ours is the first report of a resource that makes alternative expression microarray designs readily available. Using the ALEXA approach, we precomputed microarray designs representing ~100 million probe sequences for ten EnsEMBL genomes (Supplementary Table 1 online). We assessed the ALEXA approach by using a prototype human array to profile the expression of alternative mRNA isoforms in 5-fluorouracil (5-FU)sensitive and resistant colorectal cancer cell lines5 and compared the results to those from the Affymetrix ‘GeneChip Human Exon 1.0 ST’ array (see Supplementary Results, Supplementary Fig. 2 and Supplementary Table 2 online). Genes and exons differentially expressed between 5-FU–sensitive and resistant cells were identified by both platforms (with significant overlap), but ALEXA arrays provided additional information on the connectivity and boundaries of exons (Table 1). Furthermore, alternative expression events identified by ALEXA were significantly enriched for known alternative expression events represented in publicly available mRNA and expressed sequence tag (EST) databases (Supplementary Results and Supplementary Data 1 online). Finally, we demonstrated the advantage of the ALEXA approach by identifying several differentially expressed known and predicted isoforms with potential relevance to 5-FU resistance (Supplementary Fig. 3 and Supplementary Tables 3 and 4 online). The approach and resources described in this work have considerable potential to advance studies of gene regulation, transcript processing, human disease and evolutionary biology (Supplementary Discussion online). The source code, precomputed array designs and related materials to assist in the creation of custom alternative expression microarrays are available on the ALEXA website (http://www.alexaplatform.org).

Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.