During cancer progression malignant cells undergo epithelial-mesenchymal transitions (EMTs) and mesenchymal-epithelial transitions (METs) as part of a broad invasion and metastasis program. We previously observed MET events among lung metastases in a preclinical model of prostate adenocarcinoma that suggested a relationship between epithelial plasticity and metastatic spread. We thus sought to translate these findings into clinical evidence by examining the existence of EMT in circulating tumor cells (CTCs) from patients with progressive metastatic solid tumors, with a focus on men with castration-resistant prostate cancer (CRPC) and women with metastatic breast cancer (BC). We show that the majority (>80%) of these CTCs in patients with metastatic CRPC co-express epithelial proteins such as EpCAM, CK, and E-cadherin, mesenchymal proteins, including vimentin, N-cadherin, and O-cadherin, and the stem cell marker CD133. Equally, we find that over 75% of CTCs from women with metastatic BC co-express cytokeratin, vimentin, and N-cadherin. The existence and high frequency of these CTCs co-expressing epithelial, mesenchymal, and stem-cell markers in patients with progressive metastases has important implications for the application and interpretation of approved methods to detect CTCs.

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, we analyzed the splicing of the Drosophila melanogaster TAF1 pre-mRNA. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. We demonstrate that TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells was mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing was mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing.Alternative splicing is a major mechanism utilized by higher eukaryotic organisms to regulate gene expression during development and in response to stress (8,44,48,50). In fact, 35 to 74% of human genes may encode pre-mRNAs that are alternatively spliced (10,22,23,29,34). Alternative splicing can regulate whether or not a protein is produced, or it can generate pre-mRNAs that encode proteins with distinct functions (7,17). By analogy to other gene expression-regulatory mechanisms, such as transcription, it is probable that signal transduction pathways play a widespread role in controlling alternative splicing. However, documented examples of this phenomenon are limited, and a complete pathway has not been described.One of the most thoroughly understood examples of signaldependent alternative splicing is Ras signal-induced splicing of the CD44 pre-mRNA in humans (28,32,57). The Ras GTPase and the downstream mitogen-activated protein kinase (MAPK) signaling cascade specify inclusion of exon 5 (v5) in the mature CD44 mRNA. Stimuli that activate Ras lead to activation of MAPK, which in turn phosphorylates SAM68, an RNA-binding protein that interacts with an exonic splicing silencer element within v5. Phosphorylated SAM68 is then thought to interfere with the repressive activity of hnRNP A1 and allow factors bound to a v5 exonic splicing enhancer element to enhance v5 inclusion. Signal-dependent alternative splicing has also been implicated in the regulation of cellular processes, including apoptosis and the cell cycle (44, 47, 49). For instance, many genes encoding apoptotic regulators are alternatively spliced; however, littl...

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). While there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET-independent. Indeed, the MET-independent pathway was supported by reanalysis of pre-clinical and clinical data. These results provide evidence for both MET-dependent and MET-independent metastatic pathways.

Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to downregulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.Alternative splicing is a fundamental mechanism operating in metazoan organisms to regulate protein expression (1-3). In humans and Drosophila, 30 -95% of pre-mRNAs undergo alternative splicing, resulting in mature mRNAs that encode proteins with different sequences or that contain a premature stop codon and are subject to degradation (4 -11). Alternative splicing plays key roles in developmental processes, such as Drosophila sex determination, and defects in alternative splicing are commonly associated with human disorders, such as spinal muscular atrophy (12, 13). Thus, understanding the molecular details of alternative splicing mechanisms has global implications for understanding both normal and disease states.At its basic level, the choice of splicing pattern for a pre-mRNA involves the regulation of spliceosome assembly on introns to be excised. Spliceosomes contain five small nuclear ribonucleoproteins (snRNPs) 3 (U1, U2, U4, U5, and U6) and numerous regulatory proteins, including the heterodimeric complex U2AF (U2 auxiliary factor) (14 -16). Spliceosomes serve to define 5Ј and 3Ј splice sites at exon/intron boundaries and carry...

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Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 premRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 59 splice site or weak 39 splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 59 splice site or moderate 39 splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 39 splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.

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The key postulate that one gene encodes one protein has been overhauled with the discovery that one gene can generate multiple RNA transcripts through alternative mRNA processing. In this study, we describe SplicerEX, a novel and uniquely motivated algorithm designed for experimental biologists that (1) detects widespread changes in mRNA isoforms from both conventional and splice sensitive microarray data, (2) automatically categorizes mechanistic changes in mRNA processing, and (3) mitigates known technological artifacts of exon array-based detection of alternative splicing resulting from 59 and 39 signal attenuation, background detection limits, and saturation of probe set signal intensity. In this study, we used SplicerEX to compare conventional and exon-based Affymetrix microarray data in a model of EBV transformation of primary human B cells. We demonstrated superior detection of 39-located changes in mRNA processing by the Affymetrix U133 GeneChip relative to the Human Exon Array. SplicerEX-identified exon-level changes in the EBV infection model were confirmed by RT-PCR and revealed a novel set of EBV-regulated mRNA isoform changes in caspases 6, 7, and 8. Finally, SplicerEX as compared with MiDAS analysis of publicly available microarray data provided more efficiently categorized mRNA isoform changes with a significantly higher proportion of hits supported by previously annotated alternative processing events. Therefore, SplicerEX provides an important tool for the biologist interested in studying changes in mRNA isoform usage from conventional or splicesensitive microarray platforms, especially considering the expansive amount of archival microarray data generated over the past decade. SplicerEX is freely available upon request.