Cancer cells preferentially metabolize glucose by aerobic glycolysis, characterized by increased lactate production. This distinctive metabolism involves expression of the embryonic M2 isozyme of pyruvate kinase, in contrast to the M1 isozyme normally expressed in differentiated cells, and it confers a proliferative advantage to tumor cells. The M1 and M2 pyruvate-kinase isozymes are expressed from a single gene through alternative splicing of a pair of mutually exclusive exons. We measured the expression of M1 and M2 mRNA and protein isoforms in mouse tissues, tumor cell lines, and during terminal differentiation of muscle cells, and show that alternative splicing regulation is sufficient to account for the levels of expressed protein isoforms. We further show that the M1-specific exon is actively repressed in cancer-cell lines-although some M1 mRNA is expressed in cell lines derived from brain tumors -and demonstrate that the related splicing repressors hnRNP A1 and A2, as well as the polypyrimidine-tract-binding protein PTB, contribute to this control. Downregulation of these splicing repressors in cancer-cell lines using shRNAs rescues M1 isoform expression and decreases the extent of lactate production. These findings extend the links between alternative splicing and cancer, and begin to define some of the factors responsible for the switch to aerobic glycolysis.aerobic glycolysis | cancer | RNA splicing C ancer cells exhibit a metabolic phenotype characterized by increased glycolysis with lactate generation, regardless of oxygen availability-a phenomenon termed the Warburg effect. Recent work demonstrated that expression of the type II isoform of the pyruvate-kinase-M gene (PKM2, referred to here as PK-M) is a critical determinant of this metabolic phenotype, and confers a selective proliferative advantage to tumor cells in vivo (1). This finding adds to the growing body of evidence that alterations in alternative prem-RNA splicing play important roles in different aspects of cancer progression (2, 3).Pyruvate-kinase (PK) is the enzyme that catalyzes the final step in glycolysis, generating pyruvate and ATP from phosphoenolpyruvate and ADP (4). The resulting pyruvate can be converted to lactate or it can be incorporated into the tricarboxylic acid (TCA) cycle to drive oxidative phosphorylation. PK is encoded by two paralogous genes, each of which is alternatively spliced, such that four PK isoforms are expressed in mammals. The L and R isozymes, derived from the PKLR gene, show tissue-specific expression in the liver and red-blood cells, respectively. They have different first exons, defined by tissue-specific promoters (5). The PKM2 (PK-M) gene consists of 12 exons, of which exons 9 and 10 are alternatively spliced in a mutually exclusive fashion to give rise to the PK-M1 and PK-M2 isoforms, respectively (6). Exons 9 and 10 each encode a 56 amino acid variable segment that confers distinctive properties to the regulation and activity of PK-M1 and PK-M2 enzymes; as a result, PK-M1 is constitutively active,...

The immediate-early (IE) BZLF1 gene of Epstein-Barr virus (EBV) regulates the switch between latent and lytic infection by EBV. We previously showed that the cellular transcription factor ZEB1 binds to a sequence element, ZV, located at nt −17 to −12 relative to the transcription initiation site of the BZLF1 promoter, Zp, repressing transcription from Zp in a transient transfection assay. Here, we report the phenotype in the context of a whole EBV genome of a variant of EBV strain B95.8 containing a 2-bp substitution mutation in the ZV element of Zp that reduced, but did not eliminate, ZEB1 binding to Zp. Strikingly, epithelial 293 cells latently infected with the EBV ZV mutant spontaneously produced IE-, early-, and late-gene products and infectious virus, while wild-type (WT)-infected 293 cells did not and have never been reported to do so. Furthermore, treatment with the chemical inducers sodium butyrate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to an additional order-of-magnitude production of infectious virus in the ZV mutant–infected 293 cells, but still no virus in the WT-infected 293 cells. Similarly, ZV mutant–infected Burkitt's lymphoma BJAB cells accumulated at least 10-fold more EBV IE mRNAs than did WT-infected BJAB cells, with TPA or sodium butyrate treatment leading to an additional 5- to 10-fold accumulation of EBV IE mRNAs in the ZV mutant–infected cells. Thus, we conclude that ZEB1 binding to Zp plays a central role in regulating the latent-lytic switch in EBV-infected epithelial and B cells, suggesting ZEB1 as a target for lytic-induction therapies in EBV-associated malignancies.

Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, exonic, rather than intronic, cis-elements are key determinants of PK-M splicing isoform ratios. Using a systematic sub-exonic duplication approach, we identify a potent exonic splicing enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative splicing.

Alternative splicing of the pyruvate kinase M gene involves a choice between mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We previously demonstrated that splicing enhancer elements that activate exon 10 are mainly found in exon 10 itself, and deleting or mutating these elements increases the inclusion of exon 9 in cancer cells. To systematically search for new enhancer elements in exon 10 and develop an effective pharmacological method to force a switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide (ASO) screen. We found potent ASOs that target a novel enhancer in exon 10 and strongly switch the splicing of endogenous PK-M transcripts to include exon 9. We further show that the ASO-mediated switch in alternative splicing leads to apoptosis in glioblastoma cell lines, and this is caused by the downregulation of PK-M2, and not by the upregulation of PK-M1. These data highlight the potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.

Background Mutations in SCN2B, encoding voltage-gated sodium channel (VGSC) β2 subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that β2 subunits play critical roles in the establishment or maintenance of normal cardiac electrical activity in vivo. Methods and Results To understand the pathophysiological roles of β2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current density in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional re-entry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild type ventricles, as were levels of Nav1.5 protein, suggesting that similar to previous work in neurons, the major function of β2 subunits in the ventricle is to chaperone VGSC α subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared to wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than WT littermates. Conclusions Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.