When oxygen is abundant, quiescent cells efficiently extract energy from glucose primarily by oxidative phosphorylation, whereas under the same conditions tumour cells consume glucose more avidly, converting it to lactate. This long-observed phenomenon is known as aerobic glycolysis 1 , and is important for cell growth 2, 3. Because aerobic glycolysis is only useful to growing cells, it is tightly regulated in a proliferation-linked manner4. Inmammals, this is partly achieved through control of pyruvate kinase isoform expression. The embryonic pyruvate kinase isoform, PKM2, is almost universally re-expressed in cancer2, and promotes aerobic glycolysis, whereas the adult isoform, PKM1, promotes oxidative phosphorylation 2 . These two isoforms result from mutually exclusive alternative splicing of the PKM pre-mRNA, reflecting inclusion of either exon 9 (PKM1) or exon 10 (PKM2). Here we show that three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins, polypyrimidine tract binding protein (PTB, also known as hnRNPI), hnRNPA1 and hnRNPA2, bind repressively to sequences flanking exon 9, resulting in exon 10 inclusion. We also demonstrate that the oncogenic transcription factor c-Myc upregulates transcription of PTB, hnRNPA1 and hnRNPA2, ensuring a high PKM2/PKM1 ratio. Establishing a relevance to cancer, we show that human gliomas overexpress c-Myc, PTB, hnRNPA1 and hnRNPA2 in a manner that correlates with PKM2 expression. Our results thus define a pathway that regulates an alternative splicing event required for tumour cell proliferation.Alternative splicing of PKM has an important role in determining the metabolic phenotype of mammalian cells. The single exon difference imparts the enzymes produced with important functional distinctions. For example, PKM2, but not PKM1, is regulated by the binding of tyrosine phosphorylated peptides, which results in release of the allosteric activator fructose-1-6-bisphosphate and inhibition of pyruvate kinase activity 5 , a property that might allow growth-factor-initiated signalling cascades to channel glycolytic intermediates into biosynthetic processes. The importance of tumour reversion to PKM2 was underscored by experiments in which replacement of PKM2 with PKM1 in tumour cells resulted in markedly reduced growth 2 . Consistent with a critical role in proliferation, re-expression of PKM2 in tumours is robust 2 , although little is known about the regulation of this process. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 October 5. We set out to identify RNA binding proteins that might regulate PKM alternative splicing. To this end, we prepared an [α-32 P]UTP-labelled 250-nucleotide RNA spanning the exon 9 (E9) 5′ splice site (EI9), previously identified as inhibitory to E9 inclusion 6 , as well as a labelled RNA from a corresponding region of E10 (EI10) (Fig. 1b), and performed ultraviolet crosslinking assays with HeLa nuclear extracts 7 . After separation by SDS-polyacrylamide gel electrophoresis (PAGE), multiple proteins...

To test the gliomagenic potential of adult glial progenitors, we infected adult rat white matter with a retrovirus that expresses high levels of PDGF and green fluorescent protein (GFP). Tumors that closely resembled human glioblastomas formed in 100% of the animals by 14 d postinfection. Surprisingly, the tumors were composed of a heterogeneous population of cells, Ͻ20% of which expressed the retroviral reporter gene (GFP). The vast majority of both GFPϩ and GFP-tumor cells expressed markers of glial progenitors. Thus, the tumors arose from the massive expansion of both infected and uninfected glial progenitors, suggesting that PDGF was driving tumor formation via autocrine and paracrine stimulation of glial progenitor cells. To explore this possibility further, we coinjected a retrovirus expressing PDGF-IRES-DsRed with a control retrovirus expressing only GFP. The resulting tumors contained a mixture of red cells (PDGFexpressing/tumor-initiating cells) and green cells (recruited progenitors). Both populations were highly proliferative and infiltrative. In contrast, when the control GFP retrovirus was injected alone, the animals never formed tumors and the majority of infected cells differentiated along the oligodendrocyte lineage. Together, these results reveal that adult white matter progenitors not only have the capacity to give rise to gliomas, but resident progenitors are recruited to proliferate within the mitogenic environment of the tumor and in this way contribute significantly to the heterogeneous mass of cells that compose a malignant glioma.

Together, these results demonstrate that human gliomas contain multiple populations of cells with the capacity to form tumors and specifically identify a population of tumorigenic A2B5+ cells that are phenotypically distinct from CD133+ cells.

Platelet-derived growth factor (PDGF) plays a major role in regulating migration, proliferation, and differentiation of glial progenitors during normal brain development and in the abnormal proliferation and dispersion that drives the formation of malignant gliomas. To further explore the relationship between PDGF's effects on normal glial progenitors and its role in the formation of gliomas, we infected progenitor cells in the subventricular zone (SVZ) of the lateral ventricle of neonatal rat pups with a retrovirus that expresses PDGF and green fluorescent protein (GFP). At 3 days post-injection (dpi), a proliferation of PDGFRalpha+ progenitors was seen in the SVZ and white matter around the injection site and by 10 dpi the animals had large diffusely infiltrating tumors that resembled glioblastomas. The tumors contained a massive proliferation of both infected and uninfected PDGFRalpha+ progenitors, suggesting that PDGF was driving tumor formation via both autocrine and paracrine signaling. Rats co-injected with two retroviruses (one that expresses PDGF-IRES-DSRED and one that expresses only GFP) formed tumors that contained a mixture of DSRED+ cells (PDGF producers) and GFP+ cells (recruited progenitors). Time-lapse microscopy of slice cultures confirmed that both DSRED+ and GFP+ cells were highly migratory and proliferative. Furthermore, adding exogenous PDGF to slice cultures generated from nontumor-bearing brains (injected with control GFP retrovirus only) stimulated the migration and proliferation of GFP+ progenitors. These findings reveal the inherent growth factor responsiveness and tumorigenic potential of PDGFRalpha+ progenitors and highlight the importance of paracrine signaling in stimulating glioma growth and infiltration.