39510.2217/FON.12.11 © 2012 Future Medicine Ltd ISSN 1479-6694Future Oncol. (2012) 8(4), 395–398

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Summary of methods & resultsb-catenin is a multifunctional protein with roles in both cell adhesion and regulation of tran-scription. It has also been involved in numer-ous human malignancies [1]. An extraordinary diversity of transcriptional responses has been described for the b-catenin signaling pathway. How this protein controls the transcription of various genes is the subject of intense studies that could help to understand the mechanisms by which b-catenin is involved in tumor forma-tion [2]. b-catenin transactivation results from at least two independent mechanisms: the first is linked to Wnt stimulation and activating muta-tions of b-catenin partners (such as APC and axin) involving Lef/TCF-regulated transcrip-tion [3], whereas the second is a poorly under-stood mechanism in response to growth factors, in a Wnt-independent manner [4]. The results reported by Yang and colleagues provide interest-ing new data concerning this latter mechanism by showing that PKM2 acts as a nuclear coactiva-tor during EGF-induced b-catenin transactiva-tion [5]. Pyruvate kinase catalyses and regulates the rate-limiting final step of glycolysis, namely the conversion of phosphoenolpyruvate to pyru-vate with production of ATP. The PKM1 alter-natively spliced isoform is ubiquitously expressed whereas the PKM2 isoform is expressed during

embryonic development and in fast-dividing cells such as cancer cells, in which it is a key player in the reprogramming of metabolism [6,7]. Yang et al. showed that EGF stimulation induced the nuclear translocation of PKM2 (but not PKM1) in human glioblastoma multiforme (GBM), the most common and malignant primary brain tumor, or in breast and prostate cancer cell lines. Subsequently, the authors demonstrated that PKM2 was responsible for the transactiva-tion of b-catenin by EGF, with its depletion by shRNA abolishing the TCF/LEF-1 luciferase reporter (TOPFlash) activity induced by EGF in a glioblastoma cell line (U87/EGFR). The EGF-dependent PKM2–b-catenin interac-tions required phosphorylation of b-catenin on the Y333 residue by c-Src. This post-trans-lational modification is crucial for the binding of b-catenin to the phosphotyrosine-binding domain of PKM2. Indeed, a b-catenin Y333F mutant failed to bind PKM2 upon EGF recep-tor (EGFR) activation, and was thus ineffec-tive at inducing expression of well-known tar-gets of b-catenin such as cyclin D1 or c-myc. Interestingly, c-Src-dependent transactivation of b-catenin via PKM2 binding seems to regu-late a specific set of genes that differ from those induced by the canonical Wnt–b-catenin path-way. Chromatin immunoprecipitation analyses

PKM2: a new player in the b-catenin game

Frédéric Canal1 & Christine Perret*1,2,3

1Institut Cochin, Département Endocrinologie, Métabolisme et Cancer, INSERM U1016, 24 rue du Faubourg St-Jacques 75014 Paris, France 2Cnrs, UMR8104, Paris, France3Université Paris Descartes, Paris, France*Author for correspondence: Tel.: +33 1 44 41 25 64 n Fax: +33 1 44 41 24 21 n christine.perret@inserm.fr

Evaluation of: Yang W, Xia Y, Ji H et al. Nuclear PKM2 regulates b-catenin transactivation upon EGFR activation. Nature 480(7375), 118–122 (2011). b-catenin is a key player in the regulation of gene expression during morphogenesis and tumorigenesis. Although its transactivation often results from stimulation of the Wnt signaling pathway, Wnt-independent regulation of b-catenin has also been observed in cancer cells. This study discloses a new mechanism for the transactivation of b-catenin upon EGF receptor activation that relies on the binding of b-catenin to the PKM2 isoform in the nucleus. This interaction requires phosphorylation of b-catenin on the Y333 residue by c-Src and the PKM2 domain that binds phosphotyrosine. Importantly, the authors demonstrated that EGF-induced transactivation of b-catenin is necessary for brain tumor growth and that high levels of c-Src activity, Y333 b-catenin phosphorylation and nuclear localization of PKM2 altogether correlate with high aggressiveness of tumors in glioblastoma multiforme. Remarkably, this study reveals a novel role for PKM2 in cancer cells where PKM2 appears to be, in addition to its established role in aerobic glycolysis, a major coactivator of b-catenin transactivation. This nuclear function of PKM2 is shared with other transcription factors such as HIF-1a and OCT4, and highlights the nonmetabolic role of PKM2 during tumorigenesis.

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Keywords

n b-catenin n genetic reprogramming of cancer cells n glioblastoma multiforme n prognosis n pyruvate kinase M2 n tumorigenesis

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showed that the stimulation of U87/EGFR cells with EGF leads to the dissociation of the histone deacetylase HDAC3 from the CCND1 (cyclin D1) promoter region and enhances cyclin D1 and c-myc expression.

Importantly, the critical role of PKM2 in the EGF-induced b-catenin transactivation in tumorigenesis was investigated in human GBM tumors. EGFR overexpression occurs in 40–60% of human GBM, [8]. Data from Yang and col-leagues showed that brain tumor xenografts developed rapidly in nude athymic mice intacra-nially injected with U87 cells expressing a consti-tutively active EGFR [5]. Tumor growth was pre-vented by depleting either PKM2 or b-catenin, or by using a c-Src-specific inhibitor (SU6656). The effect of these depletions on tumor growth was not rescued by Y333F b-catenin. Similarly, a PKM2 mutant that is unable to bind phosphoty-rosine but retains its catalytic activity for glyco-lysis failed to rescue these tumor growth inhibi-tions, supporting an essential nonmetabolic role for PKM2 in tumorigenesis. Furthermore, in an immunohistochemical analysis of 55 human primary GBM specimens, the authors observed a correlation between c-Src activity, b-catenin Y333 phosphorylation level and nuclear PKM2 localization, and observed that grade IV GBM tumors (which have a worse prognosis) harbored the higher levels of Y333 phosphorylation and showed nuclear PKM2 localization.

DiscussionThese data are particularly relevant to cancers where PKM2 is preferentially expressed. By identifying PKM2 as a new nuclear coactiva-tor of b-catenin that contributes to tumor cell growth, the authors provide a mechanism to explain the Wnt-independent transactivation of b-catenin in response to growth factor stimula-tion that has been observed in a large proportion of cancer such as endometrial cancers (60%), melanomas (30%) and hepatocellular carcino-mas (50%) [9]. Furthermore, this study uncovers a missing link between the EGFR and b-catenin that could explain the poorly understood syner-gic activation of these pathways that have been often observed during carcinogenesis [4].

The identification of new nuclear PKM2 targets such as b-catenin confirms that PKM2 plays a far broader role in tumor cell growth than previously thought. Indeed, a similar nonmeta-bolic nuclear role of PKM2 has been identified in tumorigenesis where it coactivates two tran-scription factors, HIF1-a and OCT4, which are known to be involved in tumor progression

[10,11]. Similar to PKM2 binding to b-catenin, the HIF1-a–PKM2 interaction requires a post-translational modification in the region of the protein that is specific to the PKM2 isoform (in this case a proline hydroxylation of PKM2 via PHD3). Transactivation properties of tran-scription factors elicited through PKM2 binding thus seem to be labile events that are regulated by specific stimuli such as EGFR activation for b-catenin or hypoxia for HIF1-a.

Hence, PKM2 appears to have at least two distinct roles in tumors: the well-described meta-bolic role leading to an enhanced aerobic glyco-lytic metabolism known as the Warburg effect, and an unexpected emerging role in the control of gene expression. These two functions are syn-ergistic – PKM2 translocation to the nucleus and its decreased enzymatic activity upon bind-ing phosphotyrosine peptides will, consequently, significantly slow down its glycolytic activity [12]. This regulation of PKM2 will divert glucose metabolites from energy production towards ana-bolic processes to sustain rapid growth of cancer cells. Interestingly, we have recently shown that b-catenin signaling is a key determinant of liver metabolic activity [13,14], and the data presented in this article are another indication that the study of b-catenin in the metabolic reprogramming during tumor development merits further investigation.

Future perspectiveThe understanding of how EGFR activation is able to transactivate b-catenin opens new per-spectives for the comprehension of the molecu-lar events responsible for tumor cell growth, with both of these oncogenes being activated in numerous cancer types. It would be instruc-tive, for instance, to screen cohorts of different types of tumors bearing EGFR-activating muta-tions for nuclear PKM2 and b-catenin activa-tion. Hepatocellular carcinomas could be a good example, since both b-catenin and EGFR acti-vation have been described in a high proportion of these tumors.

Currently, the reference treatment for GBM is the surgical removal of the tumor followed by radiation therapy combined with temozolo-mide, an alkylating agent that damages DNA by methylating the O-6 position of the guanidine. Unfortunately, despite these aggressive treat-ments, the median survival reaches no more than 15 months after diagnosis, and the death of GBM patients caused by tumor recurrence occurred within a year [15]. This study provides new biomarkers for prognosis in GBM such as the nuclear localization of PMK2 and the high

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PKM2: a new player in the b-catenin game Priority Paper Evaluation

levels of Y333 b-catenin phosphorylation, which corroborate a recent report showing that high b-catenin activity is a highly predictive marker of short survival for GBM patients [16]. Notably, b-catenin, which is preferentially activated in GBM cells isolated after in vivo ionizing radia-tion, has recently been shown to be involved in GBM radioresistance [17]. In the light of the results presented by Yang and colleagues [5], it would be of particular interest to assess the role of nuclear PKM2 in glioblastoma radioresist-ance, their data making PKM2 a potential syn-thetic lethal partner of b-catenin. Since efforts to develop selective inhibitors of PKM2 are cur-rently in progress [18], PKM2 inhibition might be used as a potential radiosensitizer of a certain type of GBM cells.

Executive summary

EGF induces the PKM2–b-catenin interaction in the nucleus & transactivation of b-catenin n EGF receptor (EGFR) activation induces PKM2 (but not PKM1) translocation to the nucleus in multiple types of cancer cells. PKM2 binds

to and allows b-catenin to transactivate via molecular events distinct from those described in the Wnt–b-catenin transduction pathway. The PKM2–b-catenin interaction is required for EGFR-induced b-catenin transactivation and facilitates U87 cell proliferation. n The b-catenin Y333 residue has been identified as a new target of c-Src. This leads to the interaction between the phosphotyrosine

binding domain of PKM2 and the phosphorylated Y333 residue of b-catenin in the nucleus. This PKM2–b-catenin complex is then able to bind to the CCND1 (cyclin D1) promoter. This promotes HDAC3 dissociation from the promoter, the subsequent acetylation of histone H3 and, ultimately, induction of cyclin D1 expression, supporting tumorigenesis.

EGF-induced transactivation of b-catenin via nuclear PKM2 is required for brain tumor growth & is of particular interest for prognosis & glioblastoma multiforme treatment n The inhibition of b-catenin transactivation leads to a decrease in glioblastoma multiforme (GBM) cancer cells, an accumulation of cells

at the G1/S transition phase and a reduction of the tumor volume in mice brains. n The level of b-catenin Y333 phosphorylation and nuclear PKM2 correlates with grades of glioma malignancy. These new biomarkers

could be used for both prognosis and selection of GBM treatment.

Conclusion n Besides its well-studied role in aerobic glycolysis and particularly in the metabolic reprogramming observed during tumorigenesis

known as the Warburg effect, a new nonmetabolic function for PKM2 has clearly emerged in cancer cells. PKM2 is able to modify the transcriptional activity of several transcription factors involved in tumorigenesis. The present study also provides an important insight into the understanding of the mechanism of b-catenin transactivation in response to EGFR stimulation and opens new perspectives for GBM prognosis and treatment.

AcknowledgementsThe authors thank Sabine Colnot, Béatrice Romagnolo and Hélène Gilgenkrantz for critical reading of the manuscript.

Financial & competing interests disclosureThe authors’ work is supported by INSERM, CNRS, the Ligue Nationale contre le Cancer (équipe labelisée 2011–2013), the Institut National du Cancer and the Agence Nationale de la Recherche. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or mate-rials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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