PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in HT29 human colorectal cancer cell lines

Ajuts: Beca bianual de la Fundació Olga Torres 2008-2009Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells

Estudi del paper de la Isoforma M2 de Piruvat Quinasa (PKM2) en la resposta i resistència a oxaliplatí en línies cel∙lulars i en pacients amb càncer colorectal avançat

El càncer colorectal és la segona causa de mort per càncer al món i el tumor més freqüent a l’Estat Espanyol englobant ambdós sexes. El tractament de la malaltia avançada es basa en la combinació de fàrmacs citotòxics, citostàtics i noves molècules anti-diana, assolint-se medianes de supervivència al voltant dels 2 anys. Encara que les darreres tendències de tractament del càncer estan enfocades principalment cap al desenvolupament de molècules anti-diana, l’administració d’oxaliplatí continua essent clau i essencial en el tractament en el CCR avançat. Malgrat els grans avenços que s’han donat en el tractament del càncer de còlon metastàtic, tots ells estan al voltant de la introducció de nous medicaments i seguim indefensos pel que fa a l’aparició de resistències, que són la causa fonamental del fracàs del tractament. Per tant, l’objectiu principal d’aquesta tesi doctoral és conèixer millor els mecanismes de resistència adquirida a oxaliplatí mitjançant l’estudi de perfils d’expressió proteica en models cel·lulars in vitro per després traslladar els resultats a la selecció del tractament de primera línia en pacients amb càncer colorectal avançat. Així, a partir d’una aproximació de proteòmica comparativa, s’ha identificat la proteïna piruvat quinasa M2 (PKM2) en relació amb el procés d’adquisició de resistència a oxaliplatí en el nostre model cel·lular. PKM2 catalitza l’últim pas de la glicòlisi i regula de manera clau la glicòlisi aeròbica o efecte Warburg en les cèl·lules canceroses. Recentment, se li han atribuït altres funcions no-glicolítiques; així, pot translocar al nucli i activar la transcripció gènica interaccionant i modificant proteïnes nuclears participant en funcions tant dispars com la supervivència o la mort cel·lular. Els nostres resultats demostren que PKM2 es troba infraexpressada en les línies resistents, també a nivell d’ARNm i que després del tractament amb oxaliplatí, els nivells d’expressió disminueixen. Mitjançant tècniques d’ARN d’interferència hem demostrat que la inhibició de l’expressió de PKM2 augmenta la viabilitat de les cèl·lules sensibles, amb p53 mutada, quan es tracten amb el fàrmac en un 50% mentre que l’efecte és oposat en un model amb p53 salvatge. Tanmateix no hem pogut demostrar que aquest augment en la viabilitat sigui conseqüència d’una disminució en la mort cel·lular. D’altra banda, hem pogut observar un canvi en la compartimentació de la proteïna que només es dona en les cèl·lules sensibles a oxaliplatí, donat que PKM2 és capaç de translocar al nucli de les cèl·lules sensibles al llarg de 72 hores de tractament continu però no en les cèl·lules derivades resistents. En tercer lloc, a partir d’una aproximació de silenciament gènic i proteòmica comparativa en 2 dimensions, s’han pogut establir els perfils proteics associats a la presència/absència de PKM2 i determinar possibles proteïnes vinculades a l’expressió d’aquesta com son, FKBP4 i HSC70. Finalment, pel que fa a l’estudi en pacients, hem demostrat que el grup de pacients amb els nivells més baixos de PKM2 presenten pitjors ràtios de resposta a la quimioteràpia basada en oxaliplatí i que aquest fet també pot tenir relació amb l’estat mutacional de p53 en aquests pacients, correlacionant aquests resultats amb els obtinguts a l’estudi in vitro. Així doncs, en aquesta tesi doctoral es descriu un nou mecanisme d’adquisició de resistència a oxaliplatí i se li atribueix un nou paper a la proteïna PKM2. Tanmateix, s’obren noves vies d’investigació, tant a nivell de funcionalitat de dita proteïna com també de noves vies d’intervenir en la resistència adquirida a aquest fàrmac platinat.Colorectal cancer is the second most common cause of cancer death in the world and the most frequent tumor in Spain including both genders. Survival of the advanced disease has increased from 12 months with monotherapy to roughly 2 years with the addition of cytotoxic and cytostatic drugs combined with targeted drugs. Despite the state of the art in cancer treatment is mainly focused on the development of target molecules, oxaliplatin administration remains essential in the treatment of advanced CRC. Regardless of the great improvements achieved in the treatment of metastatic CRC, all of them are focused in the introduction of new drugs and we are still defenseless in front of resistance appearance, which is the main cause of treatment failure. According to this, the main objective of this doctoral thesis is to better know and understand oxaliplatin acquired resistance mechanisms by studying protein expression profiles in in vitro models in order to translate these results in a better selection of first line treatment in advanced CRC patients. Thus, from a comparative proteomic study, we have identified the protein M2 pyruvate kinase (PKM2) related to the oxaliplatin resistance acquisition process in our cellular model. PKM2 catalyzes the last step of glycolysis and is a key enzyme in the regulation of aerobic glycolysis (known as a Warburg effect) in cancer cells. Recently, other non-glycolytic functions have been assigned to PKM2. It has been shown this protein can translocate to the nucleus and, consequently, it activates transcription of several genes. PKM2 can also interact and modify nuclear proteins taking part in different and opposed cellular processes such as proliferation or death. Our results have demonstrated that PKM2 expression is down-regulated in oxaliplatin resistant cells as compared to sensitive cells, also at mRNA level. Moreover, we have shown that oxaliplatin treatment decreases PKM2 levels. By using RNA interference techniques, we have shown that PKM2 gene silencing increases cell proliferation of sensitive cells harboring p53 mutation in about 50% when those cells are treated with the drug, while PKM2 silencing in sensitive and wild type p53 cells has the opposite result. The effect in increased cell proliferation was not related to a cell death decrease, though. On the other hand, we could observe a change in the subcellular localization of PKM2 only in treated sensitive cells, done by the fact that this protein is able to translocate to the nucleus of these cells in response to the drug and this is not observed in oxaliplatin resistant cells. Moreover, by using gene silencing and a 2-D DIGE approach, we obtained protein profiles associated to PKM2 presence/absence and we have determined possible proteins whose expression depends on that of PKM2 such as FKBP4 and HSC70. Finally, we have demonstrated that patients with the lowest PKM2 mRNA levels showed the worst response ratios to oxaliplatin-based first line treatment. Tumors from these patients presented more frequently p53 mutations linking these results with our in vitro study. To conclude, in this doctoral thesis we describe a new mechanism of oxaliplatin resistance acquisition in which PKM2 is an important player. Further investigations are guaranteed to elucidate molecular mechanisms underlying PKM2 functionality as well as to discover new pathways to interfere in acquired resistance to this platinum agent

Estudi del paper de la isoforma M2 de Piruvat Quinasa (PKM2) en la resposta i resistència a oxaliplatí en línies cel·lulars i en pacients amb càncer colorectal avançat

Colorectal cancer is the second most common cause of cancer death in the world and the most frequent tumor in Spain including both genders. Survival of the advanced disease has increased from 12 months with monotherapy to roughly 2 years with the addition of cytotoxic and cytostatic drugs combined with targeted drugs. Despite the state of the art in cancer treatment is mainly focused on the development of target molecules, oxaliplatin administration remains essential in the treatment of advanced CRC. Regardless of the great improvements achieved in the treatment of metastatic CRC, all of them are focused in the introduction of new drugs and we are still defenseless in front of resistance appearance, which is the main cause of treatment failure. According to this, the main objective of this doctoral thesis is to better know and understand oxaliplatin acquired resistance mechanisms by studying protein expression profiles in in vitro models in order to translate these results in a better selection of first line treatment in advanced CRC patients. Thus, from a comparative proteomic study, we have identified the protein M2 pyruvate kinase (PKM2) related to the oxaliplatin resistance acquisition process in our cellular model. PKM2 catalyzes the last step of glycolysis and is a key enzyme in the regulation of aerobic glycolysis (known as a Warburg effect) in cancer cells. Recently, other non-glycolytic functions have been assigned to PKM2. It has been shown this protein can translocate to the nucleus and, consequently, it activates transcription of several genes. PKM2 can also interact and modify nuclear proteins taking part in different and opposed cellular processes such as proliferation or death. Our results have demonstrated that PKM2 expression is down-regulated in oxaliplatin resistant cells as compared to sensitive cells, also at mRNA level. Moreover, we have shown that oxaliplatin treatment decreases PKM2 levels. By using RNA interference techniques, we have shown that PKM2 gene silencing increases cell proliferation of sensitive cells harboring p53 mutation in about 50% when those cells are treated with the drug, while PKM2 silencing in sensitive and wild type p53 cells has the opposite result. The effect in increased cell proliferation was not related to a cell death decrease, though. On the other hand, we could observe a change in the subcellular localization of PKM2 only in treated sensitive cells, done by the fact that this protein is able to translocate to the nucleus of these cells in response to the drug and this is not observed in oxaliplatin resistant cells. Moreover, by using gene silencing and a 2-D DIGE approach, we obtained protein profiles associated to PKM2 presence/absence and we have determined possible proteins whose expression depends on that of PKM2 such as FKBP4 and HSC70. Finally, we have demonstrated that patients with the lowest PKM2 mRNA levels showed the worst response ratios to oxaliplatin-based first line treatment. Tumors from these patients presented more frequently p53 mutations linking these results with our in vitro study. To conclude, in this doctoral thesis we describe a new mechanism of oxaliplatin resistance acquisition in which PKM2 is an important player. Further investigations are guaranteed to elucidate molecular mechanisms underlying PKM2 functionality as well as to discover new pathways to interfere in acquired resistance to this platinum agent.El càncer colorectal és la segona causa de mort per càncer al món i el tumor més freqüent a l'Estat Espanyol englobant ambdós sexes. El tractament de la malaltia avançada es basa en la combinació de fàrmacs citotòxics, citostàtics i noves molècules anti-diana, assolint-se medianes de supervivència al voltant dels 2 anys. Encara que les darreres tendències de tractament del càncer estan enfocades principalment cap al desenvolupament de molècules anti-diana, l'administració d'oxaliplatí continua essent clau i essencial en el tractament en el CCR avançat. Malgrat els grans avenços que s'han donat en el tractament del càncer de còlon metastàtic, tots ells estan al voltant de la introducció de nous medicaments i seguim indefensos pel que fa a l'aparició de resistències, que són la causa fonamental del fracàs del tractament. Per tant, l'objectiu principal d'aquesta tesi doctoral és conèixer millor els mecanismes de resistència adquirida a oxaliplatí mitjançant l'estudi de perfils d'expressió proteica en models cel·lulars in vitro per després traslladar els resultats a la selecció del tractament de primera línia en pacients amb càncer colorectal avançat. Així, a partir d'una aproximació de proteòmica comparativa, s'ha identificat la proteïna piruvat quinasa M2 (PKM2) en relació amb el procés d'adquisició de resistència a oxaliplatí en el nostre model cel·lular. PKM2 catalitza l'últim pas de la glicòlisi i regula de manera clau la glicòlisi aeròbica o efecte Warburg en les cèl·lules canceroses. Recentment, se li han atribuït altres funcions no-glicolítiques; així, pot translocar al nucli i activar la transcripció gènica interaccionant i modificant proteïnes nuclears participant en funcions tant dispars com la supervivència o la mort cel·lular. Els nostres resultats demostren que PKM2 es troba infraexpressada en les línies resistents, també a nivell d'ARNm i que després del tractament amb oxaliplatí, els nivells d'expressió disminueixen. Mitjançant tècniques d'ARN d'interferència hem demostrat que la inhibició de l'expressió de PKM2 augmenta la viabilitat de les cèl·lules sensibles, amb p53 mutada, quan es tracten amb el fàrmac en un 50% mentre que l'efecte és oposat en un model amb p53 salvatge. Tanmateix no hem pogut demostrar que aquest augment en la viabilitat sigui conseqüència d'una disminució en la mort cel·lular. D'altra banda, hem pogut observar un canvi en la compartimentació de la proteïna que només es dona en les cèl·lules sensibles a oxaliplatí, donat que PKM2 és capaç de translocar al nucli de les cèl·lules sensibles al llarg de 72 hores de tractament continu però no en les cèl·lules derivades resistents. En tercer lloc, a partir d'una aproximació de silenciament gènic i proteòmica comparativa en 2 dimensions, s'han pogut establir els perfils proteics associats a la presència/absència de PKM2 i determinar possibles proteïnes vinculades a l'expressió d'aquesta com son, FKBP4 i HSC70. Finalment, pel que fa a l'estudi en pacients, hem demostrat que el grup de pacients amb els nivells més baixos de PKM2 presenten pitjors ràtios de resposta a la quimioteràpia basada en oxaliplatí i que aquest fet també pot tenir relació amb l'estat mutacional de p53 en aquests pacients, correlacionant aquests resultats amb els obtinguts a l'estudi in vitro. Així doncs, en aquesta tesi doctoral es descriu un nou mecanisme d'adquisició de resistència a oxaliplatí i se li atribueix un nou paper a la proteïna PKM2. Tanmateix, s'obren noves vies d'investigació, tant a nivell de funcionalitat de dita proteïna com també de noves vies d'intervenir en la resistència adquirida a aquest fàrmac platinat

PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in HT29 human colorectal cancer cell lines

Ajuts: Beca bianual de la Fundació Olga Torres 2008-2009Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells

Measurement of electrons from semileptonic heavy-flavour hadron decays at midrapidity in pp and Pb–Pb collisions at √sNN = 5.02 TeV

The differential invariant yield as a function of transverse momentum (pT) of electrons from semileptonic heavy-flavour hadron decays was measured at midrapidity in central (0–10%), semi-central (30–50%) and peripheral (60–80%) lead–lead (Pb–Pb) collisions at √sNN = 5.02 TeV in the pT intervals 0.5–26 GeV/c (0–10% and 30–50%) and 0.5–10 GeV/c (60–80%). The production cross section in proton–proton (pp) collisions at √s = 5.02 TeV was measured as well in 0.5 < pT < 10 GeV/c and it lies close to the upper band of perturbative QCD calculation uncertainties up to pT = 5 GeV/c and close to the mean value for larger pT. The modification of the electron yield with respect to what is expected for an incoherent superposition of nucleon–nucleon collisions is evaluated by measuring the nuclear modification factor RAA. The measurement of the RAA in different centrality classes allows in-medium energy loss of charm and beauty quarks to be investigated. The RAA shows a suppression with respect to unity at intermediate pT, which increases while moving towards more central collisions. Moreover, the measured RAA is sensitive to the modification of the parton distribution functions (PDF) in nuclei, like nuclear shadowing, which causes a suppression of the heavy-quark production at low pT in heavy-ion collisions at LHC

Charged-particle multiplicity fluctuations in Pb–Pb collisions at √sNN = 2.76 TeV

Measurements of event-by-event fluctuations of charged-particle multiplicities in Pb–Pb collisions at sNN−−−√ = 2.76 TeV using the ALICE detector at the CERN Large Hadron Collider (LHC) are presented in the pseudorapidity range |η|<0.8 and transverse momentum 0.2<pT<2.0 GeV/c. The amplitude of the fluctuations is expressed in terms of the variance normalized by the mean of the multiplicity distribution. The η and pT dependences of the fluctuations and their evolution with respect to collision centrality are investigated. The multiplicity fluctuations tend to decrease from peripheral to central collisions. The results are compared to those obtained from HIJING and AMPT Monte Carlo event generators as well as to experimental data at lower collision energies. Additionally, the measured multiplicity fluctuations are discussed in the context of the isothermal compressibility of the high-density strongly-interacting system formed in central Pb–Pb collisions

Enhanced deuteron coalescence probability in jets

The transverse-momentum (pT) spectra and coalescence parameters B2 of (anti)deuterons are measured in pp collisions at s√=13 TeV for the first time in and out of jets. In this measurement, the direction of the leading particle with the highest pT in the event (pleadT>5 GeV/c) is used as an approximation for the jet axis. The event is consequently divided into three azimuthal regions and the jet signal is obtained as the difference between the Toward region, that contains jet fragmentation products in addition to the underlying event (UE), and the Transverse region, which is dominated by the UE. The coalescence parameter in the jet is found to be approximately a factor of 10 larger than that in the underlying event. This experimental observation is consistent with the coalescence picture and can be attributed to the smaller average phase-space distance between nucleons inside the jet cone as compared to the underlying event. The results presented in this Letter are compared to predictions from a simple nucleon coalescence model, where the phase space distributions of nucleons are generated using PYTHIA 8 with the Monash 2013 tuning, and to predictions from a deuteron production model based on ordinary nuclear reactions with parametrized energy-dependent cross sections tuned on data. The latter model is implemented in PYTHIA 8.3. Both models reproduce the observed large difference between in-jet and out-of-jet coalescence parameters, although the almost flat trend of the BJet2 is not reproduced by the models, which instead give a decreasing trend

Measurement of the production cross section of prompt Ξ0c baryons in p–Pb collisions at √sNN = 5.02 TeV

The transverse momentum (pT) differential production cross section of the promptly-produced charm-strange baryon Ξ0c (and its charge conjugate Ξ0c¯¯¯¯¯¯) is measured at midrapidity via its hadronic decay into π+Ξ− in p−Pb collisions at a centre-of-mass energy per nucleon−nucleon collision sNN−−−√ = 5.02 TeV with the ALICE detector at the LHC. The Ξ0c nuclear modification factor (RpPb), calculated from the cross sections in pp and p−Pb collisions, is presented and compared with the RpPb of Λ+c baryons. The ratios between the pT-differential production cross section of Ξ0c baryons and those of D0 mesons and Λ+c baryons are also reported and compared with results at forward and backward rapidity from the LHCb Collaboration. The measurements of the production cross section of prompt Ξ0c baryons are compared with a model based on perturbative QCD calculations of charm-quark production cross sections, which includes only cold nuclear matter effects in p−Pb collisions, and underestimates the measurement by a factor of about 50. This discrepancy is reduced when the data is compared with a model in which hadronisation is implemented via quark coalescence. The pT-integrated cross section of prompt Ξ0c-baryon production at midrapidity extrapolated down to pT = 0 is also reported. These measurements offer insights and constraints for theoretical calculations of the hadronisation process. Additionally, they provide inputs for the calculation of the charm production cross section in p−Pb collisions at midrapidity