Several cancer cells, in order to generate ATP and sustain different anabolic processes, rely mainly on glycolysis instead of Oxidative Phosphorylation (OXPHOS). Thus, glucose assumes a critical role for cancer cell survival and proliferation. Moreover, through the penthose phospate pathway glucose leads to production of NADPH contributing to maintenance of cellular oxidative equilibrium. Besides, glucose can also enter Hexosamine Biosynthesis Pathway (HBP), sustaining lipid and protein N- and O-glycosylation that cover an important role in cancer development. Taking in consideration the essential role of glucose in cancer, one important anticancer therapeutic approach is to target its metabolism namely glycolysis and the other processes in which it is involved. On this regard, glucose deprivation and consequent analysis of cancer cell fate both at phenotypical and molecular level can be a useful strategy to unmask all mechanisms that participate to glucose-mediated cancer cell growth and survival. Such a strategy could be subsequently exploited to provide new targets and to set new anticancer therapies. Although there is evidence that tumors originate from cells with persistent defects in the mitochondrial respiratory system, inhibition of OXPHOS activity seems to be an adaptation to cancer metabolism reprogramming rather than a cause. In this scenario, reversible post-translational modifications of mitochondrial components could assume an important regulatory role. Among the most important post-translational modifications there is Ser/Thr phosphorylation and, on this regard, the protein kinase PKA has numerous mitochondrial targets being involved in the regulation of the biogenesis, the import and the activity of mitochondrial Complex I or IV as well as of mitochondrial morphology. Since it has been observed that oncogenic K-ras may lead to a depression of genes encoding for components of the cAMP/PKA signaling pathway, in K-ras-transformed cells the deregulation of cAMP/PKA pathway could cause OXPHOS depression and “glucose addiction” of cancer cells. In agreement with such a hypothesis, K-ras-transformed cells show lower PKA activity as compared to normal cells. Moreover, exogenous stimulation of PKA activity, achieved by Forskolin (FSK) treatment, protects mouse and human K-ras-transformed cells from apoptosis induced by glucose deprivation, by enhancing Complex I activity, intracellular ATP levels and mitochondrial fusion and by decreasing intracellular ROS levels. Worth noting, several of these effects are almost completely prevented by inhibition of PKA activity. Moreover, short time treatment with Mdivi-1, a molecule that favors mitochondrial fusion, strongly decreases the cellular ROS levels especially in transformed cells, indicating a close relationship between mitochondrial morphology and activity. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial Complex I decrease, reduction of ATP formation and prevalence of mitochondrial fission over fusion. Such a discovery can thereby open new approaches for the development of anticancer drugs. Given that glucose shortage is often encountered in the tumor microenvironment, it can be exploited to potentiate the action of specific agents, such as the mitochondrial OXPHOS activity modulators, that in condition of glucose deprivation could be lethal for cancer cells. Accordingly, it is shown that glucose deprivation and Complex I inhibitors, i.e., rotenone, piericidin A and capsaicin, synergize in inducing cancer cell death. In particular, low doses of Complex I inhibitors, ineffective on normal cells and on cells grown in high glucose, become specifically cytotoxic on cancer cells cultured in low glucose. Importantly, the cytotoxic effect of Complex I inhibitors is strongly enhanced when mitochondrial OXPHOS activity is stimulated by FSK. These findings demonstrate that the reactivation of the mitochondrial function associated with glucose depletion and low doses of mitochondrial Complex I inhibitors strongly affect cancer cell survival. This therapeutic approach might be valuable to eradicate cancer cells. As above indicated, glucose is implicated in numerous processes in cancer cells. Transcriptomic and proteomic analyses applied to mouse K-ras-transformed cells as compared to normal cells show that glucose deprivation modulates the expression of several genes linked to endoplasmic reticulum stress and the Unfolded Protein Response (UPR). The activation of such a response, as confirmed by mRNA and protein expression, is observed in both cell lines, but only in transformed cells is strictly associated to their death. In fact, its attenuation by protein translation inhibitor cycloheximide or chemical chaperone 4-Phenyl-butyrate specifically rescues transformed cells from death. Moreover, glucose deprivation-induced transformed cell death is also prevented by inhibition of an UPR downstream pro-apoptotic kinase, JNK, whose activation is observed specifically in transformed cells as compared to normal cells. Interestingly, UPR activation and death of transformed cells is completely prevented by addition of a specific HBP substrate, namely N-Acetyl-D-glucosamine, suggesting a strict relation between the two processes. Notably, also oncogenic K-ras expressing human glycolytic cells show similar effects after UPR modulating treatments. Thus, we show that glucose deprivation can induce an UPR-dependent transformed cell death mechanism, which is activated by harmful accumulation of unfolded proteins, probably as consequence of N-glycosylation protein reduction. The full elucidation of this response could be relevant to design new therapeutic strategies. Today the new challenge of anticancer research and therapy is the total eradication of the cancer, targeting cancer stem cells (CSCs). Considering the important role of metabolism and metabolic reprogramming in cancer development, also the definition of CSCs metabolism can be considered an important tool for future strategies targeting these cells. Recently, a human osteosarcoma 3AB-OS CSC-like line has been developed. Therefore we have decided to characterize its metabolic features as compared to the parental osteosarcoma MG63 cells, from which 3AB-OS cells were previously selected. 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, addition to the growth medium of galactose and pyruvate -mitochondrial specific substrates- instead of glucose markedly reduces 3AB-OS growth, as compared to MG63 cells. In line with these findings 3AB-OS cells, compared to MG63 cells, are strongly sensitive to glucose depletion, glycolysis inhibition and less sensitive to respiratory inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display mainly fragmented mitochondria, particularly in low glucose. Overall, these findings suggest that 3AB-OS energy metabolism is more similar either to normal stem cells or to cancer cells characterized by a glycolytic metabolism. Interestingly, the transcriptional profile of CSCs is similar to that of K-ras-transformed cells, confirming a possible similarity to glycolytic cancer cells. Therefore, some strategies developed for glucose addicted cancer cells could be used also to treat specific CSCs.

Molte cellule tumorali, al fine di generare ATP e sostenere i processi anabolici, si servono principalmente della glicolisi piuttosto che della respirazione mitocondriale. Di conseguenza, il glucosio assume un ruolo critico per la sopravvivenza e la proliferazione delle cellule tumorali. Inoltre, attraverso la via dei pentosi fosfati, il glucosio porta alla formazione di NADPH, contribuendo al mantenimento nelle cellule dell’equilibrio ossidativo. Nondimeno, il glucosio può entrare anche nel pathway biosintetico delle esosammine (HBP), sostenendo la N- e O-glicosilazione di lipidi e proteine, importante per lo sviluppo tumorale. Considerando l’essenziale ruolo del glucosio, un possibile approccio per la terapia antitumorale è l’utilizzo del metabolismo del glucosio come target, non solo attraverso la glicolisi ma sfruttando anche gli altri processi glucosio-dipendenti. A tal proposito, la deprivazione di glucosio e la seguente analisi del destino cellulare a livello fenotipico e molecolare possono costituire una strategia utile per smascherare tutti i meccanismi mediati dal glucosio che partecipano alla crescita e alla sopravvivenza delle cellule tumorali. Tale strategia potrebbe essere poi sfruttata per offrire nuovi target e progettare nuove terapie antitumorali. Sebbene alcuni dati indichino che i tumori originino da cellule con persistenti difetti alla catena respiratoria mitocondriale, l’inibizione della fosforilazione ossidativa (OXPHOS) sembra una condizione di adattamento più che una causa della riprogrammazione metabolica delle cellule tumorali. In questo scenario, i meccanismi di regolazione post-traduzionali, di natura essenzialmente reversibile, a carico di proteine mitocondriali potrebbero assumere un importante ruolo regolatorio. Una delle principali modificazioni post-traduzionali è la fosforilazione dei residui Ser/Thr e, a tal proposito, la chinasi PKA presenta numerosi target a livello mitocondriale ed è coinvolta nella regolazione di biogenesi, trasporto e attività dei Complessi I e IV e della morfologia mitocondriale. Poiché è stato osservato che K-ras può causare la diminuzione dell’espressione di geni codificanti per componenti della via cAMP/PKA, nelle cellule K-ras-trasformate la deregolazione di tale via potrebbe portare alla disfunzione mitocondriale ed allo switch metabolico caratteristico delle cellule tumorali. A conferma di questa ipotesi, le cellule K-ras-trasformate mostrano minori livelli di attività dell’enzima PKA rispetto alle cellule normali. Inoltre, la stimolazione esogena della attività della PKA, ottenuta mediante trattamento con forskolina (FSK), protegge le cellule K-ras-trasformate, sia murine sia umane, dalla morte indotta dalla deplezione di glucosio. Tale protezione è dovuta alla stimolazione dell’attività del Complesso I, all’aumento dell’ATP intracellulare e della fusione mitocondriale e alla riduzione dei livelli di ROS. L’inibizione specifica di PKA previene quasi completamente molti di questi effetti. Inoltre, il breve trattamento con Mdivi-1, molecola in grado di favorire la fusione mitocondriale, riduce fortemente i livelli di ROS specialmente nelle cellule trasformate, indicando una stretta relazione tra morfologia e attività mitocondriale. Queste osservazioni supportano l’idea che l’apoptosi indotta dalla deprivazione di glucosio nelle cellule K-ras-trasformate è associata alla deregolazione della via cAMP/PKA che a sua volta causa la diminuzione dell’attività del Complesso I, la riduzione della produzione di ATP e la prevalenza della fissione mitocondriale rispetto alla fusione. Tale scoperta può aprire nuovi scenari per lo sviluppo di farmaci antitumorali. Poiché la carenza di glucosio si può riscontrare nell’ambiente in cui cresce e si sviluppa il tumore, tale condizione può essere sfruttata per potenziare l’azione di specifici agenti, come alcuni modulatori dell’OXPHOS. Infatti, l’inibizione delle funzioni mitocondriali in condizioni di deprivazione di glucosio potrebbe risultare letale per le cellule tumorali. In accordo, in questo lavoro viene mostrato che la deprivazione di glucosio e gli inibitori del Complesso I, come rotenone, piericidina A e capsaicina, hanno un effetto sinergico nell’indurre la morte delle cellule tumorali. Nello specifico, basse dosi d’inibitori del Complesso I, inefficaci sulle cellule normali e su cellule cresciute in alto glucosio, diventano citotossiche per le cellule tumorali cresciute in basso glucosio. L’effetto citotossico degli inibitori del Complesso I sulle cellule tumorali è ulteriormente e fortemente aumentato quando l’attività OXPHOS viene stimolata tramite il trattamento con FSK. Queste osservazioni dimostrano che la riattivazione della funzione mitocondriale associata alla deplezione di glucosio e al trattamento con basse dosi di inibitori del Complesso I riduce fortemente la sopravvivenza delle cellule tumorali e potrebbe quindi essere valutato come approccio terapeutico. Come indicato in precedenza, nelle cellule tumorali il glucosio è implicato in numerosi processi. L’analisi trascrittomica e proteomica di cellule murine K-ras-trasformate e della loro controparte normale mostra che la deprivazione di glucosio modula l’espressione di molti geni legati allo stress del reticolo endoplasmatico e all’Unfolded Protein Response (UPR). L’attivazione di tale risposta si osserva in entrambe le linee cellulari ma più fortemente nelle cellule trasformate, dove è associata anche alla morte cellulare. Infatti, la sua attenuazione tramite l’inibitore della traduzione proteica, cicloesimide, o lo chaperone chimico, 4-fenil-butirrato, protegge specificatamente le cellule trasformate dalla morte cellulare in basso glucosio. Anche l’inibizione della chinasi proapoptotica JNK, attivata a valle dell’UPR, previene specificatamente la morte delle cellule trasformate. Questa osservazione è in accordo col fatto che in basso glucosio le cellule trasformate mostrano una maggiore attivazione di JNK rispetto alle cellule normali. Inoltre, l’attivazione dell’UPR e la morte glucosio-dipendente delle cellule trasformate è completamente prevenuta dall’aggiunta nel terreno di coltura di un substrato dell’HBP, N-Acetyl-D-glucosammina, cosa che suggerisce una stretta relazione tra i due processi. È interessante notare che anche cellule umane esprimenti l’oncogene K-ras e caratterizzate da un fenotipo iperglicolitico mostrano simili effetti in seguito alla modulazione dell’UPR o dell’HBP. Quindi, la deprivazione di glucosio nelle cellule K-ras-trasformate può indurre un meccanismo di morte cellulare UPR-dipendente, attivato dall’eccessivo accumulo di proteine mal foldate, probabilmente come conseguenza della riduzione della N-glicosilazione delle proteine. La piena delucidazione di questa risposta potrebbe essere importante per progettare nuove strategie terapeutiche antitumorali. Oggi la nuova sfida della ricerca e della terapia antitumorale è il totale sradicamento del tumore, uccidendo anche le cellule staminali tumorali (cancer stem cells, CSCs). Considerando l’importante ruolo del metabolismo e della sua riprogrammazione nello sviluppo tumorale, la caratterizzazione del metabolismo delle CSCs può essere considerata un importante mezzo per lo sviluppo di nuove strategie antitumorali. Recentemente, è stata ottenuta la linea cellulare staminale di osteosarcoma umano, 3AB-OS. In questo lavoro di tesi ho svolto una prima caratterizzazione del suo profilo metabolico, paragonato a quello delle cellule tumorali MG63, da cui le cellule 3AB-OS sono state selezionate. Si è osservato che le cellule 3AB-OS dipendono più fortemente dalla glicolisi rispetto alle cellule MG63. Infatti, quando cresciute in presenza di galattosio e piruvato (substrati mitocondriali) le cellule 3AB-OS riducono maggiormente la propria capacità proliferativa rispetto alle cellule MG63. Esse risultano anche essere fortemente sensibili alla deprivazione di glucosio e al trattamento con inibitori della glicolisi mentre sono insensibili all’inibizione della catena respiratoria. Inoltre, diversamente dalle cellule MG63, le cellule 3AB-OS presentano principalmente mitocondri frammentati, in particolare in basso glucosio. Tutte queste osservazioni suggeriscono che il metabolismo energetico delle cellule 3AB-OS presenti caratteristiche paragonabili a quello delle cellule staminali normali e delle cellule tumorali caratterizzate da un metabolismo glicolitico. Può essere interessante notare che il profilo trascrizionale delle cellule 3AB-OS è simile a quello delle cellule K-ras-trasformate, confermando la similitudine tra le CSCs e le cellule tumorali glicolitiche. Quindi, alcune strategie sviluppate per il trattamento delle cellule tumorali glucosio-dipendenti potrebbero essere usate anche per trattare specifiche CSCs.

(2013). K-ras cancer cell fate under glucose deprivation is influenced by alteration of bioenergetic metabolism. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).

K-ras cancer cell fate under glucose deprivation is influenced by alteration of bioenergetic metabolism

PALORINI, ROBERTA
2013

Abstract

Several cancer cells, in order to generate ATP and sustain different anabolic processes, rely mainly on glycolysis instead of Oxidative Phosphorylation (OXPHOS). Thus, glucose assumes a critical role for cancer cell survival and proliferation. Moreover, through the penthose phospate pathway glucose leads to production of NADPH contributing to maintenance of cellular oxidative equilibrium. Besides, glucose can also enter Hexosamine Biosynthesis Pathway (HBP), sustaining lipid and protein N- and O-glycosylation that cover an important role in cancer development. Taking in consideration the essential role of glucose in cancer, one important anticancer therapeutic approach is to target its metabolism namely glycolysis and the other processes in which it is involved. On this regard, glucose deprivation and consequent analysis of cancer cell fate both at phenotypical and molecular level can be a useful strategy to unmask all mechanisms that participate to glucose-mediated cancer cell growth and survival. Such a strategy could be subsequently exploited to provide new targets and to set new anticancer therapies. Although there is evidence that tumors originate from cells with persistent defects in the mitochondrial respiratory system, inhibition of OXPHOS activity seems to be an adaptation to cancer metabolism reprogramming rather than a cause. In this scenario, reversible post-translational modifications of mitochondrial components could assume an important regulatory role. Among the most important post-translational modifications there is Ser/Thr phosphorylation and, on this regard, the protein kinase PKA has numerous mitochondrial targets being involved in the regulation of the biogenesis, the import and the activity of mitochondrial Complex I or IV as well as of mitochondrial morphology. Since it has been observed that oncogenic K-ras may lead to a depression of genes encoding for components of the cAMP/PKA signaling pathway, in K-ras-transformed cells the deregulation of cAMP/PKA pathway could cause OXPHOS depression and “glucose addiction” of cancer cells. In agreement with such a hypothesis, K-ras-transformed cells show lower PKA activity as compared to normal cells. Moreover, exogenous stimulation of PKA activity, achieved by Forskolin (FSK) treatment, protects mouse and human K-ras-transformed cells from apoptosis induced by glucose deprivation, by enhancing Complex I activity, intracellular ATP levels and mitochondrial fusion and by decreasing intracellular ROS levels. Worth noting, several of these effects are almost completely prevented by inhibition of PKA activity. Moreover, short time treatment with Mdivi-1, a molecule that favors mitochondrial fusion, strongly decreases the cellular ROS levels especially in transformed cells, indicating a close relationship between mitochondrial morphology and activity. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial Complex I decrease, reduction of ATP formation and prevalence of mitochondrial fission over fusion. Such a discovery can thereby open new approaches for the development of anticancer drugs. Given that glucose shortage is often encountered in the tumor microenvironment, it can be exploited to potentiate the action of specific agents, such as the mitochondrial OXPHOS activity modulators, that in condition of glucose deprivation could be lethal for cancer cells. Accordingly, it is shown that glucose deprivation and Complex I inhibitors, i.e., rotenone, piericidin A and capsaicin, synergize in inducing cancer cell death. In particular, low doses of Complex I inhibitors, ineffective on normal cells and on cells grown in high glucose, become specifically cytotoxic on cancer cells cultured in low glucose. Importantly, the cytotoxic effect of Complex I inhibitors is strongly enhanced when mitochondrial OXPHOS activity is stimulated by FSK. These findings demonstrate that the reactivation of the mitochondrial function associated with glucose depletion and low doses of mitochondrial Complex I inhibitors strongly affect cancer cell survival. This therapeutic approach might be valuable to eradicate cancer cells. As above indicated, glucose is implicated in numerous processes in cancer cells. Transcriptomic and proteomic analyses applied to mouse K-ras-transformed cells as compared to normal cells show that glucose deprivation modulates the expression of several genes linked to endoplasmic reticulum stress and the Unfolded Protein Response (UPR). The activation of such a response, as confirmed by mRNA and protein expression, is observed in both cell lines, but only in transformed cells is strictly associated to their death. In fact, its attenuation by protein translation inhibitor cycloheximide or chemical chaperone 4-Phenyl-butyrate specifically rescues transformed cells from death. Moreover, glucose deprivation-induced transformed cell death is also prevented by inhibition of an UPR downstream pro-apoptotic kinase, JNK, whose activation is observed specifically in transformed cells as compared to normal cells. Interestingly, UPR activation and death of transformed cells is completely prevented by addition of a specific HBP substrate, namely N-Acetyl-D-glucosamine, suggesting a strict relation between the two processes. Notably, also oncogenic K-ras expressing human glycolytic cells show similar effects after UPR modulating treatments. Thus, we show that glucose deprivation can induce an UPR-dependent transformed cell death mechanism, which is activated by harmful accumulation of unfolded proteins, probably as consequence of N-glycosylation protein reduction. The full elucidation of this response could be relevant to design new therapeutic strategies. Today the new challenge of anticancer research and therapy is the total eradication of the cancer, targeting cancer stem cells (CSCs). Considering the important role of metabolism and metabolic reprogramming in cancer development, also the definition of CSCs metabolism can be considered an important tool for future strategies targeting these cells. Recently, a human osteosarcoma 3AB-OS CSC-like line has been developed. Therefore we have decided to characterize its metabolic features as compared to the parental osteosarcoma MG63 cells, from which 3AB-OS cells were previously selected. 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, addition to the growth medium of galactose and pyruvate -mitochondrial specific substrates- instead of glucose markedly reduces 3AB-OS growth, as compared to MG63 cells. In line with these findings 3AB-OS cells, compared to MG63 cells, are strongly sensitive to glucose depletion, glycolysis inhibition and less sensitive to respiratory inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display mainly fragmented mitochondria, particularly in low glucose. Overall, these findings suggest that 3AB-OS energy metabolism is more similar either to normal stem cells or to cancer cells characterized by a glycolytic metabolism. Interestingly, the transcriptional profile of CSCs is similar to that of K-ras-transformed cells, confirming a possible similarity to glycolytic cancer cells. Therefore, some strategies developed for glucose addicted cancer cells could be used also to treat specific CSCs.
CHIARADONNA, FERDINANDO
K-ras cancer cells, glucose deprivation, bioenergetic metabolism
Cellule tumorali K-ras, deprivazione di glucosio, metabolismo bioenergetico
BIO/10 - BIOCHIMICA
English
7-feb-2013
BIOTECNOLOGIE INDUSTRIALI - 15R
25
2011/2012
Il lavoro di tesi è stato svolto presso il laboratorio di biochimica cellulare guidato dal dott. Ferdinando Chiaradonna (presso Università degli Studi di Milano - Bicocca).
open
(2013). K-ras cancer cell fate under glucose deprivation is influenced by alteration of bioenergetic metabolism. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/41975
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