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Glucose regulates protein catabolism in ras-transformed fibroblasts through a lysosomal-dependent proteolytic pathway.

ABSTRACT: Transformed cells are exposed to heterogeneous microenvironments, including low D-glucose (Glc) concentrations inside tumours. The regulation of protein turnover is commonly impaired in many types of transformed cells, but the role of Glc in this regulation is unknown. In the present study we demonstrate that Glc controls protein turnover in ras-transformed fibroblasts (KBALB). The regulation by Glc of protein breakdown was correlated with modifications in the levels of lysosomal cathepsins B, L and D, while autophagic sequestration and non-lysosomal proteolytic systems (m- and mu-calpains and the zeta-subunit of the proteasome) remained unaffected. Lactacystin, a selective inhibitor of the proteasome, depressed proteolysis, but did not prevent its regulation by Glc. The sole inhibition of the cysteine endopeptidases (cathepsins B and L, and calpains) by E-64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] was also not sufficient to alter the effect of Glc on proteolysis. The Glc-dependent increase in proteolysis was, however, prevented after optimal inhibition of lysosomal cysteine and aspartic endopeptidases by methylamine. We conclude that, in transformed cells, Glc plays a critical role in the regulation of protein turnover and that the lysosomal proteolytic capacity is mainly responsible for the control of intracellular proteolysis by Glc.

SUBMITTER: Tournu C

PROVIDER: S-EPMC1221949 | biostudies-other | 2001 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundOncogene activation plays a role in metabolic reprogramming of cancer cells. We have previously shown that K-ras transformed fibroblasts have a stronger dependence on glycolysis and a reduced oxidative phosphorylation ability as compared to their normal counterparts. Another metabolic adaptation of cancer cells, that has long been established, is their propensity to exhibit increased glutamine consumption, although the effects induced by glutamine deprivation on cancer cells are still controversial.Methodology and principal findingsHere, by using nutritional perturbations and molecular physiology, we show that reduction or complete depletion of glutamine availability in K-ras transformed fibroblasts causes a strong decrease of proliferation ability and a slower re-entry of synchronized cells into the cell cycle. The reduced proliferation is accompanied by sustained expression of cyclin D and E, abortive S phase entrance and is dependent on Ras signalling deregulation, since it is rescued by expression of a dominant negative guanine nucleotide exchange factor. The growth potential of transformed cells as well as the ability to execute the G(1) to S transition is restored by adding the four deoxyribonucleotides, indicating that the arrest of proliferation of K-ras transformed cells induced by glutamine depletion is largely due to a reduced supply of DNA in the presence of signalling pathways promoting G(1) to S transition.Conclusions and significanceOur results suggest that the differential effects of glutamine and glucose on cell viability are not a property of the transformed phenotype per se, but rather depend on the specific pathway being activated in transformation. For instance, myc-overexpressing cells have been reported to die under glutamine depletion and not under glucose shortage, while the opposite holds for ras-transformed fibroblasts as shown in this paper. These different responses of transformed cells to nutritional stress should be taken into account when designing anti-cancer therapies that aim to exploit metabolic differences between normal and transformed cells.

Project description:Cancer cells, which use more glucose than normal cells and accumulate extracellular lactate even under normoxic conditions (Warburg effect), have been reported to undergo cell death under glucose deprivation, whereas normal cells remain viable. As it may be relevant to exploit the molecular mechanisms underlying this biological response to achieve new cancer therapies, in this paper we sought to identify them by using transcriptome and proteome analysis applied to an established glucose-addicted cellular model of transformation, namely, murine NIH-3T3 fibroblasts harboring an oncogenic K-RAS gene, compared with parental cells. Noteworthy is that the analyses performed in high- and low-glucose cultures indicate that reduction of glucose availability induces, especially in transformed cells, a significant increase in the expression of several unfolded protein response (UPR) hallmark genes. We show that this response is strictly associated with transformed cell death, given that its attenuation, by reducing protein translation or by increasing cell protein folding capacity, preserves the survival of transformed cells. Such an effect is also observed by inhibiting c-Jun NH2-terminal kinase, a pro-apoptotic signaling mediator set downstream of UPR. Strikingly, addition of N-acetyl-D-glucosamine, a specific substrate for the hexosamine biosynthesis pathway (HBP), to glucose-depleted cells completely prevents transformed cell death, stressing the important role of glucose in HBP fuelling to ensure UPR attenuation and increased cell survival. Interestingly, these results have been fully recognized in a human model of breast cancer, MDA-MB-231 cells. In conclusion, we show that glucose deprivation, leading to harmful accumulation of unfolded proteins in consequence of a reduction of protein glycosylation, induces a UPR-dependent cell death mechanism. These findings may open the way for new therapeutic strategies to specifically kill glycolytic cancer cells.

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Glucose regulates protein catabolism in ras-transformed fibroblasts through a lysosomal-dependent proteolytic pathway.

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