A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts

Bossù, Paola; Vanoni, Marco; Wanke,; Mp, Cesaroni; Tropea, Franco; Melillo, G.; Asti, Cinzia; Porzio, S.; Ruggiero, Paolo; Cioccio, Vito Di; Maurizi, Giovanni; A, Ciabini; Alberghina, Lilia

doi:10.1038/sj.onc.1203539

Cited by 27 publications

(28 citation statements)

References 47 publications

(43 reference statements)

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“…We used NIH-3T3 fibroblasts harboring an oncogenic K-RAS gene (G12V; transformed) compared with parental immortalized NIH-3T3 cells (normal) and a human breast cancer cell line, MDA-MB-231, carrying an oncogenic K-RAS gene (G13D) as well. 15 It is worth mentioning that both transformed cell lines, displaying a typical Warburg effect, are strongly sensitive to glucose exhaustion as, in such a condition, they show a strong increase in cell death. 10, 16 …”

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confidence: 99%

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response

et al. 2013

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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 starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response

et al. 2013

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“…Overexpression of a dominant negative Ras-specific GEF protein has been reported to phenotypically revert several phenotypes in K-ras transformed murine fibroblasts, including Ras-GTP level, morphology, anchorage independent growth, reduction of Ras-dependent tumor formation in nude mice, glucose dependence and mitochondrial dysfunction [15]–[17]. As shown in Figure 4, K-ras transformed cells expressing the dominant-negative GEF showed a complete reversion of all the phenotypes observed in transformed cells grown in all the three glutamine conditions.…”

Section: Resultsmentioning

confidence: 99%

“…As a result, K-ras transformed NIH3T3 cells are remarkably sensitive to glucose deprivation [15], a condition in which they stop growth and die. Transformation-related phenotypes of K-ras transformed cell lines can be rescued by expression of a dominant-negative guanine nucleotide exchange factor (GEF-DN) [15]–[17].…”

Section: Introductionmentioning

confidence: 99%

Glutamine Deprivation Induces Abortive S-Phase Rescued by Deoxyribonucleotides in K-Ras Transformed Fibroblasts

et al. 2009

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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 G1 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 G1 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.

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“…In this regard, expression of mutant K-Ras in mice is sufficient to cause transformation and tumor formation (7, 8). Effective abrogation of K-Ras activity reverts malignant cells to a non-malignant phenotype (9–12). Therefore, cancer therapy targeting K-Ras would be expected to produce a clinical benefit.…”

Section: Introductionmentioning

confidence: 99%

Requirement of the NF-κB Subunit p65/RelA for K-Ras–Induced Lung Tumorigenesis

et al. 2010

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K-Ras-induced lung cancer is a very common disease, for which there are currently no effective therapies. Because therapy directly targeting the activity of oncogenic Ras has been unsuccessful, a different approach for novel therapy design is to identify critical Ras downstream oncogenic targets. Given that oncogenic Ras proteins activate the transcription factor NF-κB, and the importance of NF-κB in oncogenesis, we hypothesized that NF-κB would be an important K-Ras target in lung cancer. To address this hypothesis, we generated a NF-κB-EGFP reporter mouse model of K-Ras-induced lung cancer and determined that K-Ras activates NF-κB in lung tumors in situ. Furthermore, a mouse model was generated where activation of oncogenic K-Ras in lung cells was coupled with inactivation of the NF-κB subunit p65/RelA. In this model, deletion of p65/RelA reduces the number of K-Ras-induced lung tumors both in the presence and in the absence of the tumor suppressor p53. Lung tumors with loss of p65/RelA have higher numbers of apoptotic cells, reduced spread, and lower grade. Using lung cell lines expressing oncogenic K-Ras, we show that NF-κB is activated in these cells in a K-Ras-dependent manner and that NF-κB activation by K-Ras requires inhibitor of κB kinase β (IKKβ) kinase activity. Taken together, these results show the importance of the NF-κB subunit p65/RelA in K-Ras-induced lung transformation and identify IKKβ as a potential therapeutic target for K-Ras-induced lung cancer. Cancer Res; 70(9); 3537-46. ©2010 AACR.

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A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts

Cited by 27 publications

References 47 publications

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response

Glutamine Deprivation Induces Abortive S-Phase Rescued by Deoxyribonucleotides in K-Ras Transformed Fibroblasts

Requirement of the NF-κB Subunit p65/RelA for K-Ras–Induced Lung Tumorigenesis

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