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Oncogenic KRAS sensitizes premalignant, but not malignant cells, to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway.

Conti A, Majorini MT, Elliott R, Ashworth A, Lord CJ, Cancelliere C, Bardelli A, Seneci P, Walczak H, Delia D, Lecis D - Oncotarget (2015)

Bottom Line: In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels.Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner.In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.

No MeSH data available.


Related in: MedlinePlus

Oncogenic KRAS increases sensitivity of HME cells to DNA-damaging agents and TRAIL(A) The parental human epithelial (HME) cell line and the isogenic cell lines with knock-in mutations in KRAS (G13D), PI3K (H1047R) and EGFR (delE746A750) were treated with varying doses of CPT alone (left panel) or in combination with 100 nM SM83 (right panel). Viabilities are shown after 24 h of treatment. (B) HME D13/+ cells were pre-incubated with DMSO, 50 μM z-VAD, 20 μM Nec-1 (left panel), 10 μg/ml Infliximab (IFX, middle panel) and 10 μg/ml Enbrel (right panel) for 1 h and subsequently treated with 100 nM SM83 and 1 μM CPT. Cell viability was determined after 24 h. (C, D) HME +/+ and HME D13/+ cells were mock treated and treated with 100 nM SM83, 1 μM CPT and with their combination for 6 h. Cells were lysed and subjected to western blot to detect the apoptosis markers cleaved PARP, caspase-3 and caspase-8 (C) and the SM targets cIAP1, cIAP2 and XIAP (D). Actin is the loading control, asterisks show the cleaved forms p17/p19 of caspase-3 and the pro-caspase p55/p57 forms of caspase-8, together with its cleaved forms p41/p43. One representative of two independent experiments is shown.
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Figure 1: Oncogenic KRAS increases sensitivity of HME cells to DNA-damaging agents and TRAIL(A) The parental human epithelial (HME) cell line and the isogenic cell lines with knock-in mutations in KRAS (G13D), PI3K (H1047R) and EGFR (delE746A750) were treated with varying doses of CPT alone (left panel) or in combination with 100 nM SM83 (right panel). Viabilities are shown after 24 h of treatment. (B) HME D13/+ cells were pre-incubated with DMSO, 50 μM z-VAD, 20 μM Nec-1 (left panel), 10 μg/ml Infliximab (IFX, middle panel) and 10 μg/ml Enbrel (right panel) for 1 h and subsequently treated with 100 nM SM83 and 1 μM CPT. Cell viability was determined after 24 h. (C, D) HME +/+ and HME D13/+ cells were mock treated and treated with 100 nM SM83, 1 μM CPT and with their combination for 6 h. Cells were lysed and subjected to western blot to detect the apoptosis markers cleaved PARP, caspase-3 and caspase-8 (C) and the SM targets cIAP1, cIAP2 and XIAP (D). Actin is the loading control, asterisks show the cleaved forms p17/p19 of caspase-3 and the pro-caspase p55/p57 forms of caspase-8, together with its cleaved forms p41/p43. One representative of two independent experiments is shown.

Mentions: As SMs are rarely effective in monotherapy, but sensitize cancer cells to other compounds, we searched for drugs whose cytotoxicity can be efficiently enhanced by SM83 using a high-throughput cell based screening approach. HeLa cells were exposed in vitro to SM83 and izTRAIL in addition to a combined library of about 3000 FDA-approved small molecule inhibitors and cell viability assessed (see Materials and Methods). Of the 3000 small molecule inhibitors assessed, we found that the topoisomerase I inhibitor camptothecin (CPT) most profoundly enhanced the cytotoxic effect of SM83 (Table 1). In addition to the enhancing effect of CPT, we also found that different formulations of CPT such as 10-hydroxycamptothecin also enhanced the effects of SM83, further confirming that CPT can be effectively combined with SMs and TRAIL. We then asked whether this combination is more cytotoxic in a specific genetic background and treated a panel of premalignant and cancer cell lines with izTRAIL, SM83 and CPT alone or in combination (data not shown). Viability tests showed that the immortalized human epithelial (HME) cell line bearing a KI G13D mutation in the KRAS gene (D13/+) is far more sensitive to SM83 plus CPT treatment compared to the parental HME or to HME carrying mutations activating PI3K and EGFR (Figure 1A). Moreover, HME D13/+ cells were more sensitive to izTRAIL alone or in combination with SM83 (Figure S1 upper panels), to the topoisomerase II inhibitor etoposide (ETO) and to neocarzinostatin (NCS), a DNA double strand break inducer (Figure S1 lower panel), suggesting a general enhanced sensitivity to cell death more than a specific mechanism favoring CPT-mediated death. Pre-treatment with pan-caspase inhibitor z-VAD strongly supports the idea that SM83/CPT treatment kills HME D13/+ cells through an apoptotic mechanism (Figure 1B left panel). In fact, the blocking of caspases resulted in almost complete protection from the treatment, while necroptosis inhibitor Necrostatin-1 (Nec-1) showed only a negligible effect. Importantly, as TNF is known to be a pivotal player in SM-mediated cell death, HME D13/+ were also pre-treated with the TNF-specific blockers Infliximab (Figure 1B middle panel) and Enbrel (Figure 1B right panel) which both remarkably rescued cells from the treatment, confirming the involvement of TNF in the SM83/CPT cell killing. Finally, by biochemical analysis we further confirmed that SM83 strongly increases the pro-apoptotic effect of CPT, as is evident from the substantial accumulation of cleaved PARP, caspase-8 and -3 (Figure 1C). Importantly, the altered sensitivity to treatment in cells with wild type or mutated KRAS did not stem from a diverse expression of the SM known targets cIAP1, cIAP2 and XIAP (Figure 1D), which are also depleted at the same level by SM83.


Oncogenic KRAS sensitizes premalignant, but not malignant cells, to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway.

Conti A, Majorini MT, Elliott R, Ashworth A, Lord CJ, Cancelliere C, Bardelli A, Seneci P, Walczak H, Delia D, Lecis D - Oncotarget (2015)

Oncogenic KRAS increases sensitivity of HME cells to DNA-damaging agents and TRAIL(A) The parental human epithelial (HME) cell line and the isogenic cell lines with knock-in mutations in KRAS (G13D), PI3K (H1047R) and EGFR (delE746A750) were treated with varying doses of CPT alone (left panel) or in combination with 100 nM SM83 (right panel). Viabilities are shown after 24 h of treatment. (B) HME D13/+ cells were pre-incubated with DMSO, 50 μM z-VAD, 20 μM Nec-1 (left panel), 10 μg/ml Infliximab (IFX, middle panel) and 10 μg/ml Enbrel (right panel) for 1 h and subsequently treated with 100 nM SM83 and 1 μM CPT. Cell viability was determined after 24 h. (C, D) HME +/+ and HME D13/+ cells were mock treated and treated with 100 nM SM83, 1 μM CPT and with their combination for 6 h. Cells were lysed and subjected to western blot to detect the apoptosis markers cleaved PARP, caspase-3 and caspase-8 (C) and the SM targets cIAP1, cIAP2 and XIAP (D). Actin is the loading control, asterisks show the cleaved forms p17/p19 of caspase-3 and the pro-caspase p55/p57 forms of caspase-8, together with its cleaved forms p41/p43. One representative of two independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Oncogenic KRAS increases sensitivity of HME cells to DNA-damaging agents and TRAIL(A) The parental human epithelial (HME) cell line and the isogenic cell lines with knock-in mutations in KRAS (G13D), PI3K (H1047R) and EGFR (delE746A750) were treated with varying doses of CPT alone (left panel) or in combination with 100 nM SM83 (right panel). Viabilities are shown after 24 h of treatment. (B) HME D13/+ cells were pre-incubated with DMSO, 50 μM z-VAD, 20 μM Nec-1 (left panel), 10 μg/ml Infliximab (IFX, middle panel) and 10 μg/ml Enbrel (right panel) for 1 h and subsequently treated with 100 nM SM83 and 1 μM CPT. Cell viability was determined after 24 h. (C, D) HME +/+ and HME D13/+ cells were mock treated and treated with 100 nM SM83, 1 μM CPT and with their combination for 6 h. Cells were lysed and subjected to western blot to detect the apoptosis markers cleaved PARP, caspase-3 and caspase-8 (C) and the SM targets cIAP1, cIAP2 and XIAP (D). Actin is the loading control, asterisks show the cleaved forms p17/p19 of caspase-3 and the pro-caspase p55/p57 forms of caspase-8, together with its cleaved forms p41/p43. One representative of two independent experiments is shown.
Mentions: As SMs are rarely effective in monotherapy, but sensitize cancer cells to other compounds, we searched for drugs whose cytotoxicity can be efficiently enhanced by SM83 using a high-throughput cell based screening approach. HeLa cells were exposed in vitro to SM83 and izTRAIL in addition to a combined library of about 3000 FDA-approved small molecule inhibitors and cell viability assessed (see Materials and Methods). Of the 3000 small molecule inhibitors assessed, we found that the topoisomerase I inhibitor camptothecin (CPT) most profoundly enhanced the cytotoxic effect of SM83 (Table 1). In addition to the enhancing effect of CPT, we also found that different formulations of CPT such as 10-hydroxycamptothecin also enhanced the effects of SM83, further confirming that CPT can be effectively combined with SMs and TRAIL. We then asked whether this combination is more cytotoxic in a specific genetic background and treated a panel of premalignant and cancer cell lines with izTRAIL, SM83 and CPT alone or in combination (data not shown). Viability tests showed that the immortalized human epithelial (HME) cell line bearing a KI G13D mutation in the KRAS gene (D13/+) is far more sensitive to SM83 plus CPT treatment compared to the parental HME or to HME carrying mutations activating PI3K and EGFR (Figure 1A). Moreover, HME D13/+ cells were more sensitive to izTRAIL alone or in combination with SM83 (Figure S1 upper panels), to the topoisomerase II inhibitor etoposide (ETO) and to neocarzinostatin (NCS), a DNA double strand break inducer (Figure S1 lower panel), suggesting a general enhanced sensitivity to cell death more than a specific mechanism favoring CPT-mediated death. Pre-treatment with pan-caspase inhibitor z-VAD strongly supports the idea that SM83/CPT treatment kills HME D13/+ cells through an apoptotic mechanism (Figure 1B left panel). In fact, the blocking of caspases resulted in almost complete protection from the treatment, while necroptosis inhibitor Necrostatin-1 (Nec-1) showed only a negligible effect. Importantly, as TNF is known to be a pivotal player in SM-mediated cell death, HME D13/+ were also pre-treated with the TNF-specific blockers Infliximab (Figure 1B middle panel) and Enbrel (Figure 1B right panel) which both remarkably rescued cells from the treatment, confirming the involvement of TNF in the SM83/CPT cell killing. Finally, by biochemical analysis we further confirmed that SM83 strongly increases the pro-apoptotic effect of CPT, as is evident from the substantial accumulation of cleaved PARP, caspase-8 and -3 (Figure 1C). Importantly, the altered sensitivity to treatment in cells with wild type or mutated KRAS did not stem from a diverse expression of the SM known targets cIAP1, cIAP2 and XIAP (Figure 1D), which are also depleted at the same level by SM83.

Bottom Line: In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels.Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner.In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.

No MeSH data available.


Related in: MedlinePlus