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Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis.

Goncharenko-Khaider N, Matte I, Lane D, Rancourt C, Piché A - Mol. Cancer (2012)

Bottom Line: Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression.In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, J1H 5N4, Canada.

ABSTRACT

Background: Ascites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.

Methods: Using human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.

Results: In the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.

Conclusions: These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

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ERK1/2 signaling regulates the Mcl-1 expression. (A) Immunoblot analysis of Akt, phospho-Akt, ERK1/2 and phospho-ERK1/2 in CaOV3 and OVCAR3 cells following the addition of 10% OVC508 ascites. (B) Expression levels of Akt, phospho-Akt and Mcl-1 in vehicle, Akt or control siRNA-transfected CaOV3 and OVCAR3 cells 24 h post-transfection in the presence or absence of OVC508 ascites. (C) Immunoblot analysis of ERK1/2, phospho-ERK1/2 and Mcl-1 in OVC508 ascites- and U0126-treated OVCAR3 and CaOV3 cells 24 h post treatement. (D) Real-time PCR analysis of Mcl-1 transcript levels in CaOV3 and OVCAR3 cells. Cells were treated with either ascites (OVC508), MEK1/2 inhibitor U0126 or Akt inhibitor LY294002 and RNA was extracted 4 h later. Results were standardized using primers of the housekeeping gene RPLPO. Results are expressed as fold change relative to basal levels observed in cells incubated in the absence of ascites. (E) Apoptosis was assessed 24 h following incubation with U0126 in the presence or absence of TRAIL 50 ng/ml for CaOV3 cells and 100 ng/ml for OVCAR3 cells. Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.001, ** P < 0.0001.
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Figure 3: ERK1/2 signaling regulates the Mcl-1 expression. (A) Immunoblot analysis of Akt, phospho-Akt, ERK1/2 and phospho-ERK1/2 in CaOV3 and OVCAR3 cells following the addition of 10% OVC508 ascites. (B) Expression levels of Akt, phospho-Akt and Mcl-1 in vehicle, Akt or control siRNA-transfected CaOV3 and OVCAR3 cells 24 h post-transfection in the presence or absence of OVC508 ascites. (C) Immunoblot analysis of ERK1/2, phospho-ERK1/2 and Mcl-1 in OVC508 ascites- and U0126-treated OVCAR3 and CaOV3 cells 24 h post treatement. (D) Real-time PCR analysis of Mcl-1 transcript levels in CaOV3 and OVCAR3 cells. Cells were treated with either ascites (OVC508), MEK1/2 inhibitor U0126 or Akt inhibitor LY294002 and RNA was extracted 4 h later. Results were standardized using primers of the housekeeping gene RPLPO. Results are expressed as fold change relative to basal levels observed in cells incubated in the absence of ascites. (E) Apoptosis was assessed 24 h following incubation with U0126 in the presence or absence of TRAIL 50 ng/ml for CaOV3 cells and 100 ng/ml for OVCAR3 cells. Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.001, ** P < 0.0001.

Mentions: Activation of both ERK1/2 and Akt pathways has been linked to the transcriptional regulation of Mcl-1[26,28-30,32,34]. Previous studies demonstrating Akt activation by ascites[13,17] prompted us to investigate whether Akt and ERK1/2 were involved in ascites-mediated upregulation of Mcl-1 expression. First, we examined the phosphorylation of Akt and ERK1/2 overtime and found that both Akt and ERK1/2 were activated by ascites (Figure3A and Additional file1: Figure S3). siRNA-mediated inhibition of Akt in both CaOV3 and OVCAR3 cells however did not altered ascites-mediated up-regulation of Mcl-1 expression (Figure3B). The chemical inhibitor of Akt LY294002 produced similar results (data not shown) suggesting that ascites-mediated Mcl-1 up-regulation is not dependent of Akt activation. In contrast, when ERK1/2 activation was inhibited by the specific MEK1/2 inhibitor U0126[44], ascites-mediated upregulation of Mcl-1 protein was substantially blocked in both CaOV3 and OVCAR3 cells (Figure3C). Consistent with these results, U0126 significantly blocked the transcriptional upregulation of Mcl-1 by ascites in CaOV3 and OVCAR3 cells (Figure3D). In contrast, the inhibition of Akt by LY294002 had no impact on ascites-mediated transcriptional upregulation of Mcl-1 in OC cells (Figure3D). Because Mcl-1 contributes to ascites-mediated protection from TRAIL-induced apoptosis, we examined whether ERK1/2 has a similar role. As expected, ERK1/2 inhibition by U0126 significantly blocked ascites-mediated protection from TRAIL-induced apoptosis (Figure3E). These data demonstrate that ERK1/2 activation upregulates Mcl-1 expression and contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.


Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis.

Goncharenko-Khaider N, Matte I, Lane D, Rancourt C, Piché A - Mol. Cancer (2012)

ERK1/2 signaling regulates the Mcl-1 expression. (A) Immunoblot analysis of Akt, phospho-Akt, ERK1/2 and phospho-ERK1/2 in CaOV3 and OVCAR3 cells following the addition of 10% OVC508 ascites. (B) Expression levels of Akt, phospho-Akt and Mcl-1 in vehicle, Akt or control siRNA-transfected CaOV3 and OVCAR3 cells 24 h post-transfection in the presence or absence of OVC508 ascites. (C) Immunoblot analysis of ERK1/2, phospho-ERK1/2 and Mcl-1 in OVC508 ascites- and U0126-treated OVCAR3 and CaOV3 cells 24 h post treatement. (D) Real-time PCR analysis of Mcl-1 transcript levels in CaOV3 and OVCAR3 cells. Cells were treated with either ascites (OVC508), MEK1/2 inhibitor U0126 or Akt inhibitor LY294002 and RNA was extracted 4 h later. Results were standardized using primers of the housekeeping gene RPLPO. Results are expressed as fold change relative to basal levels observed in cells incubated in the absence of ascites. (E) Apoptosis was assessed 24 h following incubation with U0126 in the presence or absence of TRAIL 50 ng/ml for CaOV3 cells and 100 ng/ml for OVCAR3 cells. Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.001, ** P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: ERK1/2 signaling regulates the Mcl-1 expression. (A) Immunoblot analysis of Akt, phospho-Akt, ERK1/2 and phospho-ERK1/2 in CaOV3 and OVCAR3 cells following the addition of 10% OVC508 ascites. (B) Expression levels of Akt, phospho-Akt and Mcl-1 in vehicle, Akt or control siRNA-transfected CaOV3 and OVCAR3 cells 24 h post-transfection in the presence or absence of OVC508 ascites. (C) Immunoblot analysis of ERK1/2, phospho-ERK1/2 and Mcl-1 in OVC508 ascites- and U0126-treated OVCAR3 and CaOV3 cells 24 h post treatement. (D) Real-time PCR analysis of Mcl-1 transcript levels in CaOV3 and OVCAR3 cells. Cells were treated with either ascites (OVC508), MEK1/2 inhibitor U0126 or Akt inhibitor LY294002 and RNA was extracted 4 h later. Results were standardized using primers of the housekeeping gene RPLPO. Results are expressed as fold change relative to basal levels observed in cells incubated in the absence of ascites. (E) Apoptosis was assessed 24 h following incubation with U0126 in the presence or absence of TRAIL 50 ng/ml for CaOV3 cells and 100 ng/ml for OVCAR3 cells. Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.001, ** P < 0.0001.
Mentions: Activation of both ERK1/2 and Akt pathways has been linked to the transcriptional regulation of Mcl-1[26,28-30,32,34]. Previous studies demonstrating Akt activation by ascites[13,17] prompted us to investigate whether Akt and ERK1/2 were involved in ascites-mediated upregulation of Mcl-1 expression. First, we examined the phosphorylation of Akt and ERK1/2 overtime and found that both Akt and ERK1/2 were activated by ascites (Figure3A and Additional file1: Figure S3). siRNA-mediated inhibition of Akt in both CaOV3 and OVCAR3 cells however did not altered ascites-mediated up-regulation of Mcl-1 expression (Figure3B). The chemical inhibitor of Akt LY294002 produced similar results (data not shown) suggesting that ascites-mediated Mcl-1 up-regulation is not dependent of Akt activation. In contrast, when ERK1/2 activation was inhibited by the specific MEK1/2 inhibitor U0126[44], ascites-mediated upregulation of Mcl-1 protein was substantially blocked in both CaOV3 and OVCAR3 cells (Figure3C). Consistent with these results, U0126 significantly blocked the transcriptional upregulation of Mcl-1 by ascites in CaOV3 and OVCAR3 cells (Figure3D). In contrast, the inhibition of Akt by LY294002 had no impact on ascites-mediated transcriptional upregulation of Mcl-1 in OC cells (Figure3D). Because Mcl-1 contributes to ascites-mediated protection from TRAIL-induced apoptosis, we examined whether ERK1/2 has a similar role. As expected, ERK1/2 inhibition by U0126 significantly blocked ascites-mediated protection from TRAIL-induced apoptosis (Figure3E). These data demonstrate that ERK1/2 activation upregulates Mcl-1 expression and contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.

Bottom Line: Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression.In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, J1H 5N4, Canada.

ABSTRACT

Background: Ascites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.

Methods: Using human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.

Results: In the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.

Conclusions: These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

Show MeSH
Related in: MedlinePlus