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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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Mcl-1 inhibition abrogates the prosurvival activity of ascites. (A) The effect of ascites on CaOV3 cell survival was assessed by long term assays where cells were exposed to TRAIL (50 ng/ml) for 24 h in the absence (control) or presence of ascites (10%). After 14 days colonies were counted and expressed as percentage of colonies obtained in the absence of TRAIL. (B) The percentage of apoptosis was measured as percentage of hypodiploid cells assessed by flow cytometry analysis during exposure to TRAIL (50 ng/ml for CaOV3; 100 ng/ml for OVCAR3) for 24 h in the presence or absence of OVC508 ascites. (C) CaOV3 and OVCAR3 cells were treated with a similar concentration of TRAIL (50 ng/ml) and apoptosis was assessed by flow cytometry 24 h later. (D) CaOV3 and (E) OVCAR3 cells were transfected with Mcl-1 or control siRNA in the presence or absence of OVC508 ascites. Mcl-1 knockdown was confirmed by immunoblot analysis 24 h after transfection and ERK1/2 was used as a loading control. Apoptosis was assessed 24 h following siRNA transfection in the presence or absence of TRAIL (50 ng/ml). Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.01, ** P < 0.001, *** P < 0.0001.
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Figure 2: Mcl-1 inhibition abrogates the prosurvival activity of ascites. (A) The effect of ascites on CaOV3 cell survival was assessed by long term assays where cells were exposed to TRAIL (50 ng/ml) for 24 h in the absence (control) or presence of ascites (10%). After 14 days colonies were counted and expressed as percentage of colonies obtained in the absence of TRAIL. (B) The percentage of apoptosis was measured as percentage of hypodiploid cells assessed by flow cytometry analysis during exposure to TRAIL (50 ng/ml for CaOV3; 100 ng/ml for OVCAR3) for 24 h in the presence or absence of OVC508 ascites. (C) CaOV3 and OVCAR3 cells were treated with a similar concentration of TRAIL (50 ng/ml) and apoptosis was assessed by flow cytometry 24 h later. (D) CaOV3 and (E) OVCAR3 cells were transfected with Mcl-1 or control siRNA in the presence or absence of OVC508 ascites. Mcl-1 knockdown was confirmed by immunoblot analysis 24 h after transfection and ERK1/2 was used as a loading control. Apoptosis was assessed 24 h following siRNA transfection in the presence or absence of TRAIL (50 ng/ml). Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.01, ** P < 0.001, *** P < 0.0001.

Mentions: Given its antiapoptotic activity, Mcl-1 could contribute to ascites-induced attenuation of TRAIL-induced apoptosis. Thus, we investigated whether Mcl-1 inhibition can alter the prosurvival activity of OC ascites. First, CaOV3 cells were incubated with ascites in the presence or absence of TRAIL (50 ng/ml) for 24 h. Long term cell survival was assessed by determining the fraction of surviving colonies after two weeks. As shown in Figure2A, the addition of OVC508 or OVC509 ascites to CaOV3 cells significantly (P < 0.0001) enhanced the fraction of survival cells. When apoptosis was determined by measuring the sub-G1 DNA content for CaOV3 and OVCAR3 cells incubated with ascites, we observed a 38% to 48% decreased of TRAIL-induced apoptosis (P < 0.001) confirming that ascites attenuate TRAIL-mediated cytotoxicity (Figure2B). These data confirmed that pretreatment with ascites attenuates TRAIL-induced apoptosis in OC cells.


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)

Mcl-1 inhibition abrogates the prosurvival activity of ascites. (A) The effect of ascites on CaOV3 cell survival was assessed by long term assays where cells were exposed to TRAIL (50 ng/ml) for 24 h in the absence (control) or presence of ascites (10%). After 14 days colonies were counted and expressed as percentage of colonies obtained in the absence of TRAIL. (B) The percentage of apoptosis was measured as percentage of hypodiploid cells assessed by flow cytometry analysis during exposure to TRAIL (50 ng/ml for CaOV3; 100 ng/ml for OVCAR3) for 24 h in the presence or absence of OVC508 ascites. (C) CaOV3 and OVCAR3 cells were treated with a similar concentration of TRAIL (50 ng/ml) and apoptosis was assessed by flow cytometry 24 h later. (D) CaOV3 and (E) OVCAR3 cells were transfected with Mcl-1 or control siRNA in the presence or absence of OVC508 ascites. Mcl-1 knockdown was confirmed by immunoblot analysis 24 h after transfection and ERK1/2 was used as a loading control. Apoptosis was assessed 24 h following siRNA transfection in the presence or absence of TRAIL (50 ng/ml). Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.01, ** P < 0.001, *** P < 0.0001.
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Related In: Results  -  Collection

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Figure 2: Mcl-1 inhibition abrogates the prosurvival activity of ascites. (A) The effect of ascites on CaOV3 cell survival was assessed by long term assays where cells were exposed to TRAIL (50 ng/ml) for 24 h in the absence (control) or presence of ascites (10%). After 14 days colonies were counted and expressed as percentage of colonies obtained in the absence of TRAIL. (B) The percentage of apoptosis was measured as percentage of hypodiploid cells assessed by flow cytometry analysis during exposure to TRAIL (50 ng/ml for CaOV3; 100 ng/ml for OVCAR3) for 24 h in the presence or absence of OVC508 ascites. (C) CaOV3 and OVCAR3 cells were treated with a similar concentration of TRAIL (50 ng/ml) and apoptosis was assessed by flow cytometry 24 h later. (D) CaOV3 and (E) OVCAR3 cells were transfected with Mcl-1 or control siRNA in the presence or absence of OVC508 ascites. Mcl-1 knockdown was confirmed by immunoblot analysis 24 h after transfection and ERK1/2 was used as a loading control. Apoptosis was assessed 24 h following siRNA transfection in the presence or absence of TRAIL (50 ng/ml). Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.01, ** P < 0.001, *** P < 0.0001.
Mentions: Given its antiapoptotic activity, Mcl-1 could contribute to ascites-induced attenuation of TRAIL-induced apoptosis. Thus, we investigated whether Mcl-1 inhibition can alter the prosurvival activity of OC ascites. First, CaOV3 cells were incubated with ascites in the presence or absence of TRAIL (50 ng/ml) for 24 h. Long term cell survival was assessed by determining the fraction of surviving colonies after two weeks. As shown in Figure2A, the addition of OVC508 or OVC509 ascites to CaOV3 cells significantly (P < 0.0001) enhanced the fraction of survival cells. When apoptosis was determined by measuring the sub-G1 DNA content for CaOV3 and OVCAR3 cells incubated with ascites, we observed a 38% to 48% decreased of TRAIL-induced apoptosis (P < 0.001) confirming that ascites attenuate TRAIL-mediated cytotoxicity (Figure2B). These data confirmed that pretreatment with ascites attenuates TRAIL-induced apoptosis in OC cells.

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