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Loss of PDCD4 contributes to enhanced chemoresistance in Glioblastoma multiforme through de-repression of Bcl-xL translation.

Liwak U, Jordan LE, Von-Holt SD, Singh P, Hanson JE, Lorimer IA, Roncaroli F, Holcik M - Oncotarget (2013)

Bottom Line: Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival.We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability.Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Canada.

ABSTRACT
Glioblastoma multiforme (GBM) is the most common and aggressive form of tumor of the central nervous system. Despite significant efforts to improve treatments, patient survival rarely exceeds 18 months largely due to the highly chemoresistant nature of these tumors. Importantly, misregulation of the apoptotic machinery plays a key role in the development of drug resistance. We previously demonstrated that Bcl-xL, an important anti-apoptotic protein, is regulated at the level of translation by the tumor suppressor programmed cell death 4 (PDCD4). We report here a strong correlation between low expression of PDCD4 and high expression of Bcl-xL in adult de novo GBM, GBM tumor initiating cells, and established GBM cell lines. Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival. We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability. Finally, we show that direct inhibition of Bcl-xL by small molecule antagonist ABT-737 sensitizes GBM cells to doxorubicin. Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

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Patient GBM tumors show a correlation between low PDCD4 and high Bcl-xL levels(A) I – Hematoxylin-eosin stain of a representative case of glioblastoma multiforme (x10); II – diffuse Bcl-xL expression in neoplastic cells (immunoperoxidase x10); III – Granular cytoplasmic immunolabeling showing mitochondrial localization of Bcl-xL (immunoperoxidase x20); IV – PDCD4 is positive in endothelial cell and intratumoral lymphocytes (immunoperoxidase x20); V – Colorectal carcinoma as positive control for Bcl-xL (immunoperoxidase x20); VI – immunoreactions with omission of the primary antibody, (immunoperoxidase x20). (B) Dot plot representation of 50 cases of adult de novo GBM with respect to PDCD4 and Bcl-xL expression (Score 0 – expression between 0-5% was considered negative, Score 1 – expression between 5-50%, Score 2 – expression over 50%). More than 70% of Bcl-xL positive tumors are PDCD4 negative (Chi square test for trend, p=0.0469). (C) Kaplan-Meier curve showing a significant correlation between high Bcl-xL expression (score 1, 2) and progression (p=0.0187, Geham-Breslow-Wilcoxon test) and (D) significant correlation between high Bcl-xL expression (score 1, 2) and poor survival (p=0.0476, Geham-Breslow-Wilcoxon test).
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Figure 1: Patient GBM tumors show a correlation between low PDCD4 and high Bcl-xL levels(A) I – Hematoxylin-eosin stain of a representative case of glioblastoma multiforme (x10); II – diffuse Bcl-xL expression in neoplastic cells (immunoperoxidase x10); III – Granular cytoplasmic immunolabeling showing mitochondrial localization of Bcl-xL (immunoperoxidase x20); IV – PDCD4 is positive in endothelial cell and intratumoral lymphocytes (immunoperoxidase x20); V – Colorectal carcinoma as positive control for Bcl-xL (immunoperoxidase x20); VI – immunoreactions with omission of the primary antibody, (immunoperoxidase x20). (B) Dot plot representation of 50 cases of adult de novo GBM with respect to PDCD4 and Bcl-xL expression (Score 0 – expression between 0-5% was considered negative, Score 1 – expression between 5-50%, Score 2 – expression over 50%). More than 70% of Bcl-xL positive tumors are PDCD4 negative (Chi square test for trend, p=0.0469). (C) Kaplan-Meier curve showing a significant correlation between high Bcl-xL expression (score 1, 2) and progression (p=0.0187, Geham-Breslow-Wilcoxon test) and (D) significant correlation between high Bcl-xL expression (score 1, 2) and poor survival (p=0.0476, Geham-Breslow-Wilcoxon test).

Mentions: In order to study the relationship between Bcl-xL and PDCD4 expression in a clinical setting, we investigated with immunohistochemistry a cohort of 50 human de novo GBMs. Twenty-six GBMs were positive for Bcl-xL, where 15 of them showed expression in more than 50% of neoplastic cells (score 2) (Figure 1A, 1B) and 11 showed focal expression (score 1). Thirty cases did not show any detectable PDCD4. Interestingly, 18 cases with no PDCD4 showed Bcl-xL positive cells and 12 PDCD4 positive cases had no Bcl-xL. Immunopositivity for Bcl-xL was cytoplasmic and granular in keeping with its mitochondrial localization (Figure 1A). Bcl-xL immunolabelling was also found in reactive astrocytes, a few microglial cells and some neurons. Sixteen tumors showed PDCD4 nuclear and/or cytoplasmic expression but it was only limited to a minority of tumor cells (score 1). In all 50 lesions, PDCD4 was present in endothelial and inflammatory cells, including perinecrotic macrophages (Figure 1A). Six out of the seven recurrent cases demonstrated diffuse Bcl-xL expression that was more intense than in the primary tumor. No change in PDCD4 was seen in recurrent tumors compared to the primary lesion. Chi square test for trend (p=0.0469) of this cohort suggested that PDCD4 likely regulates Bcl-xL protein in human samples, therefore confirming our previous in vitro data. The distribution of Bcl-xL and PDCD4 positive cases is represented in Figure 1B.


Loss of PDCD4 contributes to enhanced chemoresistance in Glioblastoma multiforme through de-repression of Bcl-xL translation.

Liwak U, Jordan LE, Von-Holt SD, Singh P, Hanson JE, Lorimer IA, Roncaroli F, Holcik M - Oncotarget (2013)

Patient GBM tumors show a correlation between low PDCD4 and high Bcl-xL levels(A) I – Hematoxylin-eosin stain of a representative case of glioblastoma multiforme (x10); II – diffuse Bcl-xL expression in neoplastic cells (immunoperoxidase x10); III – Granular cytoplasmic immunolabeling showing mitochondrial localization of Bcl-xL (immunoperoxidase x20); IV – PDCD4 is positive in endothelial cell and intratumoral lymphocytes (immunoperoxidase x20); V – Colorectal carcinoma as positive control for Bcl-xL (immunoperoxidase x20); VI – immunoreactions with omission of the primary antibody, (immunoperoxidase x20). (B) Dot plot representation of 50 cases of adult de novo GBM with respect to PDCD4 and Bcl-xL expression (Score 0 – expression between 0-5% was considered negative, Score 1 – expression between 5-50%, Score 2 – expression over 50%). More than 70% of Bcl-xL positive tumors are PDCD4 negative (Chi square test for trend, p=0.0469). (C) Kaplan-Meier curve showing a significant correlation between high Bcl-xL expression (score 1, 2) and progression (p=0.0187, Geham-Breslow-Wilcoxon test) and (D) significant correlation between high Bcl-xL expression (score 1, 2) and poor survival (p=0.0476, Geham-Breslow-Wilcoxon test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3824522&req=5

Figure 1: Patient GBM tumors show a correlation between low PDCD4 and high Bcl-xL levels(A) I – Hematoxylin-eosin stain of a representative case of glioblastoma multiforme (x10); II – diffuse Bcl-xL expression in neoplastic cells (immunoperoxidase x10); III – Granular cytoplasmic immunolabeling showing mitochondrial localization of Bcl-xL (immunoperoxidase x20); IV – PDCD4 is positive in endothelial cell and intratumoral lymphocytes (immunoperoxidase x20); V – Colorectal carcinoma as positive control for Bcl-xL (immunoperoxidase x20); VI – immunoreactions with omission of the primary antibody, (immunoperoxidase x20). (B) Dot plot representation of 50 cases of adult de novo GBM with respect to PDCD4 and Bcl-xL expression (Score 0 – expression between 0-5% was considered negative, Score 1 – expression between 5-50%, Score 2 – expression over 50%). More than 70% of Bcl-xL positive tumors are PDCD4 negative (Chi square test for trend, p=0.0469). (C) Kaplan-Meier curve showing a significant correlation between high Bcl-xL expression (score 1, 2) and progression (p=0.0187, Geham-Breslow-Wilcoxon test) and (D) significant correlation between high Bcl-xL expression (score 1, 2) and poor survival (p=0.0476, Geham-Breslow-Wilcoxon test).
Mentions: In order to study the relationship between Bcl-xL and PDCD4 expression in a clinical setting, we investigated with immunohistochemistry a cohort of 50 human de novo GBMs. Twenty-six GBMs were positive for Bcl-xL, where 15 of them showed expression in more than 50% of neoplastic cells (score 2) (Figure 1A, 1B) and 11 showed focal expression (score 1). Thirty cases did not show any detectable PDCD4. Interestingly, 18 cases with no PDCD4 showed Bcl-xL positive cells and 12 PDCD4 positive cases had no Bcl-xL. Immunopositivity for Bcl-xL was cytoplasmic and granular in keeping with its mitochondrial localization (Figure 1A). Bcl-xL immunolabelling was also found in reactive astrocytes, a few microglial cells and some neurons. Sixteen tumors showed PDCD4 nuclear and/or cytoplasmic expression but it was only limited to a minority of tumor cells (score 1). In all 50 lesions, PDCD4 was present in endothelial and inflammatory cells, including perinecrotic macrophages (Figure 1A). Six out of the seven recurrent cases demonstrated diffuse Bcl-xL expression that was more intense than in the primary tumor. No change in PDCD4 was seen in recurrent tumors compared to the primary lesion. Chi square test for trend (p=0.0469) of this cohort suggested that PDCD4 likely regulates Bcl-xL protein in human samples, therefore confirming our previous in vitro data. The distribution of Bcl-xL and PDCD4 positive cases is represented in Figure 1B.

Bottom Line: Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival.We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability.Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Canada.

ABSTRACT
Glioblastoma multiforme (GBM) is the most common and aggressive form of tumor of the central nervous system. Despite significant efforts to improve treatments, patient survival rarely exceeds 18 months largely due to the highly chemoresistant nature of these tumors. Importantly, misregulation of the apoptotic machinery plays a key role in the development of drug resistance. We previously demonstrated that Bcl-xL, an important anti-apoptotic protein, is regulated at the level of translation by the tumor suppressor programmed cell death 4 (PDCD4). We report here a strong correlation between low expression of PDCD4 and high expression of Bcl-xL in adult de novo GBM, GBM tumor initiating cells, and established GBM cell lines. Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival. We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability. Finally, we show that direct inhibition of Bcl-xL by small molecule antagonist ABT-737 sensitizes GBM cells to doxorubicin. Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

Show MeSH
Related in: MedlinePlus