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Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas.

Oliva CR, Markert T, Gillespie GY, Griguer CE - Oncotarget (2015)

Bottom Line: Using The Cancer Genome Atlas database and a retrospective data set from patients with glioblastoma multiforme, we found that BMI1 expression levels positively correlated with COX4-1 expression and overall survival.We also demonstrate that COX4-1 attenuates mitochondrial reactive oxygen species (ROS) production, which is required for COX4-1-mediated effects on BMI1 expression and cell proliferation.Taken together, these findings identify a novel COX4-1-mitochondrial ROS axis, in which differential expression of COX4 isoforms regulates mitochondrial ROS production and controls BMI1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
Nuclear-encoded cytochrome c oxidase subunit 4 (COX4) is a key regulatory subunit of mammalian cytochrome c oxidase, and recent studies have demonstrated that COX4 isoform 1 (COX4-1) could have a role in glioma chemoresistance. The Polycomb complex protein BMI1 is a stem cell regulatory gene implicated in the pathogenesis of many aggressive cancers, including glioma. This study sought to determine if COX4 regulates BMI1 and modulates tumor cell proliferation. Using The Cancer Genome Atlas database and a retrospective data set from patients with glioblastoma multiforme, we found that BMI1 expression levels positively correlated with COX4-1 expression and overall survival. Whereas COX4-1 promoted cell growth by increasing BMI1 expression, COX4-2 inhibited cell growth even in cells overexpressing BMI1. We also demonstrate that COX4-1 attenuates mitochondrial reactive oxygen species (ROS) production, which is required for COX4-1-mediated effects on BMI1 expression and cell proliferation. Notably, mice bearing COX4-1-expressing glioma cell xenografts quickly developed invasive tumors characterized by the presence of multiple lesions positive for Ki-67, BMI1, and COX4-1, whereas mice bearing COX4-2-expressing xenografts rarely developed tumors by this point. COX4-1 also promoted the self-renewal of glioma stem-like cells, consistent with the reported role of BMI1 in stem cell growth. Taken together, these findings identify a novel COX4-1-mitochondrial ROS axis, in which differential expression of COX4 isoforms regulates mitochondrial ROS production and controls BMI1 expression.

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COX4-1 and BMI1 co-expression is required to promote cell proliferation(A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.
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Figure 4: COX4-1 and BMI1 co-expression is required to promote cell proliferation(A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.

Mentions: To gain insight into the mechanism by which COX4 isoform expression regulates tumor proliferation and phenotypic changes, we determined whether reduction of BMI1 levels affects the growth of glioma cells. Treatment of U251-TgCOX4-1 cells with PTC-209, a small-molecule BMI1 inhibitor [21], reduced BMI1 expression (Figure 4A) and cell proliferation (Figure 4B). To more directly establish a role for BMI1 in cell proliferation, we generated U251-TgCOX4-1 cells with BMI1 knockdown. A total of four different lentivirus-encoded shRNAs for BMI1 were used to knock down BMI1, with each shRNA yielding different results. Clone 1 shRNA-infected cells expressed BMI1 mRNA levels similar to the scramble-shRNA-control cells and showed similar rates of proliferation. Clone 2 shRNA-infected cells (<80% knockdown of BMI1) progressively lost the ability to grow in vitro, and cells expressing shRNA clones 3 and 4 (<40–60% knockdown of BMI1) displayed a 2-fold reduction in cell proliferation compared with cells expressing shRNA-control (Figure 4C–4E). To investigate the effect of BMI1 on the aggressiveness of COX4-2 glioma cells, U251 cells stably overexpressing BMI1 were established (Figure 4F inset). The proliferation rate of cells overexpressing BMI1 was 2.5-fold lower than that of control cells (p < 0.0001) (Figure 4F). Collectively, these data indicate that GBM cells require both COX4-1 and BMI1 expression to promote cell growth in vitro.


Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas.

Oliva CR, Markert T, Gillespie GY, Griguer CE - Oncotarget (2015)

COX4-1 and BMI1 co-expression is required to promote cell proliferation(A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414193&req=5

Figure 4: COX4-1 and BMI1 co-expression is required to promote cell proliferation(A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.
Mentions: To gain insight into the mechanism by which COX4 isoform expression regulates tumor proliferation and phenotypic changes, we determined whether reduction of BMI1 levels affects the growth of glioma cells. Treatment of U251-TgCOX4-1 cells with PTC-209, a small-molecule BMI1 inhibitor [21], reduced BMI1 expression (Figure 4A) and cell proliferation (Figure 4B). To more directly establish a role for BMI1 in cell proliferation, we generated U251-TgCOX4-1 cells with BMI1 knockdown. A total of four different lentivirus-encoded shRNAs for BMI1 were used to knock down BMI1, with each shRNA yielding different results. Clone 1 shRNA-infected cells expressed BMI1 mRNA levels similar to the scramble-shRNA-control cells and showed similar rates of proliferation. Clone 2 shRNA-infected cells (<80% knockdown of BMI1) progressively lost the ability to grow in vitro, and cells expressing shRNA clones 3 and 4 (<40–60% knockdown of BMI1) displayed a 2-fold reduction in cell proliferation compared with cells expressing shRNA-control (Figure 4C–4E). To investigate the effect of BMI1 on the aggressiveness of COX4-2 glioma cells, U251 cells stably overexpressing BMI1 were established (Figure 4F inset). The proliferation rate of cells overexpressing BMI1 was 2.5-fold lower than that of control cells (p < 0.0001) (Figure 4F). Collectively, these data indicate that GBM cells require both COX4-1 and BMI1 expression to promote cell growth in vitro.

Bottom Line: Using The Cancer Genome Atlas database and a retrospective data set from patients with glioblastoma multiforme, we found that BMI1 expression levels positively correlated with COX4-1 expression and overall survival.We also demonstrate that COX4-1 attenuates mitochondrial reactive oxygen species (ROS) production, which is required for COX4-1-mediated effects on BMI1 expression and cell proliferation.Taken together, these findings identify a novel COX4-1-mitochondrial ROS axis, in which differential expression of COX4 isoforms regulates mitochondrial ROS production and controls BMI1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT
Nuclear-encoded cytochrome c oxidase subunit 4 (COX4) is a key regulatory subunit of mammalian cytochrome c oxidase, and recent studies have demonstrated that COX4 isoform 1 (COX4-1) could have a role in glioma chemoresistance. The Polycomb complex protein BMI1 is a stem cell regulatory gene implicated in the pathogenesis of many aggressive cancers, including glioma. This study sought to determine if COX4 regulates BMI1 and modulates tumor cell proliferation. Using The Cancer Genome Atlas database and a retrospective data set from patients with glioblastoma multiforme, we found that BMI1 expression levels positively correlated with COX4-1 expression and overall survival. Whereas COX4-1 promoted cell growth by increasing BMI1 expression, COX4-2 inhibited cell growth even in cells overexpressing BMI1. We also demonstrate that COX4-1 attenuates mitochondrial reactive oxygen species (ROS) production, which is required for COX4-1-mediated effects on BMI1 expression and cell proliferation. Notably, mice bearing COX4-1-expressing glioma cell xenografts quickly developed invasive tumors characterized by the presence of multiple lesions positive for Ki-67, BMI1, and COX4-1, whereas mice bearing COX4-2-expressing xenografts rarely developed tumors by this point. COX4-1 also promoted the self-renewal of glioma stem-like cells, consistent with the reported role of BMI1 in stem cell growth. Taken together, these findings identify a novel COX4-1-mitochondrial ROS axis, in which differential expression of COX4 isoforms regulates mitochondrial ROS production and controls BMI1 expression.

Show MeSH
Related in: MedlinePlus