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Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival.

Choudhry H, Albukhari A, Morotti M, Haider S, Moralli D, Smythies J, Schödel J, Green CM, Camps C, Buffa F, Ratcliffe P, Ragoussis J, Harris AL, Mole DR - Oncogene (2014)

Bottom Line: Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing.Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis.Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.

ABSTRACT
Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

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Hypoxic induction of NEAT1 accelerates tumor cell proliferation, inhibits apoptosis and is associated with adverse clinical outcome in patients with breast cancer(A) Cell proliferation rates (normalized to control ASO), (B) Colony formation rate and (C) Annexin V staining for normoxic and hypoxic MCF-7 cells treated with either control ASO or NEAT1 ASO showing reduced proliferation, reduced colony formation and increased apoptosis following NEAT1 depletion. Each experiment was performed with three biological replicates (*P<0.05, **p≤0.009, ***p≤0.0009, Student’s t-test). (D) Kaplan-Meier plot for 2000 breast cancer patients from the Metabric trial stratified according to expression of NEAT1 mRNA (above versus below median) showing that high tumor NEAT1 levels are associated with increased patient mortality.
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Figure 5: Hypoxic induction of NEAT1 accelerates tumor cell proliferation, inhibits apoptosis and is associated with adverse clinical outcome in patients with breast cancer(A) Cell proliferation rates (normalized to control ASO), (B) Colony formation rate and (C) Annexin V staining for normoxic and hypoxic MCF-7 cells treated with either control ASO or NEAT1 ASO showing reduced proliferation, reduced colony formation and increased apoptosis following NEAT1 depletion. Each experiment was performed with three biological replicates (*P<0.05, **p≤0.009, ***p≤0.0009, Student’s t-test). (D) Kaplan-Meier plot for 2000 breast cancer patients from the Metabric trial stratified according to expression of NEAT1 mRNA (above versus below median) showing that high tumor NEAT1 levels are associated with increased patient mortality.

Mentions: We first examined the effects of NEAT1 on cell proliferation and colony formation in normoxia and in hypoxia. MCF-7 cells were pre-treated with either control or NEAT1 ASOs for 48 hours prior to incubation in either normoxia or 1% hypoxia for a further 24 hours and cell proliferation rates were then determined. Suppression of NEAT1 reduced cell proliferation rates in both normoxia and hypoxia (Figure 5A). However, the effect of NEAT1 suppression was greater in hypoxia indicating that NEAT1 has a larger effect on cell proliferation in hypoxia than it does in normoxia. Suppression of NEAT1 also reduced colony formation in both normoxia and hypoxia (Figure 5B). Again the effect of NEAT1 suppression was larger in hypoxia, when NEAT1 levels are higher, than it was in normoxia. Similar results were observed in ZR-75-1 cells (Supplemental Figures 7A and 7B).


Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival.

Choudhry H, Albukhari A, Morotti M, Haider S, Moralli D, Smythies J, Schödel J, Green CM, Camps C, Buffa F, Ratcliffe P, Ragoussis J, Harris AL, Mole DR - Oncogene (2014)

Hypoxic induction of NEAT1 accelerates tumor cell proliferation, inhibits apoptosis and is associated with adverse clinical outcome in patients with breast cancer(A) Cell proliferation rates (normalized to control ASO), (B) Colony formation rate and (C) Annexin V staining for normoxic and hypoxic MCF-7 cells treated with either control ASO or NEAT1 ASO showing reduced proliferation, reduced colony formation and increased apoptosis following NEAT1 depletion. Each experiment was performed with three biological replicates (*P<0.05, **p≤0.009, ***p≤0.0009, Student’s t-test). (D) Kaplan-Meier plot for 2000 breast cancer patients from the Metabric trial stratified according to expression of NEAT1 mRNA (above versus below median) showing that high tumor NEAT1 levels are associated with increased patient mortality.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4430310&req=5

Figure 5: Hypoxic induction of NEAT1 accelerates tumor cell proliferation, inhibits apoptosis and is associated with adverse clinical outcome in patients with breast cancer(A) Cell proliferation rates (normalized to control ASO), (B) Colony formation rate and (C) Annexin V staining for normoxic and hypoxic MCF-7 cells treated with either control ASO or NEAT1 ASO showing reduced proliferation, reduced colony formation and increased apoptosis following NEAT1 depletion. Each experiment was performed with three biological replicates (*P<0.05, **p≤0.009, ***p≤0.0009, Student’s t-test). (D) Kaplan-Meier plot for 2000 breast cancer patients from the Metabric trial stratified according to expression of NEAT1 mRNA (above versus below median) showing that high tumor NEAT1 levels are associated with increased patient mortality.
Mentions: We first examined the effects of NEAT1 on cell proliferation and colony formation in normoxia and in hypoxia. MCF-7 cells were pre-treated with either control or NEAT1 ASOs for 48 hours prior to incubation in either normoxia or 1% hypoxia for a further 24 hours and cell proliferation rates were then determined. Suppression of NEAT1 reduced cell proliferation rates in both normoxia and hypoxia (Figure 5A). However, the effect of NEAT1 suppression was greater in hypoxia indicating that NEAT1 has a larger effect on cell proliferation in hypoxia than it does in normoxia. Suppression of NEAT1 also reduced colony formation in both normoxia and hypoxia (Figure 5B). Again the effect of NEAT1 suppression was larger in hypoxia, when NEAT1 levels are higher, than it was in normoxia. Similar results were observed in ZR-75-1 cells (Supplemental Figures 7A and 7B).

Bottom Line: Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing.Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis.Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.

ABSTRACT
Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

Show MeSH
Related in: MedlinePlus