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Epigenetic regulation of glucose transporters in non-small cell lung cancer.

O'Byrne KJ, Baird AM, Kilmartin L, Leonard J, Sacevich C, Gray SG - Cancers (Basel) (2011)

Bottom Line: Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect.Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes.Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James´s Hospital, Dublin 8, Ireland. sgray@stjames.ie.

ABSTRACT
Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy.

No MeSH data available.


Related in: MedlinePlus

Direct chromatin remodeling of the Glut-4 promoter. (a) Pre-treatment with cycloheximide is unable to prevent TSA mediated induction of Glut-4 indicating that de novo protein synthesis is not required for activation that induction is via direct chromatin remodeling. (b) A chromatin immunoprecipitation (ChIP) assay demonstrates that TSA treatment results in an increase in the acetylation of histone H3 and H4 at the Glut-4 promoter. A549 cells were cultured in the presence or absence of TSA (250 ng/mL) for a period of 16 h. Subsequently a ChIP assay was performed using the following antibodies; pan acetylated histone H3 (Ac H3) and H4 (AcH4), histone H3 acetylated at lysine 9 and 14 (H3K9/K14Ac), histone H3 acetylated at lysine 9 (H3K9Ac), histone H3 tri-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 4 (H3K4me2), histone H3 mono-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 9 (H3K9me2). Input represents a positive control consisting of 1/10th of the original fixed chromatin prior to immunoprecipitation as recommended by the manufacturer (Diagenode). A no-antibody control was included to test for non-specific carriage of DNA with histones. (UT, untreated; CHX, cycloheximide; TSA, Trichostatin A).
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f4-cancers-03-01550: Direct chromatin remodeling of the Glut-4 promoter. (a) Pre-treatment with cycloheximide is unable to prevent TSA mediated induction of Glut-4 indicating that de novo protein synthesis is not required for activation that induction is via direct chromatin remodeling. (b) A chromatin immunoprecipitation (ChIP) assay demonstrates that TSA treatment results in an increase in the acetylation of histone H3 and H4 at the Glut-4 promoter. A549 cells were cultured in the presence or absence of TSA (250 ng/mL) for a period of 16 h. Subsequently a ChIP assay was performed using the following antibodies; pan acetylated histone H3 (Ac H3) and H4 (AcH4), histone H3 acetylated at lysine 9 and 14 (H3K9/K14Ac), histone H3 acetylated at lysine 9 (H3K9Ac), histone H3 tri-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 4 (H3K4me2), histone H3 mono-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 9 (H3K9me2). Input represents a positive control consisting of 1/10th of the original fixed chromatin prior to immunoprecipitation as recommended by the manufacturer (Diagenode). A no-antibody control was included to test for non-specific carriage of DNA with histones. (UT, untreated; CHX, cycloheximide; TSA, Trichostatin A).

Mentions: To see if the induction/upregulation of Glut-4 was a direct consequence of HDAC inhibition, cells were exposed to cycloheximide prior to TSA treatments. Pretreatment with cycloheximide did not prevent the induction of Glut-4 mRNA (Figure 4a), suggesting that de novo protein synthesis was not required for the upregulation of Glut-4, and that direct chromatin remodeling of the Glut-4 promoter was occurring. To confirm that the observed effects of HDAC inhibition were due to increased histone hyperacetylation at the promoter of Glut-4, we carried out chromatin immunoprecipitation (ChIP) analysis at the Glut-4 promoter in A549 cells treated with TSA. A no-antibody control was included to test for non-specific carriage of DNA with histones. As can be seen in Figure 4b, treatment with TSA results in an increase in the amount of PCR product for Glut-4 indicating an increase in histone hyperacetylation around its promoter. Using specific antibodies we show that lysine 9 and lysine 14 are hyperacetylated in this region following treatment with TSA, and clearly demonstrate that chromatin remodeling is directly involved with the activation of Glut-4. Interestingly, no enhancement of histone trimethylation on lysine 4 of histone H3 was observed in response to promoter activation. H3K4me3 is considered to be a mark for transcriptionally active chromatin [18], and recent findings suggest that methylated H3K4 acts to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components to H3K4me3 via CHD1 [19]. Unusually, we observe a strong increase in the levels of histone H3 lysine 4 dimethylation (H3K4me2) a modification generally perceived to be associated with repressive chromatin [20], at the Glut-4 promoter following activation of transcription via HDACi (Figure 4). However, this mark has also been found to be enriched in the cis-regulatory regions of active promoters as well as at developmentally poised genes [21]. We also observe a loss of an activating mark for transcription, histone lysine 4 monomethylation (H3K4me) [22], following activation of Glut-4 in our cells. In agreement with the current literature, we observe the presence of a repressive mark H3K9Me2 at the Glut-4 promoter prior to activation via HDACi, and levels of this histone modification are lost following activation [20,22]. This would therefore indicate that much more detailed studies of the histone post-translational modifications will be required to functionally elucidate the “histone code” regulating this promoter region.


Epigenetic regulation of glucose transporters in non-small cell lung cancer.

O'Byrne KJ, Baird AM, Kilmartin L, Leonard J, Sacevich C, Gray SG - Cancers (Basel) (2011)

Direct chromatin remodeling of the Glut-4 promoter. (a) Pre-treatment with cycloheximide is unable to prevent TSA mediated induction of Glut-4 indicating that de novo protein synthesis is not required for activation that induction is via direct chromatin remodeling. (b) A chromatin immunoprecipitation (ChIP) assay demonstrates that TSA treatment results in an increase in the acetylation of histone H3 and H4 at the Glut-4 promoter. A549 cells were cultured in the presence or absence of TSA (250 ng/mL) for a period of 16 h. Subsequently a ChIP assay was performed using the following antibodies; pan acetylated histone H3 (Ac H3) and H4 (AcH4), histone H3 acetylated at lysine 9 and 14 (H3K9/K14Ac), histone H3 acetylated at lysine 9 (H3K9Ac), histone H3 tri-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 4 (H3K4me2), histone H3 mono-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 9 (H3K9me2). Input represents a positive control consisting of 1/10th of the original fixed chromatin prior to immunoprecipitation as recommended by the manufacturer (Diagenode). A no-antibody control was included to test for non-specific carriage of DNA with histones. (UT, untreated; CHX, cycloheximide; TSA, Trichostatin A).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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f4-cancers-03-01550: Direct chromatin remodeling of the Glut-4 promoter. (a) Pre-treatment with cycloheximide is unable to prevent TSA mediated induction of Glut-4 indicating that de novo protein synthesis is not required for activation that induction is via direct chromatin remodeling. (b) A chromatin immunoprecipitation (ChIP) assay demonstrates that TSA treatment results in an increase in the acetylation of histone H3 and H4 at the Glut-4 promoter. A549 cells were cultured in the presence or absence of TSA (250 ng/mL) for a period of 16 h. Subsequently a ChIP assay was performed using the following antibodies; pan acetylated histone H3 (Ac H3) and H4 (AcH4), histone H3 acetylated at lysine 9 and 14 (H3K9/K14Ac), histone H3 acetylated at lysine 9 (H3K9Ac), histone H3 tri-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 4 (H3K4me2), histone H3 mono-methylated at lysine 4 (H3K4me3), histone H3 di-methylated at lysine 9 (H3K9me2). Input represents a positive control consisting of 1/10th of the original fixed chromatin prior to immunoprecipitation as recommended by the manufacturer (Diagenode). A no-antibody control was included to test for non-specific carriage of DNA with histones. (UT, untreated; CHX, cycloheximide; TSA, Trichostatin A).
Mentions: To see if the induction/upregulation of Glut-4 was a direct consequence of HDAC inhibition, cells were exposed to cycloheximide prior to TSA treatments. Pretreatment with cycloheximide did not prevent the induction of Glut-4 mRNA (Figure 4a), suggesting that de novo protein synthesis was not required for the upregulation of Glut-4, and that direct chromatin remodeling of the Glut-4 promoter was occurring. To confirm that the observed effects of HDAC inhibition were due to increased histone hyperacetylation at the promoter of Glut-4, we carried out chromatin immunoprecipitation (ChIP) analysis at the Glut-4 promoter in A549 cells treated with TSA. A no-antibody control was included to test for non-specific carriage of DNA with histones. As can be seen in Figure 4b, treatment with TSA results in an increase in the amount of PCR product for Glut-4 indicating an increase in histone hyperacetylation around its promoter. Using specific antibodies we show that lysine 9 and lysine 14 are hyperacetylated in this region following treatment with TSA, and clearly demonstrate that chromatin remodeling is directly involved with the activation of Glut-4. Interestingly, no enhancement of histone trimethylation on lysine 4 of histone H3 was observed in response to promoter activation. H3K4me3 is considered to be a mark for transcriptionally active chromatin [18], and recent findings suggest that methylated H3K4 acts to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components to H3K4me3 via CHD1 [19]. Unusually, we observe a strong increase in the levels of histone H3 lysine 4 dimethylation (H3K4me2) a modification generally perceived to be associated with repressive chromatin [20], at the Glut-4 promoter following activation of transcription via HDACi (Figure 4). However, this mark has also been found to be enriched in the cis-regulatory regions of active promoters as well as at developmentally poised genes [21]. We also observe a loss of an activating mark for transcription, histone lysine 4 monomethylation (H3K4me) [22], following activation of Glut-4 in our cells. In agreement with the current literature, we observe the presence of a repressive mark H3K9Me2 at the Glut-4 promoter prior to activation via HDACi, and levels of this histone modification are lost following activation [20,22]. This would therefore indicate that much more detailed studies of the histone post-translational modifications will be required to functionally elucidate the “histone code” regulating this promoter region.

Bottom Line: Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect.Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes.Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James´s Hospital, Dublin 8, Ireland. sgray@stjames.ie.

ABSTRACT
Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy.

No MeSH data available.


Related in: MedlinePlus