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Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.

Li J, Taich ZJ, Goyal A, Gonda D, Akers J, Adhikari B, Patel K, Vandenberg S, Yan W, Bao Z, Carter BS, Wang R, Mao Y, Jiang T, Chen CC - Oncotarget (2014)

Bottom Line: The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood.Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406).These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions.

View Article: PubMed Central - PubMed

Affiliation: Center for Theoretical and Applied Neuro-Oncology, Division of Neurosurgery, University of California, San Diego. Contributed equally to this work.

ABSTRACT
The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients.

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Suppression of H-Ras expression in G-CIMP+ glioblastomas(A) Ectopic expression of EGFR or EGFRvIII failed to restore pERK accumulation in G-CIMP+ Ink4a-Arf−/− glioblastoma cells. Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1 were stably transduced with retrovirus carrying vector, EGFR or EGFRvIII expression construct, separately. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, EGFR and Tubulin (as loading control). (B) Decreased H-Ras mRNA level in G-CIMP+ TCGA glioblastomas. G-CIMP status in the TCGA dataset is determined using global genomic methylation profiles as described by Noushmehr et al. [6]. (C) Induction of G-CIMP+ status suppressed H-Ras expression. Human U87 MG glioblastoma line was stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H. Cells were propagated for >10 passages. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against H-Ras, Flag (for IDH1), and Tubulin (as loading control). (D) Induction of G-CIMP+ state increased deposition of repressive histone markers H3K9me3 and H3K27me3 in the H-Ras promoter region. U87 cells stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H mutant were lysed after crosslinking with formaldehyde. Chromatin was extracted, fragmented, and immunoprecipitated with antibodies against Histone H3, H3K9me3 and H3K27me3. Relative abundance of H3K9me3 and H3K27me3 at H-Ras promoter region was shown as fold change compared to the cells transduced with empty vector. Error bars represent standard deviation. (E) Ectopic expression of H-Ras restored pERK accumulation in G-CIMP+ Ink4a-Arf−/− EGFRvIII glioblastoma cells. H-Ras expression constructs were stably transfected into Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, H-Ras, Flag (for IDH1), and Tubulin (as loading control). (F) G-CIMP+ state lowered glioblastoma sensitivity to the EGFR inhibitor, Gefitinib. Murine Ink4a-Arf−/− cells expressing wild type IDH1 or IDH1-R132H were stably transduced with empty vector or EGFRvIII and treated with vehicle or Gefitinib at 10 μM for 14 days. Clonogenic survival was determined. ** indicates statistical significance at p<0.01.
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Figure 5: Suppression of H-Ras expression in G-CIMP+ glioblastomas(A) Ectopic expression of EGFR or EGFRvIII failed to restore pERK accumulation in G-CIMP+ Ink4a-Arf−/− glioblastoma cells. Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1 were stably transduced with retrovirus carrying vector, EGFR or EGFRvIII expression construct, separately. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, EGFR and Tubulin (as loading control). (B) Decreased H-Ras mRNA level in G-CIMP+ TCGA glioblastomas. G-CIMP status in the TCGA dataset is determined using global genomic methylation profiles as described by Noushmehr et al. [6]. (C) Induction of G-CIMP+ status suppressed H-Ras expression. Human U87 MG glioblastoma line was stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H. Cells were propagated for >10 passages. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against H-Ras, Flag (for IDH1), and Tubulin (as loading control). (D) Induction of G-CIMP+ state increased deposition of repressive histone markers H3K9me3 and H3K27me3 in the H-Ras promoter region. U87 cells stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H mutant were lysed after crosslinking with formaldehyde. Chromatin was extracted, fragmented, and immunoprecipitated with antibodies against Histone H3, H3K9me3 and H3K27me3. Relative abundance of H3K9me3 and H3K27me3 at H-Ras promoter region was shown as fold change compared to the cells transduced with empty vector. Error bars represent standard deviation. (E) Ectopic expression of H-Ras restored pERK accumulation in G-CIMP+ Ink4a-Arf−/− EGFRvIII glioblastoma cells. H-Ras expression constructs were stably transfected into Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, H-Ras, Flag (for IDH1), and Tubulin (as loading control). (F) G-CIMP+ state lowered glioblastoma sensitivity to the EGFR inhibitor, Gefitinib. Murine Ink4a-Arf−/− cells expressing wild type IDH1 or IDH1-R132H were stably transduced with empty vector or EGFRvIII and treated with vehicle or Gefitinib at 10 μM for 14 days. Clonogenic survival was determined. ** indicates statistical significance at p<0.01.

Mentions: If suppression of EGFR expression is the sole mechanism by which G-CIMP+ glioblastomas down-regulate EGFR signaling, then exogenous expression of EGFR should restore this signaling. We tested this hypothesis. Surprisingly, exogenous expression of neither a wild type EGFR nor a hyperactive, oncogenic form of EGFR, EGFRvIII restored pERK accumulation in the G-CIMP+ Ink4-Arf−/−glioblastoma line (Figure 5A). This result suggests that G-CIMP+ status may additionally suppress the expression of genes down-stream of EGFR that are required for EGFR signaling.


Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.

Li J, Taich ZJ, Goyal A, Gonda D, Akers J, Adhikari B, Patel K, Vandenberg S, Yan W, Bao Z, Carter BS, Wang R, Mao Y, Jiang T, Chen CC - Oncotarget (2014)

Suppression of H-Ras expression in G-CIMP+ glioblastomas(A) Ectopic expression of EGFR or EGFRvIII failed to restore pERK accumulation in G-CIMP+ Ink4a-Arf−/− glioblastoma cells. Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1 were stably transduced with retrovirus carrying vector, EGFR or EGFRvIII expression construct, separately. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, EGFR and Tubulin (as loading control). (B) Decreased H-Ras mRNA level in G-CIMP+ TCGA glioblastomas. G-CIMP status in the TCGA dataset is determined using global genomic methylation profiles as described by Noushmehr et al. [6]. (C) Induction of G-CIMP+ status suppressed H-Ras expression. Human U87 MG glioblastoma line was stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H. Cells were propagated for >10 passages. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against H-Ras, Flag (for IDH1), and Tubulin (as loading control). (D) Induction of G-CIMP+ state increased deposition of repressive histone markers H3K9me3 and H3K27me3 in the H-Ras promoter region. U87 cells stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H mutant were lysed after crosslinking with formaldehyde. Chromatin was extracted, fragmented, and immunoprecipitated with antibodies against Histone H3, H3K9me3 and H3K27me3. Relative abundance of H3K9me3 and H3K27me3 at H-Ras promoter region was shown as fold change compared to the cells transduced with empty vector. Error bars represent standard deviation. (E) Ectopic expression of H-Ras restored pERK accumulation in G-CIMP+ Ink4a-Arf−/− EGFRvIII glioblastoma cells. H-Ras expression constructs were stably transfected into Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, H-Ras, Flag (for IDH1), and Tubulin (as loading control). (F) G-CIMP+ state lowered glioblastoma sensitivity to the EGFR inhibitor, Gefitinib. Murine Ink4a-Arf−/− cells expressing wild type IDH1 or IDH1-R132H were stably transduced with empty vector or EGFRvIII and treated with vehicle or Gefitinib at 10 μM for 14 days. Clonogenic survival was determined. ** indicates statistical significance at p<0.01.
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Figure 5: Suppression of H-Ras expression in G-CIMP+ glioblastomas(A) Ectopic expression of EGFR or EGFRvIII failed to restore pERK accumulation in G-CIMP+ Ink4a-Arf−/− glioblastoma cells. Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1 were stably transduced with retrovirus carrying vector, EGFR or EGFRvIII expression construct, separately. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, EGFR and Tubulin (as loading control). (B) Decreased H-Ras mRNA level in G-CIMP+ TCGA glioblastomas. G-CIMP status in the TCGA dataset is determined using global genomic methylation profiles as described by Noushmehr et al. [6]. (C) Induction of G-CIMP+ status suppressed H-Ras expression. Human U87 MG glioblastoma line was stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H. Cells were propagated for >10 passages. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against H-Ras, Flag (for IDH1), and Tubulin (as loading control). (D) Induction of G-CIMP+ state increased deposition of repressive histone markers H3K9me3 and H3K27me3 in the H-Ras promoter region. U87 cells stably transduced with retrovirus carrying empty vector, IDH1 or IDH1-R132H mutant were lysed after crosslinking with formaldehyde. Chromatin was extracted, fragmented, and immunoprecipitated with antibodies against Histone H3, H3K9me3 and H3K27me3. Relative abundance of H3K9me3 and H3K27me3 at H-Ras promoter region was shown as fold change compared to the cells transduced with empty vector. Error bars represent standard deviation. (E) Ectopic expression of H-Ras restored pERK accumulation in G-CIMP+ Ink4a-Arf−/− EGFRvIII glioblastoma cells. H-Ras expression constructs were stably transfected into Ink4a-Arf−/− glioblastoma lines expressing wild type or mutated (R132H) IDH1. Whole cell lysates were extracted and analyzed with Western blotting using antibodies against pERK, ERK, H-Ras, Flag (for IDH1), and Tubulin (as loading control). (F) G-CIMP+ state lowered glioblastoma sensitivity to the EGFR inhibitor, Gefitinib. Murine Ink4a-Arf−/− cells expressing wild type IDH1 or IDH1-R132H were stably transduced with empty vector or EGFRvIII and treated with vehicle or Gefitinib at 10 μM for 14 days. Clonogenic survival was determined. ** indicates statistical significance at p<0.01.
Mentions: If suppression of EGFR expression is the sole mechanism by which G-CIMP+ glioblastomas down-regulate EGFR signaling, then exogenous expression of EGFR should restore this signaling. We tested this hypothesis. Surprisingly, exogenous expression of neither a wild type EGFR nor a hyperactive, oncogenic form of EGFR, EGFRvIII restored pERK accumulation in the G-CIMP+ Ink4-Arf−/−glioblastoma line (Figure 5A). This result suggests that G-CIMP+ status may additionally suppress the expression of genes down-stream of EGFR that are required for EGFR signaling.

Bottom Line: The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood.Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406).These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions.

View Article: PubMed Central - PubMed

Affiliation: Center for Theoretical and Applied Neuro-Oncology, Division of Neurosurgery, University of California, San Diego. Contributed equally to this work.

ABSTRACT
The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients.

Show MeSH
Related in: MedlinePlus