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G9a is essential for EMT-mediated metastasis and maintenance of cancer stem cell-like characters in head and neck squamous cell carcinoma.

Liu S, Ye D, Guo W, Yu W, He Y, Hu J, Wang Y, Zhang L, Liao Y, Song H, Zhong S, Xu D, Yin H, Sun B, Wang X, Liu J, Wu Y, Zhou BP, Zhang Z, Deng J - Oncotarget (2015)

Bottom Line: We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9).Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers.Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer with poor prognosis, largely due to lymph node metastasis and local recurrence. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics. However, the mechanisms underlying metastasis to lymph nodes in HNSCC is poorly defined. In this study, we show that E-cadherin repression correlates with cancer metastasis and poor prognosis in HNSCC. We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9). Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers. Our study demonstrates that the G9a protein is essential for the induction of EMT and CSC-like properties in HNSCC. Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

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Related in: MedlinePlus

H3K9 methylation at the E-cadherin promoter is associated with TGF-β–induced EMT in HNSCC(A) HN4 cells were treated with TGF-β1 (5 ng/ml) for 3, 6, 9, 12 and 15 days, respectively; morphologic changes associated with EMT at 12 days are shown in the phase contrast images. Immunofluorescence (IF) staining for E-cadherin, N-cadherin and vimentin is presented with nuclei stained with DAPI (blue). Scale bar = 200 μm. (B) Migration of HN4 cells exposed to TGF-β (5 ng/ml) for 6 days before assay with representative images shown. Scale bar = 200 μm. (C) Graph demonstrates the mean ± SD percent of migrated cells from 3 separate experiments. (D) Western blot analysis of E-cadherin, N-cadherin and Snail in HN4 cells treated with TGF-β (5 ng/ml) for the indicated time periods. (E) ChIP analysis of H3K9me2 and H3K9Ac at the E-cadherin promoter of HN4 cells treated with TGF-β (5 ng/ml).
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Figure 4: H3K9 methylation at the E-cadherin promoter is associated with TGF-β–induced EMT in HNSCC(A) HN4 cells were treated with TGF-β1 (5 ng/ml) for 3, 6, 9, 12 and 15 days, respectively; morphologic changes associated with EMT at 12 days are shown in the phase contrast images. Immunofluorescence (IF) staining for E-cadherin, N-cadherin and vimentin is presented with nuclei stained with DAPI (blue). Scale bar = 200 μm. (B) Migration of HN4 cells exposed to TGF-β (5 ng/ml) for 6 days before assay with representative images shown. Scale bar = 200 μm. (C) Graph demonstrates the mean ± SD percent of migrated cells from 3 separate experiments. (D) Western blot analysis of E-cadherin, N-cadherin and Snail in HN4 cells treated with TGF-β (5 ng/ml) for the indicated time periods. (E) ChIP analysis of H3K9me2 and H3K9Ac at the E-cadherin promoter of HN4 cells treated with TGF-β (5 ng/ml).

Mentions: It appears that HN12 cells are ‘locked’ in the mesenchymal state since the E-cadherin promoter is methylated in these cells; this situation makes it difficult to study the initial and dynamic events of EMT in HNSCC. To overcome this technical issue, we investigated the mechanisms underlying histone modification and DNA methylation during TGF-β-induced EMT in HN4 cells. We found that, TGF-β treatment (5 ng/ml) induced EMT in HN4 cell as characterized by acquisition of fibroblastic mesenchymal morphology (Figure 4A), downregulation of the epithelial marker E-cadherin, and upregulation of mesenchymal markers N-cadherin and vimentin (Figure 4A and 4D). The transwell migration assay showed that TGF-β treatment significantly increased the number of migrating cells, suggesting that TGF-β increased the motility of HN4 cells (Figure 4B and 4C). Moreover, ChIP analysis showed that TGF-β treatment significantly increased H3K9me2, and decreased H3K9 acetylation at the E-cadherin promoter in HN4 cells (Figure 4E). Taken together, the results suggest that H3K9me2 is involved in TGF-β-induced EMT in HNSCC. Because the methyltransferase activity of G9a is specific, the elevated level of H3K9me2 on the E-cadherin promoter implies that G9a might be involved in regulation of E-cadherin expression. To determine if the G9a-Snail complex in the E-cadherin promoter is essential for E-cadherin repression in HNSCC, we examined the effects of an inhibitor of G9a, BIX01294, on the H3K9me2 methyltransferase activity in the TGF-β-induced EMT model. BIX01294 treatment reversed the EMT phenotype induced by TGF-β treatment: the fibroblastic mesenchymal morphology reverted to the polygonal epithelial morphology (Figure 5A). Moreover, BIX01294 treatment upregulated E-cadherin expression (Figure 5B) and reduced the wound healing activity in the TGF-β-induced EMT model (Figure 5C–5D). Consistently, ChIP analysis showed that BIX treatment significantly decreased the level of H3K9me2 and increased the level of H3K9Ac on the E-cadherin promoter (Figure 5E), which accounts for the upregulated E-cadherin in BIX-treated cells. Thus, G9a is essential for the formation of H3K9me2 on the E-cadherin promoter, which mediates TGF-β-induced EMT in HNSCC cells.


G9a is essential for EMT-mediated metastasis and maintenance of cancer stem cell-like characters in head and neck squamous cell carcinoma.

Liu S, Ye D, Guo W, Yu W, He Y, Hu J, Wang Y, Zhang L, Liao Y, Song H, Zhong S, Xu D, Yin H, Sun B, Wang X, Liu J, Wu Y, Zhou BP, Zhang Z, Deng J - Oncotarget (2015)

H3K9 methylation at the E-cadherin promoter is associated with TGF-β–induced EMT in HNSCC(A) HN4 cells were treated with TGF-β1 (5 ng/ml) for 3, 6, 9, 12 and 15 days, respectively; morphologic changes associated with EMT at 12 days are shown in the phase contrast images. Immunofluorescence (IF) staining for E-cadherin, N-cadherin and vimentin is presented with nuclei stained with DAPI (blue). Scale bar = 200 μm. (B) Migration of HN4 cells exposed to TGF-β (5 ng/ml) for 6 days before assay with representative images shown. Scale bar = 200 μm. (C) Graph demonstrates the mean ± SD percent of migrated cells from 3 separate experiments. (D) Western blot analysis of E-cadherin, N-cadherin and Snail in HN4 cells treated with TGF-β (5 ng/ml) for the indicated time periods. (E) ChIP analysis of H3K9me2 and H3K9Ac at the E-cadherin promoter of HN4 cells treated with TGF-β (5 ng/ml).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: H3K9 methylation at the E-cadherin promoter is associated with TGF-β–induced EMT in HNSCC(A) HN4 cells were treated with TGF-β1 (5 ng/ml) for 3, 6, 9, 12 and 15 days, respectively; morphologic changes associated with EMT at 12 days are shown in the phase contrast images. Immunofluorescence (IF) staining for E-cadherin, N-cadherin and vimentin is presented with nuclei stained with DAPI (blue). Scale bar = 200 μm. (B) Migration of HN4 cells exposed to TGF-β (5 ng/ml) for 6 days before assay with representative images shown. Scale bar = 200 μm. (C) Graph demonstrates the mean ± SD percent of migrated cells from 3 separate experiments. (D) Western blot analysis of E-cadherin, N-cadherin and Snail in HN4 cells treated with TGF-β (5 ng/ml) for the indicated time periods. (E) ChIP analysis of H3K9me2 and H3K9Ac at the E-cadherin promoter of HN4 cells treated with TGF-β (5 ng/ml).
Mentions: It appears that HN12 cells are ‘locked’ in the mesenchymal state since the E-cadherin promoter is methylated in these cells; this situation makes it difficult to study the initial and dynamic events of EMT in HNSCC. To overcome this technical issue, we investigated the mechanisms underlying histone modification and DNA methylation during TGF-β-induced EMT in HN4 cells. We found that, TGF-β treatment (5 ng/ml) induced EMT in HN4 cell as characterized by acquisition of fibroblastic mesenchymal morphology (Figure 4A), downregulation of the epithelial marker E-cadherin, and upregulation of mesenchymal markers N-cadherin and vimentin (Figure 4A and 4D). The transwell migration assay showed that TGF-β treatment significantly increased the number of migrating cells, suggesting that TGF-β increased the motility of HN4 cells (Figure 4B and 4C). Moreover, ChIP analysis showed that TGF-β treatment significantly increased H3K9me2, and decreased H3K9 acetylation at the E-cadherin promoter in HN4 cells (Figure 4E). Taken together, the results suggest that H3K9me2 is involved in TGF-β-induced EMT in HNSCC. Because the methyltransferase activity of G9a is specific, the elevated level of H3K9me2 on the E-cadherin promoter implies that G9a might be involved in regulation of E-cadherin expression. To determine if the G9a-Snail complex in the E-cadherin promoter is essential for E-cadherin repression in HNSCC, we examined the effects of an inhibitor of G9a, BIX01294, on the H3K9me2 methyltransferase activity in the TGF-β-induced EMT model. BIX01294 treatment reversed the EMT phenotype induced by TGF-β treatment: the fibroblastic mesenchymal morphology reverted to the polygonal epithelial morphology (Figure 5A). Moreover, BIX01294 treatment upregulated E-cadherin expression (Figure 5B) and reduced the wound healing activity in the TGF-β-induced EMT model (Figure 5C–5D). Consistently, ChIP analysis showed that BIX treatment significantly decreased the level of H3K9me2 and increased the level of H3K9Ac on the E-cadherin promoter (Figure 5E), which accounts for the upregulated E-cadherin in BIX-treated cells. Thus, G9a is essential for the formation of H3K9me2 on the E-cadherin promoter, which mediates TGF-β-induced EMT in HNSCC cells.

Bottom Line: We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9).Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers.Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer with poor prognosis, largely due to lymph node metastasis and local recurrence. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics. However, the mechanisms underlying metastasis to lymph nodes in HNSCC is poorly defined. In this study, we show that E-cadherin repression correlates with cancer metastasis and poor prognosis in HNSCC. We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9). Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers. Our study demonstrates that the G9a protein is essential for the induction of EMT and CSC-like properties in HNSCC. Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

Show MeSH
Related in: MedlinePlus