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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: Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics.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).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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Knockdown of G9a inhibit tumorsphere formation and suppress the expression of CD44(A) Western blot analysis of CD44 expression in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (B) Representative image of tumorsphere formation in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (C) The tumorsphere formation was measured in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). Mean ± SD from 3 separate experiments. (D) Western blot analysis of CD44 expression in HN12 cells stably expressing control vector or G9a shRNA. (E) Image of immunofluorescence staining for CD44 expression. (F) Image of tumorsphere formation in HN12 cells stably expressing control vector or G9a shRNA. Scale bar = 200 μm. (G) Graph demonstrates the mean ± SD for tumorsphere number in HN12 cells stably expressing control vector or G9a shRNA from 3 separate experiments.
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Figure 8: Knockdown of G9a inhibit tumorsphere formation and suppress the expression of CD44(A) Western blot analysis of CD44 expression in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (B) Representative image of tumorsphere formation in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (C) The tumorsphere formation was measured in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). Mean ± SD from 3 separate experiments. (D) Western blot analysis of CD44 expression in HN12 cells stably expressing control vector or G9a shRNA. (E) Image of immunofluorescence staining for CD44 expression. (F) Image of tumorsphere formation in HN12 cells stably expressing control vector or G9a shRNA. Scale bar = 200 μm. (G) Graph demonstrates the mean ± SD for tumorsphere number in HN12 cells stably expressing control vector or G9a shRNA from 3 separate experiments.

Mentions: Since G9a is essential for TGF-β-induced EMT, and EMT induces CSC-like characteristics, we asked whether G9a is essential for induction of CSC-like characteristics in HNSCC. TGF-β treatment induced expression of the CD44 marker in HN4 cells (Figure 8A). However, addition of a G9a inhibitor BIX01294 suppressed CD44 expression, suggesting that a functional G9a is required for TGF-β-induced CD44 expression in these cells. Consistently, BIX01294 also suppressed TGF-β-induced tumorsphere-formation (Figure 8B and 8C). These results suggest that a functional G9a is required for TGF-β-induced CSC-like characters. To determine if G9a is essential for maintenance of CSC-like characteristics, we examined and compared the biochemical and biological characteristics of stable shRNA-G9a transfectants (knockdown of G9a expression) with HN12 cells. Knockdown of G9a expression significantly suppressed CD44 expression when levels were compared to control HN12 cells, suggesting that G9a is required for CD44 expression (Figure 8D, 8E and Figure S3). Moreover, knockdown of G9a also suppressed tumorsphere formation in the transfected cells compared with formation in control HN12 cells (Figure 8F and 8G). Taken together, these observations demonstrate that G9a is essential for induction and maintenance of EMT-related CSC-like characteristics in HNSCC.


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)

Knockdown of G9a inhibit tumorsphere formation and suppress the expression of CD44(A) Western blot analysis of CD44 expression in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (B) Representative image of tumorsphere formation in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (C) The tumorsphere formation was measured in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). Mean ± SD from 3 separate experiments. (D) Western blot analysis of CD44 expression in HN12 cells stably expressing control vector or G9a shRNA. (E) Image of immunofluorescence staining for CD44 expression. (F) Image of tumorsphere formation in HN12 cells stably expressing control vector or G9a shRNA. Scale bar = 200 μm. (G) Graph demonstrates the mean ± SD for tumorsphere number in HN12 cells stably expressing control vector or G9a shRNA from 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Knockdown of G9a inhibit tumorsphere formation and suppress the expression of CD44(A) Western blot analysis of CD44 expression in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (B) Representative image of tumorsphere formation in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). (C) The tumorsphere formation was measured in HN4 cells treated without or with BIX (2.5 μM) followed by treatment with TGF-β (5 ng/ml). Mean ± SD from 3 separate experiments. (D) Western blot analysis of CD44 expression in HN12 cells stably expressing control vector or G9a shRNA. (E) Image of immunofluorescence staining for CD44 expression. (F) Image of tumorsphere formation in HN12 cells stably expressing control vector or G9a shRNA. Scale bar = 200 μm. (G) Graph demonstrates the mean ± SD for tumorsphere number in HN12 cells stably expressing control vector or G9a shRNA from 3 separate experiments.
Mentions: Since G9a is essential for TGF-β-induced EMT, and EMT induces CSC-like characteristics, we asked whether G9a is essential for induction of CSC-like characteristics in HNSCC. TGF-β treatment induced expression of the CD44 marker in HN4 cells (Figure 8A). However, addition of a G9a inhibitor BIX01294 suppressed CD44 expression, suggesting that a functional G9a is required for TGF-β-induced CD44 expression in these cells. Consistently, BIX01294 also suppressed TGF-β-induced tumorsphere-formation (Figure 8B and 8C). These results suggest that a functional G9a is required for TGF-β-induced CSC-like characters. To determine if G9a is essential for maintenance of CSC-like characteristics, we examined and compared the biochemical and biological characteristics of stable shRNA-G9a transfectants (knockdown of G9a expression) with HN12 cells. Knockdown of G9a expression significantly suppressed CD44 expression when levels were compared to control HN12 cells, suggesting that G9a is required for CD44 expression (Figure 8D, 8E and Figure S3). Moreover, knockdown of G9a also suppressed tumorsphere formation in the transfected cells compared with formation in control HN12 cells (Figure 8F and 8G). Taken together, these observations demonstrate that G9a is essential for induction and maintenance of EMT-related CSC-like characteristics in HNSCC.

Bottom Line: Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics.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).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