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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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Lymph node metastatic HNSCC cells exhibit EMT characters(A) Morphology and staining for E-cadherin, N-cadherin and vimentin in HN-4 and HN12 cells. Scale bar = 200 μm. (B) Western blot analysis of E-cadherin, N-cadherin, Claudin-1, vimentin and Snail protein levels in HN4 and HN12 cell lines. (C) The transwell migration assay identified the migration capability of HN4 and HN12 cells with representative images shown. Scale bar = 200 μm. (D) Graph demonstrates the mean ± SD for the percent of migrated cells from 3 separate experiments.
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Figure 2: Lymph node metastatic HNSCC cells exhibit EMT characters(A) Morphology and staining for E-cadherin, N-cadherin and vimentin in HN-4 and HN12 cells. Scale bar = 200 μm. (B) Western blot analysis of E-cadherin, N-cadherin, Claudin-1, vimentin and Snail protein levels in HN4 and HN12 cell lines. (C) The transwell migration assay identified the migration capability of HN4 and HN12 cells with representative images shown. Scale bar = 200 μm. (D) Graph demonstrates the mean ± SD for the percent of migrated cells from 3 separate experiments.

Mentions: To investigate the molecular mechanisms involved in HNSCC metastasis to lymph nodes, we selected HN4 and HN12 as a paired cell line for further characterization, since HN12 and HN4 cells were derived from the same patient, with HN12 a nodal metastatic subclone from the HN4 primary tumor [37]. HN4 cells exhibit the typical polygonal morphology for epithelial cells (Figure 2A). Immunofluorescent analysis showed high expression levels of the epithelial marker E-cadherin and low levels of mesenchymal markers N-cadherin and vimentin in HN4 cells (Figure 2A). In contrast, HN12 cells were scattered throughout the plate surface, displayed a fibroblast-like morphology, and expressed low levels of E-cadherin and high levels of the N-cadherin and vimentin (Figure 2A and 2B). Immunoblot analysis confirmed the molecular features of these two cell lines (Figure 2B). Next, we examined the migratory capabilities of HN4 and HN12 cells, an EMT-associated biological activity, using a transwell migration assay. HN12 cells exhibited a significantly higher motility than did the HN4 cells (Figure 2C and 2D). Taken together, these results indicate that HN12 cells gain EMT-related molecular and functional phenotypic changes relative to their companion HN4 cells. Thus, EMT may play a key role in metastasis to lymph nodes 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)

Lymph node metastatic HNSCC cells exhibit EMT characters(A) Morphology and staining for E-cadherin, N-cadherin and vimentin in HN-4 and HN12 cells. Scale bar = 200 μm. (B) Western blot analysis of E-cadherin, N-cadherin, Claudin-1, vimentin and Snail protein levels in HN4 and HN12 cell lines. (C) The transwell migration assay identified the migration capability of HN4 and HN12 cells with representative images shown. Scale bar = 200 μm. (D) Graph demonstrates the mean ± SD for the percent of migrated cells from 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466657&req=5

Figure 2: Lymph node metastatic HNSCC cells exhibit EMT characters(A) Morphology and staining for E-cadherin, N-cadherin and vimentin in HN-4 and HN12 cells. Scale bar = 200 μm. (B) Western blot analysis of E-cadherin, N-cadherin, Claudin-1, vimentin and Snail protein levels in HN4 and HN12 cell lines. (C) The transwell migration assay identified the migration capability of HN4 and HN12 cells with representative images shown. Scale bar = 200 μm. (D) Graph demonstrates the mean ± SD for the percent of migrated cells from 3 separate experiments.
Mentions: To investigate the molecular mechanisms involved in HNSCC metastasis to lymph nodes, we selected HN4 and HN12 as a paired cell line for further characterization, since HN12 and HN4 cells were derived from the same patient, with HN12 a nodal metastatic subclone from the HN4 primary tumor [37]. HN4 cells exhibit the typical polygonal morphology for epithelial cells (Figure 2A). Immunofluorescent analysis showed high expression levels of the epithelial marker E-cadherin and low levels of mesenchymal markers N-cadherin and vimentin in HN4 cells (Figure 2A). In contrast, HN12 cells were scattered throughout the plate surface, displayed a fibroblast-like morphology, and expressed low levels of E-cadherin and high levels of the N-cadherin and vimentin (Figure 2A and 2B). Immunoblot analysis confirmed the molecular features of these two cell lines (Figure 2B). Next, we examined the migratory capabilities of HN4 and HN12 cells, an EMT-associated biological activity, using a transwell migration assay. HN12 cells exhibited a significantly higher motility than did the HN4 cells (Figure 2C and 2D). Taken together, these results indicate that HN12 cells gain EMT-related molecular and functional phenotypic changes relative to their companion HN4 cells. Thus, EMT may play a key role in metastasis to lymph nodes in HNSCC.

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