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Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition.

Ma SR, Wang WM, Huang CF, Zhang WF, Sun ZJ - Oncotarget (2015)

Bottom Line: Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role.We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa.These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, Wuhan University, Wuhan, China.

ABSTRACT
Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/ or lost function of Pten. In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

No MeSH data available.


Related in: MedlinePlus

Knock down of AGR2 induces cell apoptosis and reduces colony formation in CAL27 cell line(A) Annexin V-FITC/PI dual labeling assay showed AGR2 siRNA enhance apoptosis in CAL27 cell lines by using flow cytometry; (B) The morphologic changes of CAL27 cell lines transfected with AGR2 siRNA was observed by fluorescence microscopy with DAPI staining; (C) Knock down of AGR2 in CAL27 cell line reduced anchor dependent colony formation; (D) Knock down of AGR2 in CAL27 cell line reduced sphere formation; Scale bar=100μm;(E) Western blot analysis revealed that the protein level of Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog, Sox2 and OCT4 were reduced in different degrees after AGR2 konck down for 48h. Quantification is performed using Image J by pixel analysis of band by normalized of β-actin as a loading control. Neg siRNA, negative siRNA,si AGR2, AGR2 siRNA.
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Figure 5: Knock down of AGR2 induces cell apoptosis and reduces colony formation in CAL27 cell line(A) Annexin V-FITC/PI dual labeling assay showed AGR2 siRNA enhance apoptosis in CAL27 cell lines by using flow cytometry; (B) The morphologic changes of CAL27 cell lines transfected with AGR2 siRNA was observed by fluorescence microscopy with DAPI staining; (C) Knock down of AGR2 in CAL27 cell line reduced anchor dependent colony formation; (D) Knock down of AGR2 in CAL27 cell line reduced sphere formation; Scale bar=100μm;(E) Western blot analysis revealed that the protein level of Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog, Sox2 and OCT4 were reduced in different degrees after AGR2 konck down for 48h. Quantification is performed using Image J by pixel analysis of band by normalized of β-actin as a loading control. Neg siRNA, negative siRNA,si AGR2, AGR2 siRNA.

Mentions: To direct identify the function of AGR2, we used siRNA to knockdown AGR2 in human HNSCC CAL27 and FaDu cell lines. By using Annexin V/PI double staining analysis, cell apoptosis was counted by flow cytometry. Annexin V+/PI− apoptotic cell population increased in AGR2 siRNA-treated CAL27 cell line compared with the untreated and negative control siRNA counterpart (Fig. 5A). Hoechst staining of siAGR2 increased the nuclear fragment as shown in Fig. 5B. The colony formation ability and sphere formation assay is widely used as a surrogate for self-renewal and colony formation property of CSCs. Therefore, we performed the sphere-forming assay to investigate the potential role of AGR2 knock down in the self-renewal capacity of CAL27 cells. Indeed, AGR2 siRNA reduced the colony formation (Fig. 5C) when compared with the untreated group and negative group. AGR2 siRNA treated CAL27 cells was found to form less and smaller spheres than the counterpart (Fig. 5D) indicating its possible inhibition of self-renewal of HNSCC cells in vitro. The apoptotic assay and sphere formation assay are quiet repeatable in FaDu cell line (Supplementary Fig. S4 A-C)


Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition.

Ma SR, Wang WM, Huang CF, Zhang WF, Sun ZJ - Oncotarget (2015)

Knock down of AGR2 induces cell apoptosis and reduces colony formation in CAL27 cell line(A) Annexin V-FITC/PI dual labeling assay showed AGR2 siRNA enhance apoptosis in CAL27 cell lines by using flow cytometry; (B) The morphologic changes of CAL27 cell lines transfected with AGR2 siRNA was observed by fluorescence microscopy with DAPI staining; (C) Knock down of AGR2 in CAL27 cell line reduced anchor dependent colony formation; (D) Knock down of AGR2 in CAL27 cell line reduced sphere formation; Scale bar=100μm;(E) Western blot analysis revealed that the protein level of Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog, Sox2 and OCT4 were reduced in different degrees after AGR2 konck down for 48h. Quantification is performed using Image J by pixel analysis of band by normalized of β-actin as a loading control. Neg siRNA, negative siRNA,si AGR2, AGR2 siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496185&req=5

Figure 5: Knock down of AGR2 induces cell apoptosis and reduces colony formation in CAL27 cell line(A) Annexin V-FITC/PI dual labeling assay showed AGR2 siRNA enhance apoptosis in CAL27 cell lines by using flow cytometry; (B) The morphologic changes of CAL27 cell lines transfected with AGR2 siRNA was observed by fluorescence microscopy with DAPI staining; (C) Knock down of AGR2 in CAL27 cell line reduced anchor dependent colony formation; (D) Knock down of AGR2 in CAL27 cell line reduced sphere formation; Scale bar=100μm;(E) Western blot analysis revealed that the protein level of Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog, Sox2 and OCT4 were reduced in different degrees after AGR2 konck down for 48h. Quantification is performed using Image J by pixel analysis of band by normalized of β-actin as a loading control. Neg siRNA, negative siRNA,si AGR2, AGR2 siRNA.
Mentions: To direct identify the function of AGR2, we used siRNA to knockdown AGR2 in human HNSCC CAL27 and FaDu cell lines. By using Annexin V/PI double staining analysis, cell apoptosis was counted by flow cytometry. Annexin V+/PI− apoptotic cell population increased in AGR2 siRNA-treated CAL27 cell line compared with the untreated and negative control siRNA counterpart (Fig. 5A). Hoechst staining of siAGR2 increased the nuclear fragment as shown in Fig. 5B. The colony formation ability and sphere formation assay is widely used as a surrogate for self-renewal and colony formation property of CSCs. Therefore, we performed the sphere-forming assay to investigate the potential role of AGR2 knock down in the self-renewal capacity of CAL27 cells. Indeed, AGR2 siRNA reduced the colony formation (Fig. 5C) when compared with the untreated group and negative group. AGR2 siRNA treated CAL27 cells was found to form less and smaller spheres than the counterpart (Fig. 5D) indicating its possible inhibition of self-renewal of HNSCC cells in vitro. The apoptotic assay and sphere formation assay are quiet repeatable in FaDu cell line (Supplementary Fig. S4 A-C)

Bottom Line: Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role.We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa.These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, Wuhan University, Wuhan, China.

ABSTRACT
Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/ or lost function of Pten. In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

No MeSH data available.


Related in: MedlinePlus