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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

Increased expression of AGR2 is associated with Tgfbr1 deletion(A) Immunohistochemistry of AGR2 in tongue of the wild type (WT) mice, the tumor of Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice (Scale bar+25μm); (B) Quantitative of histoscore of AGR2 in wild type mice, Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice. Expression of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly higher than the wild type mice (P < 0.001); (C) Quantitative Real-time PCR revealed the mRNA level of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly increased when compared with the wild type mice; (D) Western blot analysis of AGR2 48h after knocking down PTEN, TGFBR1, and combined TGFBR1/PTEN by using siRNA; (E) Quantitative analysis showed the protein level of AGR2 in TGFBR1 siRNA group and TGFBR1/PTEN combined siRNA group were significantly higher than the control group (P < 0.001). Mean±SEM, ***, P<0.001.
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Figure 7: Increased expression of AGR2 is associated with Tgfbr1 deletion(A) Immunohistochemistry of AGR2 in tongue of the wild type (WT) mice, the tumor of Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice (Scale bar+25μm); (B) Quantitative of histoscore of AGR2 in wild type mice, Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice. Expression of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly higher than the wild type mice (P < 0.001); (C) Quantitative Real-time PCR revealed the mRNA level of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly increased when compared with the wild type mice; (D) Western blot analysis of AGR2 48h after knocking down PTEN, TGFBR1, and combined TGFBR1/PTEN by using siRNA; (E) Quantitative analysis showed the protein level of AGR2 in TGFBR1 siRNA group and TGFBR1/PTEN combined siRNA group were significantly higher than the control group (P < 0.001). Mean±SEM, ***, P<0.001.

Mentions: Recent studies revealed that TGFβ signaling plays a pivotal role in EMT and in cancer stem cell self-renewal [27]. Our previous work showed that mice with tissue specific deletion of tumor suppressor gene Pten in epithalia had spontaneous HNSCC (Fig. 6A). Pathologically, Pten conditional kock out (Pten cKO) mice tumor present as well differentiation keratinizing squamous cell carcinoma (8/8) with frequently observed extracellular keratin, similar to human Grade I SCC (Fig. 6B and high magnification in Fig. 6C). Tgfbr1conditional knock out (Tgfbr1cKO) mice Fig. 6D) tumor histologically present as aggressive strand or island growth pattern (Fig.6E). The transitional epithelial cell present spindle-type with extensive inflammation, abundant stromal cells, intratumor necrosis and low amount of keratin pearl formation (High magnification in Fig. 6F), which similar to human Grades II and III squamous cell carcinoma. Combined deletion of Tgfbr1/Pten in mice epithelia would lead to rapid tumor formation (Fig. 6G) with pathological infiltrating growth pattern (Fig. 6H) and poorly differentiated SCC (High magnification in Fig. 6I). To determine whether over-expression of AGR2 was related to high-grade squamous cell carcinoma in mice, we performed AGR2 immunohistochemistry and found that AGR2 was located mostly in the membrane and cytoplasm of the cancer cell of Tgfbr1 cKO mice and Tgfbr1/Pten 2cKO mice (n = 5, respectively). However, AGR2 staining was negative in wild type mice mucosa and focal weakly positive in Pten cKO mice HNSCC (Fig. 7A with quantification histoscore in Fig. 7B). The mRNA levels of AGR2 in Tgfbr1 cKO mice and Tgfbr1/Pten mice were significantly higher as compared with wild type mice mucosa (P < 0.001 respectively, Fig. 7C). We further knockdown TGFBR1 and/or PTEN in human HNSCC CAL27 and FaDu cell lines. After transfection with PTEN and/or TGFBR1 siRNA for 48 h, the protein was extracted and analyzed by Western blot. The protein level of AGR2 in PTEN, TGFBR1 as well as in combined PTEN/TGFBR1 knock down was significantly higher than scramble siRNA in CAL27 cell line (Fig. 7D and quantification in Fig. 7E) and quiet repeatable in FaDu cell line Supplementary Fig. S4E and 4F). The discovery above suggested that increased AGR2 protein level may be associated with lost function of TGFBR1.


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)

Increased expression of AGR2 is associated with Tgfbr1 deletion(A) Immunohistochemistry of AGR2 in tongue of the wild type (WT) mice, the tumor of Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice (Scale bar+25μm); (B) Quantitative of histoscore of AGR2 in wild type mice, Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice. Expression of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly higher than the wild type mice (P < 0.001); (C) Quantitative Real-time PCR revealed the mRNA level of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly increased when compared with the wild type mice; (D) Western blot analysis of AGR2 48h after knocking down PTEN, TGFBR1, and combined TGFBR1/PTEN by using siRNA; (E) Quantitative analysis showed the protein level of AGR2 in TGFBR1 siRNA group and TGFBR1/PTEN combined siRNA group were significantly higher than the control group (P < 0.001). Mean±SEM, ***, P<0.001.
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Figure 7: Increased expression of AGR2 is associated with Tgfbr1 deletion(A) Immunohistochemistry of AGR2 in tongue of the wild type (WT) mice, the tumor of Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice (Scale bar+25μm); (B) Quantitative of histoscore of AGR2 in wild type mice, Pten conditional knock out mice, Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice. Expression of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly higher than the wild type mice (P < 0.001); (C) Quantitative Real-time PCR revealed the mRNA level of AGR2 in Tgfbr1 conditional knock out mice and Pten/Tgfbr1 conditional knock out mice was significantly increased when compared with the wild type mice; (D) Western blot analysis of AGR2 48h after knocking down PTEN, TGFBR1, and combined TGFBR1/PTEN by using siRNA; (E) Quantitative analysis showed the protein level of AGR2 in TGFBR1 siRNA group and TGFBR1/PTEN combined siRNA group were significantly higher than the control group (P < 0.001). Mean±SEM, ***, P<0.001.
Mentions: Recent studies revealed that TGFβ signaling plays a pivotal role in EMT and in cancer stem cell self-renewal [27]. Our previous work showed that mice with tissue specific deletion of tumor suppressor gene Pten in epithalia had spontaneous HNSCC (Fig. 6A). Pathologically, Pten conditional kock out (Pten cKO) mice tumor present as well differentiation keratinizing squamous cell carcinoma (8/8) with frequently observed extracellular keratin, similar to human Grade I SCC (Fig. 6B and high magnification in Fig. 6C). Tgfbr1conditional knock out (Tgfbr1cKO) mice Fig. 6D) tumor histologically present as aggressive strand or island growth pattern (Fig.6E). The transitional epithelial cell present spindle-type with extensive inflammation, abundant stromal cells, intratumor necrosis and low amount of keratin pearl formation (High magnification in Fig. 6F), which similar to human Grades II and III squamous cell carcinoma. Combined deletion of Tgfbr1/Pten in mice epithelia would lead to rapid tumor formation (Fig. 6G) with pathological infiltrating growth pattern (Fig. 6H) and poorly differentiated SCC (High magnification in Fig. 6I). To determine whether over-expression of AGR2 was related to high-grade squamous cell carcinoma in mice, we performed AGR2 immunohistochemistry and found that AGR2 was located mostly in the membrane and cytoplasm of the cancer cell of Tgfbr1 cKO mice and Tgfbr1/Pten 2cKO mice (n = 5, respectively). However, AGR2 staining was negative in wild type mice mucosa and focal weakly positive in Pten cKO mice HNSCC (Fig. 7A with quantification histoscore in Fig. 7B). The mRNA levels of AGR2 in Tgfbr1 cKO mice and Tgfbr1/Pten mice were significantly higher as compared with wild type mice mucosa (P < 0.001 respectively, Fig. 7C). We further knockdown TGFBR1 and/or PTEN in human HNSCC CAL27 and FaDu cell lines. After transfection with PTEN and/or TGFBR1 siRNA for 48 h, the protein was extracted and analyzed by Western blot. The protein level of AGR2 in PTEN, TGFBR1 as well as in combined PTEN/TGFBR1 knock down was significantly higher than scramble siRNA in CAL27 cell line (Fig. 7D and quantification in Fig. 7E) and quiet repeatable in FaDu cell line Supplementary Fig. S4E and 4F). The discovery above suggested that increased AGR2 protein level may be associated with lost function of TGFBR1.

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