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Human epididymis protein 4 in association with Annexin II promotes invasion and metastasis of ovarian cancer cells.

Zhuang H, Tan M, Liu J, Hu Z, Liu D, Gao J, Zhu L, Lin B - Mol. Cancer (2014)

Bottom Line: Annexin II was identified as an HE4 interacting protein.HE4 gene interference downregulated the expression of MAPK and the FOCAL adhesion signaling pathway-associated molecules MKNK2 and LAMB2, and HE4 protein supplementation reversed this effect.The binding interaction between HE4 and annexin II activates the MAPK and FOCAL adhesion signaling pathways, promoting ovarian cancer cell invasion and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, China Medical University Shengjing Hospital, No, 36 Sanhao Street, Heping District, Shenyang, Liaoning Province 110004, P,R, China. linbei88@hotmail.com.

ABSTRACT

Background: The objective of the present study was to identify human epididymis protein 4 (HE4) interacting proteins and explore the mechanisms underlying their effect on ovarian cancer cell invasion and metastasis.

Methods: HE4 interacting proteins were identified by mass spectrometry and validated by co-immunoprecipitation and pull-down assays. The scratch test, the Transwell assay and animal experiments were used to assess the invasive and metastatic abilities of ovarian cancer cells before and after transfection and HE4 protein treatment. HE4 and annexin II protein expression in epithelial ovarian tissues was detected by immunohistochemistry, and the relation between their expression levels was examined.

Results: Annexin II was identified as an HE4 interacting protein. HE4 and annexin II binding interaction promoted ovarian cancer cell invasion and metastasis. HE4 and annexin II expression levels were significantly higher in malignant epithelial ovarian tissues than in benign and normal epithelial ovarian tissues, and they were higher in tissues with lymph node metastases than in those without. HE4 gene interference downregulated the expression of MAPK and the FOCAL adhesion signaling pathway-associated molecules MKNK2 and LAMB2, and HE4 protein supplementation reversed this effect.

Conclusion: The binding interaction between HE4 and annexin II activates the MAPK and FOCAL adhesion signaling pathways, promoting ovarian cancer cell invasion and metastasis.

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Related in: MedlinePlus

Expression Patterns of HE4 and ANXA2 in ovarian tissues. A, Cells at a density of 1 × 106 in 200 μl were injected into nude mice through the tail vein. Macroscopic view of lung metastatic nodes after inoculation with cancer cells. B, H&E staining in lung tissues of nude mice. High expression (a, original magnification × 200), low expression (b), and mock (c) groups are shown. C, Immunohistochemical staining of lung tissues of nude mice. High expression (a1, a2; original magnification × 200), low expression (b1, b2), and mock (c1, c2) groups are shown. D, Representative images of the peritoneal metastasis results. High expression (a), low expression (b), and mock (c) groups are shown. E, Real time PCR results show the expression of MKNK2 and LAMB2 in ovarian cancer CaoV-3 cells. Lane 1, mock group; lane 2, HE4 low expression groups; lane 3, HE4 low expression cells treated with HE4 active protein; lane 4, HE4 high expression groups. F, Immunohistochemical staining of ovarian malignant tumors (serous tumors, 1 and 2; mucinous tumors, 3 and 4; borderline tumors, 5 and 6; benign tumors, 7 and 8); and normal ovarian tissues, 9 and 10). HE4 (1, 3, 5, 7 and 9) and ANXA2 (2, 4, 6, 8 and 10; original magnification × 200).
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Fig5: Expression Patterns of HE4 and ANXA2 in ovarian tissues. A, Cells at a density of 1 × 106 in 200 μl were injected into nude mice through the tail vein. Macroscopic view of lung metastatic nodes after inoculation with cancer cells. B, H&E staining in lung tissues of nude mice. High expression (a, original magnification × 200), low expression (b), and mock (c) groups are shown. C, Immunohistochemical staining of lung tissues of nude mice. High expression (a1, a2; original magnification × 200), low expression (b1, b2), and mock (c1, c2) groups are shown. D, Representative images of the peritoneal metastasis results. High expression (a), low expression (b), and mock (c) groups are shown. E, Real time PCR results show the expression of MKNK2 and LAMB2 in ovarian cancer CaoV-3 cells. Lane 1, mock group; lane 2, HE4 low expression groups; lane 3, HE4 low expression cells treated with HE4 active protein; lane 4, HE4 high expression groups. F, Immunohistochemical staining of ovarian malignant tumors (serous tumors, 1 and 2; mucinous tumors, 3 and 4; borderline tumors, 5 and 6; benign tumors, 7 and 8); and normal ovarian tissues, 9 and 10). HE4 (1, 3, 5, 7 and 9) and ANXA2 (2, 4, 6, 8 and 10; original magnification × 200).

Mentions: In vivo studies were performed to analyze the metastasis of ovarian cancer. An autopsy revealed a large number of lung metastatic nodules in the ANXA2 high expression group, whereas almost no metastatic nodules were observed in the low expression groups (Figure 5A). Few metastatic nodules were detected in the mock groups (Figure 5A). The results of H&E staining showed that the mice in all the high expression groups were metastasized, whereas only 2 mice were metastasized among the mock groups. There was no metastasis in the low expression groups during the experiment. The matastasis rates in the ANXA2 high expression, mock and low expression groups were 100.00%, 40% and 0%, respectively. The high expression groups displayed the highest matastasis rates, which were significantly higher than those of the mock groups (P < 0.05, Figure 5B). Matastasis rates in the mock groups were markedly higher than those in the low expression groups (P < 0.05). The results of immunohistochemistry were consistent with those of H&E staining (Figure 5C). The peritoneal metastasis results are all the same as the results of lung metastasis (Figure 5D). The matastasis rates in the ANXA2 high expression, mock and low expression groups were 100.00%, 40% and 0%, respectively (P < 0.05). The upregulation of annexin II promoted the metastasis of ovarian cancer, whereas the downregulation of annexin II decreased metastasis.Figure 5


Human epididymis protein 4 in association with Annexin II promotes invasion and metastasis of ovarian cancer cells.

Zhuang H, Tan M, Liu J, Hu Z, Liu D, Gao J, Zhu L, Lin B - Mol. Cancer (2014)

Expression Patterns of HE4 and ANXA2 in ovarian tissues. A, Cells at a density of 1 × 106 in 200 μl were injected into nude mice through the tail vein. Macroscopic view of lung metastatic nodes after inoculation with cancer cells. B, H&E staining in lung tissues of nude mice. High expression (a, original magnification × 200), low expression (b), and mock (c) groups are shown. C, Immunohistochemical staining of lung tissues of nude mice. High expression (a1, a2; original magnification × 200), low expression (b1, b2), and mock (c1, c2) groups are shown. D, Representative images of the peritoneal metastasis results. High expression (a), low expression (b), and mock (c) groups are shown. E, Real time PCR results show the expression of MKNK2 and LAMB2 in ovarian cancer CaoV-3 cells. Lane 1, mock group; lane 2, HE4 low expression groups; lane 3, HE4 low expression cells treated with HE4 active protein; lane 4, HE4 high expression groups. F, Immunohistochemical staining of ovarian malignant tumors (serous tumors, 1 and 2; mucinous tumors, 3 and 4; borderline tumors, 5 and 6; benign tumors, 7 and 8); and normal ovarian tissues, 9 and 10). HE4 (1, 3, 5, 7 and 9) and ANXA2 (2, 4, 6, 8 and 10; original magnification × 200).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Expression Patterns of HE4 and ANXA2 in ovarian tissues. A, Cells at a density of 1 × 106 in 200 μl were injected into nude mice through the tail vein. Macroscopic view of lung metastatic nodes after inoculation with cancer cells. B, H&E staining in lung tissues of nude mice. High expression (a, original magnification × 200), low expression (b), and mock (c) groups are shown. C, Immunohistochemical staining of lung tissues of nude mice. High expression (a1, a2; original magnification × 200), low expression (b1, b2), and mock (c1, c2) groups are shown. D, Representative images of the peritoneal metastasis results. High expression (a), low expression (b), and mock (c) groups are shown. E, Real time PCR results show the expression of MKNK2 and LAMB2 in ovarian cancer CaoV-3 cells. Lane 1, mock group; lane 2, HE4 low expression groups; lane 3, HE4 low expression cells treated with HE4 active protein; lane 4, HE4 high expression groups. F, Immunohistochemical staining of ovarian malignant tumors (serous tumors, 1 and 2; mucinous tumors, 3 and 4; borderline tumors, 5 and 6; benign tumors, 7 and 8); and normal ovarian tissues, 9 and 10). HE4 (1, 3, 5, 7 and 9) and ANXA2 (2, 4, 6, 8 and 10; original magnification × 200).
Mentions: In vivo studies were performed to analyze the metastasis of ovarian cancer. An autopsy revealed a large number of lung metastatic nodules in the ANXA2 high expression group, whereas almost no metastatic nodules were observed in the low expression groups (Figure 5A). Few metastatic nodules were detected in the mock groups (Figure 5A). The results of H&E staining showed that the mice in all the high expression groups were metastasized, whereas only 2 mice were metastasized among the mock groups. There was no metastasis in the low expression groups during the experiment. The matastasis rates in the ANXA2 high expression, mock and low expression groups were 100.00%, 40% and 0%, respectively. The high expression groups displayed the highest matastasis rates, which were significantly higher than those of the mock groups (P < 0.05, Figure 5B). Matastasis rates in the mock groups were markedly higher than those in the low expression groups (P < 0.05). The results of immunohistochemistry were consistent with those of H&E staining (Figure 5C). The peritoneal metastasis results are all the same as the results of lung metastasis (Figure 5D). The matastasis rates in the ANXA2 high expression, mock and low expression groups were 100.00%, 40% and 0%, respectively (P < 0.05). The upregulation of annexin II promoted the metastasis of ovarian cancer, whereas the downregulation of annexin II decreased metastasis.Figure 5

Bottom Line: Annexin II was identified as an HE4 interacting protein.HE4 gene interference downregulated the expression of MAPK and the FOCAL adhesion signaling pathway-associated molecules MKNK2 and LAMB2, and HE4 protein supplementation reversed this effect.The binding interaction between HE4 and annexin II activates the MAPK and FOCAL adhesion signaling pathways, promoting ovarian cancer cell invasion and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, China Medical University Shengjing Hospital, No, 36 Sanhao Street, Heping District, Shenyang, Liaoning Province 110004, P,R, China. linbei88@hotmail.com.

ABSTRACT

Background: The objective of the present study was to identify human epididymis protein 4 (HE4) interacting proteins and explore the mechanisms underlying their effect on ovarian cancer cell invasion and metastasis.

Methods: HE4 interacting proteins were identified by mass spectrometry and validated by co-immunoprecipitation and pull-down assays. The scratch test, the Transwell assay and animal experiments were used to assess the invasive and metastatic abilities of ovarian cancer cells before and after transfection and HE4 protein treatment. HE4 and annexin II protein expression in epithelial ovarian tissues was detected by immunohistochemistry, and the relation between their expression levels was examined.

Results: Annexin II was identified as an HE4 interacting protein. HE4 and annexin II binding interaction promoted ovarian cancer cell invasion and metastasis. HE4 and annexin II expression levels were significantly higher in malignant epithelial ovarian tissues than in benign and normal epithelial ovarian tissues, and they were higher in tissues with lymph node metastases than in those without. HE4 gene interference downregulated the expression of MAPK and the FOCAL adhesion signaling pathway-associated molecules MKNK2 and LAMB2, and HE4 protein supplementation reversed this effect.

Conclusion: The binding interaction between HE4 and annexin II activates the MAPK and FOCAL adhesion signaling pathways, promoting ovarian cancer cell invasion and metastasis.

Show MeSH
Related in: MedlinePlus