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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

Interaction of HE4 and recombinant annexin II proteins. A, immunoprecipitation (IP) of annexin II/HE4 complex by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1and 2, control proteins; lanes 3, 4 and 5, IP by anti-HE4 antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. IB, immunoblotting. B, immunoprecipitation of annexin II/ HE4 complex by anti-annexin II antibody and Western blot analysis with anti- HE4 antibody. Lanes 1 and 2, control proteins; lanes 3, 4 and 5, IP by anti-annexin II antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. Lane6, negative control (Ig G). C, IP of membrane and cytoplasmic proteins by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. D, IP of membrane and cytoplasmic proteins by anti-annexin II antibody and Western blot analysis with anti-HE4 antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. Lane 5, negative control (Ig G). E, binding of truncated annexin II mutants, A2-del15 and A2-del26, to GST-HE4. Complexes were immunoprecipitated by anti-HE4 antibody , followed by Western blot analysis with anti-His-tagged antibody. Full-size annexin II was used in lanes 1, A2-del15 was used in lanes 2, and A2-del26 was used in lanes 3. All the plus sign under the panel mean antibodies used.
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Fig2: Interaction of HE4 and recombinant annexin II proteins. A, immunoprecipitation (IP) of annexin II/HE4 complex by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1and 2, control proteins; lanes 3, 4 and 5, IP by anti-HE4 antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. IB, immunoblotting. B, immunoprecipitation of annexin II/ HE4 complex by anti-annexin II antibody and Western blot analysis with anti- HE4 antibody. Lanes 1 and 2, control proteins; lanes 3, 4 and 5, IP by anti-annexin II antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. Lane6, negative control (Ig G). C, IP of membrane and cytoplasmic proteins by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. D, IP of membrane and cytoplasmic proteins by anti-annexin II antibody and Western blot analysis with anti-HE4 antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. Lane 5, negative control (Ig G). E, binding of truncated annexin II mutants, A2-del15 and A2-del26, to GST-HE4. Complexes were immunoprecipitated by anti-HE4 antibody , followed by Western blot analysis with anti-His-tagged antibody. Full-size annexin II was used in lanes 1, A2-del15 was used in lanes 2, and A2-del26 was used in lanes 3. All the plus sign under the panel mean antibodies used.

Mentions: To identify specific HE4 binding proteins, co-immunoprecipitation assays were performed in the ovarian cancer cell line OVCAR-3. Proteins co-immunoprecipitating with HE4 were separated by electrophoresis and detected by Coomassie brilliant blue staining (Figure 1A). Protein bands were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which resulted in the identification of ANXA2 as the band with the highest Mascot score. Binding between HE4 and ANXA2 was validated via in vivo and in vitro experiments. Firstly, OVCAR-3, ES-2 and CaoV-3 ovarian cancer cells lysates were precipitated with antibodies specific to HE4 and ANXA2, and the structures of ANXA2 and HE4 in ovarian cancer cells were examined. The results of these experiments shown in Figure 2 demonstrate that HE4 and annexin II form a complex that can be precipitated with either anti-HE4 or anti-annexin II antibodies and detected by Western blot with anti-annexin II (Figure 2A) or anti-HE4 (Figure 2B) antibodies, respectively. To determine the distribution of HE4 and ANXA2 in ES-2 and CaoV-3 ovarian cancer cells, membrane and cytoplasmic proteins were isolated and subjected to co-immunoprecipitation analysis. The results showed the presence of HE4 and ANXA2 in the membrane and plasma and confirmed that they are binding partners (Figure 2C, D).Figure 1


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)

Interaction of HE4 and recombinant annexin II proteins. A, immunoprecipitation (IP) of annexin II/HE4 complex by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1and 2, control proteins; lanes 3, 4 and 5, IP by anti-HE4 antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. IB, immunoblotting. B, immunoprecipitation of annexin II/ HE4 complex by anti-annexin II antibody and Western blot analysis with anti- HE4 antibody. Lanes 1 and 2, control proteins; lanes 3, 4 and 5, IP by anti-annexin II antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. Lane6, negative control (Ig G). C, IP of membrane and cytoplasmic proteins by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. D, IP of membrane and cytoplasmic proteins by anti-annexin II antibody and Western blot analysis with anti-HE4 antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. Lane 5, negative control (Ig G). E, binding of truncated annexin II mutants, A2-del15 and A2-del26, to GST-HE4. Complexes were immunoprecipitated by anti-HE4 antibody , followed by Western blot analysis with anti-His-tagged antibody. Full-size annexin II was used in lanes 1, A2-del15 was used in lanes 2, and A2-del26 was used in lanes 3. All the plus sign under the panel mean antibodies used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232681&req=5

Fig2: Interaction of HE4 and recombinant annexin II proteins. A, immunoprecipitation (IP) of annexin II/HE4 complex by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1and 2, control proteins; lanes 3, 4 and 5, IP by anti-HE4 antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. IB, immunoblotting. B, immunoprecipitation of annexin II/ HE4 complex by anti-annexin II antibody and Western blot analysis with anti- HE4 antibody. Lanes 1 and 2, control proteins; lanes 3, 4 and 5, IP by anti-annexin II antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. Lane6, negative control (Ig G). C, IP of membrane and cytoplasmic proteins by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. D, IP of membrane and cytoplasmic proteins by anti-annexin II antibody and Western blot analysis with anti-HE4 antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. Lane 5, negative control (Ig G). E, binding of truncated annexin II mutants, A2-del15 and A2-del26, to GST-HE4. Complexes were immunoprecipitated by anti-HE4 antibody , followed by Western blot analysis with anti-His-tagged antibody. Full-size annexin II was used in lanes 1, A2-del15 was used in lanes 2, and A2-del26 was used in lanes 3. All the plus sign under the panel mean antibodies used.
Mentions: To identify specific HE4 binding proteins, co-immunoprecipitation assays were performed in the ovarian cancer cell line OVCAR-3. Proteins co-immunoprecipitating with HE4 were separated by electrophoresis and detected by Coomassie brilliant blue staining (Figure 1A). Protein bands were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which resulted in the identification of ANXA2 as the band with the highest Mascot score. Binding between HE4 and ANXA2 was validated via in vivo and in vitro experiments. Firstly, OVCAR-3, ES-2 and CaoV-3 ovarian cancer cells lysates were precipitated with antibodies specific to HE4 and ANXA2, and the structures of ANXA2 and HE4 in ovarian cancer cells were examined. The results of these experiments shown in Figure 2 demonstrate that HE4 and annexin II form a complex that can be precipitated with either anti-HE4 or anti-annexin II antibodies and detected by Western blot with anti-annexin II (Figure 2A) or anti-HE4 (Figure 2B) antibodies, respectively. To determine the distribution of HE4 and ANXA2 in ES-2 and CaoV-3 ovarian cancer cells, membrane and cytoplasmic proteins were isolated and subjected to co-immunoprecipitation analysis. The results showed the presence of HE4 and ANXA2 in the membrane and plasma and confirmed that they are binding partners (Figure 2C, D).Figure 1

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