Nasopharyngeal carcinoma (NPC) has a highly increased incidence rate (20/100,000) in Southern regions of China, while being rare in the rest of the world. NPC is a malignant type of cancer due to its high occurrence rate of metastasis; however, biomarkers for effective diagnosis and treatment are yet to be identified. Annexin A1 is a glucocorticoid‑regulated member of a large superfamily of calcium and phospholipid‑binding proteins and has been shown to have important roles in tumor development and progression, and was demonstrated to be a prognostic biomarker for head and neck cancer types. A previous study by our group showed that Annexin A1 was decreased in NPC tissue as compared with normal adjacent tissue. To investigate whether Annexin A1 is a potential biomarker for NPC, the present study assessed the effect of the Annexin A1 on the biological behavior (i.e., invasion and metastasis) of the highly metastatic NPC cell line 5‑8F and the non‑metastatic NPC cell line 6‑10B. The expression levels of Annexin A1 in the above two cell lines were determined by western blot analysis. Next, the recombinant plasmid pEGFP‑C1‑Annexin A1 and the small interfering (si)RNA plasmid pRNAT‑U6.1‑Annexin A1 were used and stably transfected into 5‑8F and 6‑10B cells, respectively. These established recombinant cell lines were then used to study the up- and downregulation of Annexin A1, respectively. The correlation of Annexin A1 expression levels with the biological behavior of NPC cell lines was analyzed using a cell proliferation assay, flow cytometry, soft agar colony formation assay, as well as Transwell invasion and migration assays. The results demonstrated that upregulation of Annexin A1 suppressed the proliferation, invasion and migration of NPC cells, while downregulation of Annexin A1 promoted the proliferation, invasion and migration of NPC cells. These findings suggested that Annexin A1 may be a potential biomarker for the development and prognosis of NPC, and its dysregulation may have an important role in its underlying pathogenesis.
Background: Cathepsin D is a lysosomal aspartyl protease. Our previous studies have suggested that cathepsin D plays an important role in invasion and metastasis of nasopharyngeal carcinoma (NPC).Methods: To identify proteins that interact with cathepsin D and gain an insight into the role of cathepsin D in invasion and metastasis of NPC, co-immunoprecipitation (co-IP) combined with mass spectrometry (MS)-based proteomics approach was used. Cathepsin D associated proteins were identified by MS. Protein-protein interaction network were analyzed by bioinformatics including gene ontology (GO), function clustering. And co-IP and western blotting confirmation were for protein-protein interaction. The expression of cathepsin D, epidermal growth factor receptor (EGFR) and heat-shock protein 90A (HSP90A) in NPC were detected by immunohistochemistry (IHC). And the invasion and metastasis capability were detected by Transwell invasion assay.Results: One hundred and forty-one cathepsin D associated proteins were identified, including EGFR and HSP90A, proteins clearly associated with tumor invasion and metastasis. The interaction of these two proteins with cathepsin D was further validated by co-IP followed by western blotting. The 141 proteins were classified into 12 function-related groups. Protein-protein interaction network analysis indicated that cathepsin D might have a significance on invasion and metastasis of NPC by interacting with EGFR and HSP90A. Indeed, cathepsin D/EGFR/HSP90A could form complexes in NPC cells. In addition, overexpression of cathepsin D in NPC cells not only up-regulated EGFR and HSP90A, but also increased the invasive ability of NPC cells.Conclusions: Cathepsin D could enhance the invasion and metastasis capability of NPC cells may through binding to EGFR and HSP90A and triggering the activation of the signaling pathways.
Background: Cathepsin D is a lysosomal aspartyl protease. Our previous studies have suggested that cathepsin D plays an important role in invasion and metastasis of nasopharyngeal carcinoma (NPC). Methods: To identify proteins that interact with cathepsin D and gain an insight into the role of cathepsin D in invasion and metastasis of NPC, co-immunoprecipitation (co-IP) combined with mass spectrometry (MS)-based proteomics approach was used. Cathepsin D associated proteins were identified by MS. Protein-protein interaction network were analyzed by bioinformatics including gene ontology (GO), function clustering. And co-IP and western blotting confirmation were for protein-protein interaction. The expression of cathepsin D, epidermal growth factor receptor (EGFR) and heat-shock protein 90A (HSP90A) in NPC were detected by immunohistochemistry (IHC). And the invasion and metastasis capability were detected by Transwell invasion assay. Results: One hundred and forty-one cathepsin D associated proteins were identified, including EGFR and HSP90A, proteins clearly associated with tumor invasion and metastasis. The interaction of these two proteins with cathepsin D was further validated by co-IP followed by western blotting. The 141 proteins were classified into 12 function-related groups. Protein-protein interaction network analysis indicated that cathepsin D might have a significance on invasion and metastasis of NPC by interacting with EGFR and HSP90A. Indeed, cathepsin D/EGFR/HSP90A could form complexes in NPC cells. In addition, overexpression of cathepsin D in NPC cells not only up-regulated EGFR and HSP90A, but also increased the invasive ability of NPC cells.
Conclusions: Cathepsin D could enhance the invasion and metastasis capability of NPC cells may through binding to EGFR and HSP90A and triggering the activation of the signaling pathways.
Annexin A1 is a member of a large superfamily of glucocorticoid-regulated, calcium- and phospholipid-binding proteins. Our previous studies have shown that the abnormal expression of Annexin A1 is related to the occurrence and development of nasopharyngeal carcinoma (NPC). To understand the roles of Annexin A1 in the tumorigenesis of NPC, targeted proteomic analysis was performed on Annexin A1-associated proteins from NPC cells. We identified 436 proteins associated with Annexin A1, as well as two Annexin A1-interacted key proteins, S100A9 and Vimentin, which were confirmed by co-immunoprecipitation. Gene function classification revealed that the Annexin A1-associated proteins can be grouped into 21 clusters based on their molecular functions. Protein–protein interaction analysis indicated that Annexin A1 /S100A9/Vimentin interactions may be involved in the invasion and metastasis of NPC because they can form complexes in NPC cells. The down-regulation of Annexin A1 in NPC may lead to the overexpression of S100A9/Vimentin, which may increase the possibility of the invasion ability of NPC cells by adjusting the function of cytoskeleton proteins. Results suggested that the biological functions of Annexin A1 in NPC were diverse, and that Annexin A1 can inhibit the in vitro invasive ability of NPC cells through Annexin A1 /S100A9/Vimentin interaction.
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