Abstract
Cancer metastasis is the main cause of mortality associated with non-small-cell lung cancer (NSCLC), accounting for up to 70% of deaths among patients. The mechanisms underlying distal metastasis remain largely unknown. Golgi phosphoprotein 3 (GOLPH3) correlates negatively with overall survival in multiple tumors. In this study, we evaluated the function of GOLPH3 in NSCLC distal metastasis. GOLPH3 was expressed at high levels in samples from patients with NSCLC and was positively associated with clinicopathologic characteristics including clinical stage (P < 0.001), T (P = 0.001), N (P = 0.007), and M (P = 0.001) classification. Functionally, Transwell and wound-healing assays suggested that GOLPH3 overexpression enhances NSCLC cell migration and invasion abilities. Tumor-sphere formation and flow cytometry assays demonstrated that GOLPH3 overexpression enhances a stem cell-like phenotype of NSCLC cells. Metastasis models established by tail vein and intracardiac injection confirmed the pro-metastatic function of GOLPH3 in vivo. A subcutaneous tumor formation model confirmed that GOLPH3 overexpression increased the tumorigenicity of NSCLC cells. Mechanistically, gene set enrichment analysis revealed a positive association of GOLPH3 mRNA expression with WNT-activated gene signatures. Luciferase-reporter and nuclear extract assays showed that GOLPH3 overexpression enhances metastasis and tumorigenicity through activation of the WNT/β-catenin pathway. Immunoprecipitation-mass spectrometry and gene ontology analysis demonstrated that GOLPH3 interacts with cytoskeleton-associated protein 4 (CKAP4) in exosome-mediated distal metastasis. We found that GOLPH3 decreased the amount of plasma membrane-localized CKAP4 and increased the amount of exosome-localized CKAP4 to promote the formation of CKAP4-containing exosomes. Furthermore, we demonstrated that CKAP4 binds exosomal WNT3A to enhance its secretion. Therefore, the GOLPH3/CKAP4 axis plays a crucial role in promoting exosomal-WNT3A secretion to enhance and maintain the stem-like phenotype and metastasis in NSCLC, thus indicating the therapeutic potential of GOLPH3 in patients with NSCLC metastasis.
Highlights
Lung cancer has a high rate of mortality and remains the second most commonly diagnosed cancer worldwide [1]
We found that Golgi phosphoprotein 3 (GOLPH3) decreased the amount of plasma membrane-localized cytoskeleton-associated protein 4 (CKAP4) and increased the amount of exosome-localized CKAP4 to promote the formation of CKAP4-containing exosomes
To clarify the role of GOLPH3 in non-small cell lung cancer (NSCLC) metastasis in vivo, we Analysis of a publicly available NSCLC dataset (NCBI/GEO/ GSE75037) revealed that GOLPH3 mRNA levels were significantly higher in NSCLC cancer tissues compared with those in normal established a mouse model by intravenous injection of A549-lucvector, A549-luc-GOLPH3, A549-luc-RNAi-vector, or A549-lucGOLPH3-short hairpin RNA (shRNA)#2 cells (1 × 106) into the tail vein
Summary
Lung cancer has a high rate of mortality and remains the second most commonly diagnosed cancer worldwide [1]. Based on clinical histology, ~85% of lung cancer cases are diagnosed as non-small cell lung cancer (NSCLC) with an overall 5-year survival rate of only 15% [2]. It has been reported that metastasis is responsible for >70% of NSCLC deaths, and the majority of patients with advanced-stage NSCLC survive for only 18 months after diagnosis [3]. Determining the mechanism of distal metastasis is important to improve the poor prognosis of patients with NSCLC and to identify biomarkers or drug targets. Wang et al found that exosomal miR-105 targets the mRNA encoding the tight junction protein ZO-1 to induce destruction of tight junctions, which act as natural barriers to the movement of cells
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