Agarwal Akhil Kumar, Srinivasan Nithya, Godbole Rashmi, More Shyam K, Budnar Srikanth, Gude Rajiv P, Kalraiya Rajiv D
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Kharghar, Navi Mumbai, 410210, India.
J Cancer Res Clin Oncol. 2015 Sep;141(9):1563-74. doi: 10.1007/s00432-015-1917-2. Epub 2015 Jan 23.
Expression of lysosome-associated membrane protein-1 (LAMP1) on the surface correlates with metastatic potential of B16 melanoma cells. Downregulation of their expression in high metastatic (B16F10) cells reduced their surface expression and metastatic potential. Present investigations explore if overexpression of LAMP1 on the surface of low metastatic (B16F1) cells augment their metastatic ability, and if so, how?
B16F1 cells were transduced with lentiviral vector carrying mutant-LAMP1 (Y386A) (mutLAMP1). Surface expression of LAMP1 and carbohydrates was analyzed by flow cytometry, immunofluorescence and/or immunoprecipitation and Western blotting. Cell spreading and motility were assessed on components of extracellular matrix (ECM) (fibronectin) and basement membrane (BM) (matrigel), and galectin-3-coated coverslips/plates. Metastatic potential was assessed using experimental metastasis assay.
Pre-incubation with anti-LAMP1 antibodies significantly reduced lung metastasis of B16F10 cells. Overexpression of mutLAMP1 significantly increased its surface expression on B16F1 cells, resulting in increased cellular spreading and motility on fibronectin and matrigel. LAMP1 is the major carrier of poly-N-acetyllactosamine (polyLacNAc) on B16F10 cells. However, significantly higher expression of mutLAMP1 had no effect on galectin-3 binding on cell surface or on spreading or motility of cells on galectin-3-coated coverslips/plates. These cells also failed to show any gain in metastatic ability. This could be because LAMP1 from these cells carried significantly lower levels of polyLacNAc in comparison with B16F10 cells.
PolyLacNAc on B16F10 cells and galectin-3 on lungs are the major participants in melanoma metastasis. Although surface LAMP1 promotes interactions with organ ECM and BM, carbohydrates on LAMP1 play a decisive role in dictating lung metastasis.
溶酶体相关膜蛋白1（LAMP1）在表面的表达与B16黑色素瘤细胞的转移潜能相关。在高转移（B16F10）细胞中下调其表达可降低其表面表达和转移潜能。目前的研究探讨低转移（B16F1）细胞表面LAMP1的过表达是否会增强其转移能力，若如此，其机制如何？
用携带突变型LAMP1（Y386A）（mutLAMP1）的慢病毒载体转导B16F1细胞。通过流式细胞术、免疫荧光和/或免疫沉淀以及蛋白质印迹分析LAMP1和碳水化合物的表面表达。在细胞外基质（ECM）（纤连蛋白）和基底膜（BM）（基质胶）以及半乳糖凝集素-3包被的盖玻片/平板上评估细胞铺展和运动能力。使用实验性转移测定评估转移潜能。
用抗LAMP1抗体预孵育可显著降低B16F10细胞的肺转移。mutLAMP1的过表达显著增加其在B16F1细胞表面的表达，导致细胞在纤连蛋白和基质胶上的铺展和运动能力增强。LAMP1是B16F10细胞上多聚N-乙酰乳糖胺（polyLacNAc）的主要载体。然而，mutLAMP1的显著更高表达对细胞表面半乳糖凝集素-3的结合或细胞在半乳糖凝集素-3包被的盖玻片/平板上的铺展或运动能力没有影响。这些细胞也未显示出转移能力的任何增强。这可能是因为与B16F10细胞相比，这些细胞的LAMP1携带的polyLacNAc水平显著更低。
B16F10细胞上的polyLacNAc和肺上的半乳糖凝集素-3是黑色素瘤转移的主要参与者。虽然表面LAMP1促进与器官ECM和BM的相互作用，但LAMP1上的碳水化合物在决定肺转移方面起决定性作用。
