pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigen.
pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigen.
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DOI：
10.1016/j.bbrc.2012.01.086
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发表时间：
2012-03
影响因子：
3.1
通讯作者：
Yasuyuki Matsumoto;Qing Zhang;Kaoru Akita;H. Nakada;K. Hamamura;N. Tokuda;A. Tsuchida;T. Matsubara-T.-Mats
中科院分区：
文献类型：
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作者：
Yasuyuki Matsumoto;Qing Zhang;Kaoru Akita;H. Nakada;K. Hamamura;N. Tokuda;A. Tsuchida;T. Matsubara-T.-Mats
为了分析癌症转移的机制，通过将小鼠刘易斯肺癌亚系H7和C4重复注射到C57 BL/6小鼠中来获得高转移亚系（H7-A、H7-Lu、H7-O、C4-sc和C4-ly）。这些亚系在体外表现出增加的增殖和侵袭活性。神经节苷脂档案表现出较低的表达GM1在高转移的亚系比亲本系。然后，我们通过在H7细胞中稳定沉默GM1合酶来建立GM1-Si-1和GM1-Si-2。这些GM1敲低的克隆表现出增加的增殖和侵袭。然后，我们通过DNA微阵列在C4与C4-ly或H7与H7（GM1-Si）的组合中探索表达水平显著改变的基因。因此，pp-GalNAc-T13基因被鉴定为在高转移亚系中上调的基因。将pp-GalNAc-T13 cDNA稳定转染入C4（T13-TF）导致侵袭和运动增加。然后，使用各种抗体和凝集素进行免疫印迹和流式细胞术。只有抗三聚体Tn抗体（mAb MLS128）显示T13-TF克隆中三聚体Tn抗原的表达水平增加。此外，通过mAb MLS128进行免疫沉淀/免疫印迹，导致鉴定出携带三聚体Tn抗原（即Syndecan-1）的80 kDa条带。内源性pp-GalNAc-T13在C4-sc（T13-KD）中的稳定沉默揭示了通过皮下注射T13-KD克隆产生的原发性肿瘤显示出与筋膜和腹膜的较低聚结，并且与对照克隆相比显著减少了肺转移。这些数据表明pp-GalNAc-T13基因的高表达在Syndecan-1上产生三聚体Tn抗原，导致转移增强。
In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.