pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigen.
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 80 kDa 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.
Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan.
Yasuyuki
Y
Matsumoto
Qing
Q
Zhang
Kaoru
K
Akita
Hiroshi
H
Nakada
Kazunori
K
Hamamura
Noriyo
N
Tokuda
Akiko
A
Tsuchida
Takeshi
T
Matsubara
Tomoko
T
Hori
Tetsuya
T
Okajima
Keiko
K
Furukawa
Takeshi
T
Urano
Koichi
K
Furukawa
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000378:metabolism*; D018827:Carcinoma, Lewis Lung / Q000201:enzymology / Q000235:genetics / Q000556:secondary*; D045744:Cell Line, Tumor; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D004338:Drug Combinations; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D007797:Laminin / Q000737:chemistry / Q000378:metabolism; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D051379:Mice; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D055503:Protein Multimerization; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism; D053668:Syndecan-1 / Q000737:chemistry / Q000378:metabolism
D015295:Antigens, Tumor-Associated, Carbohydrate; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D053668:Syndecan-1; C040286:Tn antigen; C059485:matrigel; D003094:Collagen; D017350:N-Acetylgalactosaminyltransferases; C470848:UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetylgalactosaminyltransferase 13, mouse