Regulation of RON tyrosine kinase-mediated invasion of breast cancer cells.
RON (recepteur d'origine nantais), a tyrosine kinase receptor for macrophage-stimulating protein (MSP) was implicated in tumor progression. However, it was not investigated how this important oncogene is regulated. We show that MSP promotes invasion of MDA MB 231 and MDA MB 468 but not MCF-7 breast cancer cells. Reverse transcription-PCR and Western analysis indicated the expression of RON message and protein, respectively, in MDA MB 231 and MDA MB 468 cells but not in MCF-7 cells. RON expression correlated with Sp1 expression. Initial analysis of a 1.2-kb and 400-bp RON promoter in MDA MB 231 and MDA MB 468 cells suggested the presence of all the necessary regulatory elements within 400 bp from the transcription start site. Site-directed mutagenesis of the 400-bp RON promoter revealed that the overlapping Sp1 sites at-94 (Sp1-3/4) and Sp1 site at -113 (Sp1-5) are essential for RON gene transcription. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis indicated that Sp1 binding to these sites is required for RON promoter activity. Ectopic Sp1 expression in Sp1 null SL2 cells confirmed the involvement of these Sp1 sites in the regulation of oncogenic RON tyrosine kinase. Treatment of MDA MB 231 cells with mithramycin A, an inhibitor of Sp1 binding, or siRNA knock-down of Sp1 blocked RON gene expression and MSP-mediated invasion of MDA MB 231 cells. This is the first report demonstrating a clear link between Sp1-dependent RON tyrosine kinase expression and invasion of breast carcinoma cells.
Thangasamy
Amalraj
A
Department of Medicine, Division of Hematology and Medical Oncology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Rogge
Jessica
J
Ammanamanchi
Sudhakar
S
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D004330:Drosophila; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D008926:Plicamycin / Q000031:analogs & derivatives / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; C066851:mithramycin A; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases; D008926:Plicamycin