Regulation of RON Tyrosine Kinase-mediated Invasion of Breast Cancer Cells

Amalraj Thangasamy,Jessica Rogge,Sudhakar Ammanamanchi

doi:10.1074/jbc.m706957200

Amalraj Thangasamy, Jessica Rogge + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m706957200

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Abstract

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.

Highlights

The results presented in this manuscript show that macrophage-stimulating protein (MSP) stimulates the invasion of MDA MB 231 and MDA MB 468 breast cancer cells, which is correlated to the expression of MSP receptor, RON tyrosine kinase
We demonstrate that all other regulatory elements are dispensable except the overlapping Sp1 sites at Ϫ94 bp and another Sp1 site at Ϫ113 bp for RON promoter activity and RON gene expression in breast cancer cells
MSP Promotes Invasion of Breast Cancer Cells—To determine whether MSP stimulates the invasive phenotype of breast cancer cells, we have carried out in vitro Matrigel assay using MCF-7, MDA MB 231, and MDA MB 468 breast cancer cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—MCF-7, MDA MB 231, and MDA MB 468 breast cancer cells were obtained from American Type Culture Collection. To determine the effect of siRNA Sp1 knock-down on RON expression, MDA MB 231 cells were transfected with 100 nM scrambled or Sp1 siRNA, and Western analysis was performed 48 h following transfection. To verify whether similar levels of Sp1 were expressed in the ectopic Sp1transfected SL2 cells, Western analysis using Sp1 and actin antibodies was performed on the total cell lysates from control pGl3, wild type, and mutant RON promoter-luciferase construct-transfected SL2 cells. The wild type or mutant RON-Luc constructs (1 ␮g) or control null vector without the RON promoter insert (pGL3) were transiently transfected into MDA MB 231 and MDA MB 468 breast cancer cells using Lipofectamine (Invitrogen). To determine the ectopic Sp1 effects on wild type/ mutant RON promoter, SL2 cells were transiently transfected with the RON promoter-luciferase reporter constructs along with pPac empty vector or pPac-Sp1 using cellfectin (Invitrogen) as a reagent. The following primers were used for PCR to generate a 293-bp fragment covering the Sp1-binding sites on the RON promoter: forward, 5Ј-CTC CAA GGG CCG GAA GAG TCG GAT GG-3Ј; and reverse, 5Ј-TTA AGC AGC GGT CCC GAC AGC CCC AA-3Ј

RESULTS

Wild Mutant

DISCUSSION

ADDITIONS AND CORRECTIONS