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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Lucia Knopfová, Petr Beneš, Lucie Pekarčíková, Markéta Hermanová, Michal Masařík, Zuzana Pernicová, Karel Souček, Jan Šmarda
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-15) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LK and PB carried out experiments in vitro and analyzed results as well as took part in writing the manuscript. LP participated in performing transactivation assays. MM, LK, KS and ZP participated in in vivo experiments. MH provided histological analysis. JS supervised the experimental work, participated in data analysis and interpretation of results and revised the manuscript. All authors read and approved the manuscript.

Abstract

Background

The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells.

Results

Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP) 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner.

Conclusions

This study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.
Zusatzmaterial
Additional file 1: Figure S1 Kinetics of c-Myb-induced migration and Matrigel invasion of MDA-MB-231MYBup cells. Cell migration/invasion was analyzed in real time by the xCELLigence RTCA as described in Figure 3. The panels show the average cell indexes at indicated time points from seven (migration) and five (invasion) independent experiments. Error bars indicate standard deviations. Asterisks indicate significant (p < 0.05) differences in the migration/Matrigel invasion of the myb-less vector-transfected cells and MYBup cells (M2, M5) as determined by the t-test. (PDF 119 KB)
Additional file 2: Figure S2 siRNA-mediated c- myb silencing reduces migration and Matrigel invasion activities of MDA-MB-231MYBup cells. MDA-MB-231MYBup cells were transfected with c-myb (MYB) or control (ctrl) siRNAs as described in the Material and Methods. The level of c-Myb protein in these cells was determined by immunoblotting. A representative western blot is presented (A). Migration and invasion activities of the same cells were determined by the xCELLigence RTCA. The chart shows the representative outcomes of the kinetics analysis of cell migration (B). The average cell indexes at the 6-h (migration, left) and 12-h (invasion, right) time points, respectively, from four independent measurements are shown (C). Error bars indicate standard deviations. Asterisks indicate significant (p < 0.05) differences in the migration/invasion rates of the cells transfected with c-myb siRNA and control siRNA as determined by the t-test. (PDF 131 KB)
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Additional file 3: Figure S3 c-Myb-induced Matrigel invasion is sensitive to the MMP inhibitor GM6001. MDA-MB-231MYBup cells (M2, M5) were starved in the serum-free medium for 8 h and processed for cell invasion assay as described in the Material and Methods. The membranes were coated with the diluted Matrigel. The broad-spectrum MMP inhibitor GM6001 (Sigma) at final concentration of 20 μM or DMSO were added to the bottom and upper chambers of indicated wells of CIM-plate 16. The bottom chambers were filled with complete medium containing 10% FCS as a chemoattractant. (A) The chart shows the representative outcomes of kinetics analysis of Matrigel invasion. (B) The columns show the average cell indexes in 12 h time point from three independent measurements. Error bars indicate standard deviations. Asterisks indicate significant (p < 0.05) differences in Matrigel invasion of the GM6001-treated MDA-MB-231MYBup cells and DMSO-treated MDA-MB-231MYBup controls as determined by the t-test. (PDF 578 KB)
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Additional file 4: Figure S4 Histological examination of lungs, liver and bones of BALB/c mice orthotopically injected with c- myb overexpressing (MYBup) and control 4T1 cells. Lungs were fixed in Bouin's solution. Liver and bones were harvested and fixed in 10% buffered formalin. Tissues were processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin. Bones were decalcified overnight before embedding. (A) The mean number of pulmonary metastatic lesions as determined by histological examination. Every 10th consecutive section was examined for the presence of metastases (mts). (B) Semi-quantitative evaluation of liver metastasis: + rare microscopic neoplastic lesions, ++ numerous microscopic lesion, +++ numerous extensive neoplastic lesions. (C) Semi-quantitative evaluation of skeletal metastasis: + epithelial tumor cell infiltration and bone destruction, ++ tumor cell infiltration and bone destruction with the invasion of soft tissues. (PDF 66 KB)
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Additional file 5: Figure S5 c-Myb upregulates Slug in MDA-MB-231MYBup cells. The nontransfected (wt), the myb-less vector-transfected (vector) and MYBup (M2, M5) cells were harvested. Protein extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti-Slug (9585, Cell Signaling), anti-vimentin (13.2, Sigma) and anti-N-cadherin (610920, BD Biosciences) antibodies. To control for sample loading, the blots were probed with the β-actin-specific antibody. (PDF 77 KB)
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Additional file 6: Figure S6 c-Myb induces the cathepsin D transcription independently on Slug. MDA-MB-231 cells were transiently cotransfected with either c-myb (MYBup), Slug cDNA (SLUGup) or the empty vector (vector) and the reporter plasmids (the 2.85 kb- or 0.82 kb pGL3-CD containing segments of cathepsin D promoter of indicated lengths upstream of the luciferase gene, and CMV-βgal) and harvested 48 h later. (A) The amounts of c-Myb and Slug proteins were determined by immunoblotting. (B) The luciferase activity of each sample was expressed in relative light units and normalized for transfection efficiency according to the β-galactosidase activity. The columns show the average relative luciferase activity from three independent measurements. Error bars indicate the standard deviations. Asterisks indicate significant (p < 0.05) differences in the luciferase activity in the empty vector-transfected cells and cells transfected with c-myb cDNA as determined by the t-test. (PDF 128 KB)
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Additional file 8: Figure S8a c-Myb down-regulates interstitial collagenase Mmp1a in 4T1 cells. The control (wt, vector) and the MYBup (M-M5, M-M8) cell protein extracts were analyzed by immunoblotting. To control for sample loading, the blots were probed with a β-actin-specific antibody. The secretion of Mmp1a was determined in the cell-conditioned medium. The cells were cultured in a serum-free medium for 8 h, and the medium was harvested and concentrated using the Amicon Ultra centrifugal filter units. Equal amounts of total proteins were loaded. Figure S8b Expression of c-myb and mmp1a mRNAs in 4T1 MYBup and control tumors. Total RNA was isolated from mouse mammary tumors. The relative amounts of c-myb and mmp1a mRNAs in the MYBup and control tumors were determined by qRT-PCR. GAPDH was used as an internal control. Asterisks indicate significant (p < 0.05) differences in the relative amounts of c-myb and mmp1a mRNAs in the control and MYBup tumors as determined by the t-test. (PDF 112 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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Authors’ original file for figure 7
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