Elevated STAT3 Signaling-Mediated Upregulation of MMP-2/9 Confers Enhanced Invasion Ability in Multidrug-Resistant Breast Cancer Cells

Abstract

The development of multidrug resistance greatly impedes effective cancer therapy. Recent advances in cancer research have demonstrated that acquisition of multidrug resistance by cancer cells is usually accompanied by enhanced cell invasiveness. Several lines of evidence indicated that cross activation of other signaling pathways during development of drug resistance may increase invasive potential of multidrug-resistant (MDR) cancer cells. However, the accurate mechanism of this process is largely undefined. In this study, to better understand the associated molecular pathways responsible for cancer progression induced by drug resistance, a MDR human breast cancer cell line SK-BR-3/EPR with P-glycoprotein overexpression was established using stepwise long-term exposure to increasing concentration of epirubicin. The SK-BR-3/EPR cell line exhibited decreased cell proliferative activity, but enhanced cell invasive capacity. We showed that the expression of metastasis-related matrix metalloproteinase (MMP)-2/9 was elevated in SK-BR-3/EPR cells. Moreover, SK-BR-3/EPR cells showed elevated activation of STAT3. Activation of STAT3 signaling is responsible for enhanced invasiveness of SK-BR-3/EPR cells through upregulation of MMP-2/9. STAT3 is a well-known oncogene and is frequently implicated in tumorigenesis and chemotherapeutic resistance. Our findings augment insight into the mechanism underlying the functional association between MDR and cancer invasiveness.

Keywords: MMP-2; MMP-9; STAT3; breast cancer; invasion; multidrug-resistant (MDR).

Figure 1

Establishment of a multi-drug resistant human breast cancer cell line SK-BR-3/EPR. (A–C) Drug sensitivity assay of SK-BR-3 and SK-BR-3/EPR cells to epirubicin, paclitaxel and 5-Fluorouracil (5-FU). Cell viability assay was determined using a Cell Counting Kit-8 (CCK-8) according to the manufacturer’s protocol. In brief, cells were seeded in 96 well plate at a density of 5 × 10³ cells per well and cultured for 24 h, then different concentration of indicated drugs were added into each well and further cultured for 72 h. After removal of culture medium, 10 μL of CCK-8 reagent in 200 μL medium were added into each well and incubated for 3 h, then the cell viability were calculated by measuring the absorbance at 450 nm on a micro-ELISA reader. The assays were performed using five replicates for each concentration and repeated three times. IC₅₀ is defined as the concentration of drug causing half inhibition of cell growth compared with the cell growth of the control group. The IC₅₀ was calculated by the Graphpad Prism 6.00 software.

Figure 2

P-glycoprotein is upregulated in SK-BR-3/EPR cells. (A) Quantitative PCR analysis of expression of MDR1, MRP1, BCRP mRNA in SK-BR-3 and SK-BR-3 cells. The experiments were performed using three replicates for each group and repeated three times; (B) Further analysis of PCR products by agarose gel electrophoresis confirmed the upregulated expression of MDR1 mRNA in SK-BR-3/EPR cells; (C) Western blotting analysis of P-glycoprotein expression in SK-BR-3 and SK-BR-3/EPR cells; (D) Confocal immunofluorescence microscopy analysis showed positive expression of P-glycoprotein in SK-BR-3/EPR cells, but not in drug sensitive SK-BR-3 cells (×600 magnification); (E) SK-BR-3/EPR cells showed markedly reduced retention of intracellular Rh123 compared with SK-BR-3 cells as measured by rhodamine efflux assay.

Figure 2

P-glycoprotein is upregulated in SK-BR-3/EPR cells. (A) Quantitative PCR analysis of expression of MDR1, MRP1, BCRP mRNA in SK-BR-3 and SK-BR-3 cells. The experiments were performed using three replicates for each group and repeated three times; (B) Further analysis of PCR products by agarose gel electrophoresis confirmed the upregulated expression of MDR1 mRNA in SK-BR-3/EPR cells; (C) Western blotting analysis of P-glycoprotein expression in SK-BR-3 and SK-BR-3/EPR cells; (D) Confocal immunofluorescence microscopy analysis showed positive expression of P-glycoprotein in SK-BR-3/EPR cells, but not in drug sensitive SK-BR-3 cells (×600 magnification); (E) SK-BR-3/EPR cells showed markedly reduced retention of intracellular Rh123 compared with SK-BR-3 cells as measured by rhodamine efflux assay.

Figure 3

The SK-BR-3/EPR cells maintained MDR phenotype after sequential passages in epirubicin-free medium for six weeks. (A) Drug sensitivity assay of SK-BR-3 and SK-BR-3/EPR cells to epirubicin and paclitaxel. The SK-BR-3/EPR cells were cultured in epirubicin-free medium and sequential passages for six-consecutive weeks, then IC₅₀ assay was performed by using the CCK-8 based method; (B) Western blotting analysis of P-glycoprotein expression (P-gp) in SK-BR-3, SK-BR-3/EPR, and SK-BR-3/EPR cells cultured in drug-free condition for indicated times. The numbers above the blots show the relative values from the densitometric analysis. The levels of P-gp was normalized to β-actin, values are expressed as fold changes compared to SK-BR-3/EPR cells; (C) Confocal immunofluorescence microscopy analysis of P-glycoprotein expression in SK-BR-3 and SK-BR-3/EPR cells cultured in epirubicin-free medium for six-consecutive weeks (×600 magnification).

Figure 3

The SK-BR-3/EPR cells maintained MDR phenotype after sequential passages in epirubicin-free medium for six weeks. (A) Drug sensitivity assay of SK-BR-3 and SK-BR-3/EPR cells to epirubicin and paclitaxel. The SK-BR-3/EPR cells were cultured in epirubicin-free medium and sequential passages for six-consecutive weeks, then IC₅₀ assay was performed by using the CCK-8 based method; (B) Western blotting analysis of P-glycoprotein expression (P-gp) in SK-BR-3, SK-BR-3/EPR, and SK-BR-3/EPR cells cultured in drug-free condition for indicated times. The numbers above the blots show the relative values from the densitometric analysis. The levels of P-gp was normalized to β-actin, values are expressed as fold changes compared to SK-BR-3/EPR cells; (C) Confocal immunofluorescence microscopy analysis of P-glycoprotein expression in SK-BR-3 and SK-BR-3/EPR cells cultured in epirubicin-free medium for six-consecutive weeks (×600 magnification).

Figure 4

The SK-BR-3/EPR cells exhibited a marked reduction in cell proliferation rate, and enhancement in cell invasion ability. (A) The SK-BR-3/EPR cells showed decreased cell proliferation rate in the presence or absence of epirubicin in comparison with that of drug sensitive SK-BR-3 cells; (B) The SK-BR-3/EPR cells showed a remarkably reduction in the formation of cell colonies compared with that of in drug sensitive SK-BR-3 cells. Cells were seeded in 35-mm dishes at a density of 500 cells per dish and then cultured for 10–14 days, the number of colonies was counted under an inverted microscope, and only colonies of more than 50 individual cells were counted (×20 magnification). The assays were performed in triplicate for each group and repeated three times; (C) The SK-BR-3/EPR cells exhibited enhanced invasive ability compared with that of drug sensitive SK-BR-3 cells. The cells were cultured to 80% confluence, and harvested by trypsinization, then the cell suspension at a density of 4 × 105 cells/mL were added into the matrigel pre-coated transwell insert. The lower insert was filled with 10% FBS-containing medium. After incubation for 24 h at 37 °C, the invaded cells were fixed, stained, and counted. The assay was performed in triplicate and repeated three times (×200 magnification).

Figure 5

Expression of MMP-2 and -9 as well as phosphorylation of STAT3 was elevated in SK-BR-3/EPR cells. (A) Quantitative PCR analysis of expression of MMP-2 and -9 mRNA in SK-BR-3 and SK-BR-3/EPR cells. The experiments were performed using three replicates for each group and repeated three times; (B) Western blotting analysis of expression of MMP-2 and -9 protein in SK-BR-3 and SK-BR-3/EPR cells. The numbers above the blots show the relative values from the densitometric analysis. The levels of MMP-2 and -9 were normalized to β-actin, values are expressed as fold changes compared to SK-BR-3 cells; (C) Phosphorylation and activation of STAT3 was increased in MDR SK-BR-3/EPR cells. Western blotting analysis of expression of phosphorylation of Erk1/2 and STAT3 in SK-BR-3 and SK-BR-3/EPR cells. SK-BR-3 and SK-BR-3/EPR cells were grown to 90% confluence, then total cellular proteins were extracted and subjected to Western blotting analysis. The numbers above the blots show the relative values from the densitometric analysis. The level of Stat3 was normalized to β-actin, p-Erk1/2 and p-Stat3 were normalized to the corresponding total Erk1/2 and Stat3, values are expressed as fold changes compared to SK-BR-3 cells. * p < 0.05.

Figure 6

Knockdown of STAT3 inhibited cell invasion and downregulated expression of MMP-2 and -9 in SK-BR-3/EPR cells. (A) Knockdown of STAT3 significantly reduced the expression levels of MMP-2 and -9 protein in SK-BR-3/EPR cells. Western blotting analysis of STAT3, MMP-2, and -9 expression in SK-BR-3/EPR cells and SK-BR-3/EPR cells transfected with control or STAT3 specific siRNAs. The numbers above the blots show the relative values from the densitometric analysis. The levels of MMP-2 and -9 were normalized to β-actin, values are expressed as fold changes compared to SK-BR-3/EPR cells; (B) Knockdown of STAT3 in SK-BR-3/EPR cells slightly increased the sensitivity to epirubicin in comparison with control cells; (C) Knockdown of STAT3 remarkably inhibited the invasive ability of SK-BR-3/EPR cells as measured by transwell based assay (×200 magnification). * p < 0.05.