Signal transducers and activators of transcription-3 up-regulates tissue inhibitor of metalloproteinase-1 expression and decreases invasiveness of breast cancer - PubMed (original) (raw)

Signal transducers and activators of transcription-3 up-regulates tissue inhibitor of metalloproteinase-1 expression and decreases invasiveness of breast cancer

Jennifer Dien et al. Am J Pathol. 2006 Aug.

Abstract

Signal transducers and activators of transcription (STAT)-3 is an oncogenic protein that is constitutively activated in many human cancers, including 30 to 60% of primary breast cancer. The biological significance of STAT3 activation in breast cancer is not fully understood. We have previously shown that STAT3 up-regulates tissue inhibitors of metalloproteinase (TIMP)-1, a cytokine known to block metalloproteinases and decrease invasiveness in certain cancer cell types. We hypothesize that STAT3 activation may modulate tumor invasiveness of breast cancer by regulating TIMP1 expression. Using MCF-7 cells transfected with tetracycline-off STAT3C (constitutively active STAT3), we generated an in vitro system in which STAT3C levels can be tightly controlled in breast cancer cells. Increasing tetracycline levels gradually decreased STAT3C and TIMP1 in a dose-dependent manner, and down-regulation of these proteins led to a reciprocal decrease in invasiveness of these cells in Matrigel. Addition of a neutralizing anti-TIMP1 antibody increased invasiveness in the same experimental system. Using immunohistochemistry and 142 primary breast tumors, we found a significant association between the expression of the phosphorylated/active form of STAT3 (pSTAT3) and that of TIMP1. Importantly, STAT3 activation correlated significantly with a lower frequency of vascular and lymphatic invasion (P = 0.015 and P = 0.0002, respectively). Our data support the concept that STAT3 activation significantly modulates the biological and clinical behavior of breast cancer.

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Figures

Figure 1-6917

Figure 1-6917

A: Western blot analysis of STAT3, FLAG, TIMP1, cyclin D1, Bcl-2, and Bcl-xL expression in MCF-7 cells transfected with tTA-TRE-STAT3C and treated with increasing concentrations of tetracycline for 24 hours. There was a gradual decrease in STAT3 and FLAG with increasing concentrations of tetracycline, with FLAG being completely undetectable at 60 ng/ml. Down-regulation of cyclin D1, Bcl-2, Bcl-xL, and TIMP1 expression was identified in the same experiment. MCF-7 cells transfected with tTA alone served as negative controls. B: Western blot analysis of FLAG and TIMP1 expression in MDA-MB-436 cells transfected with tTA-TRE-STAT3C and treated with increasing concentrations of tetracycline for 24 hours. A gradual decrease in FLAG and TIMP1 was observed. MDA-MB-436 cells transfected with tTA alone served as negative controls. C: Immunofluorescence staining and confocal microscopy studies revealed that STAT3 was localized to the nucleus in MCF-7 cells transfected with tTA-TRE-STAT3C, as illustrated by the white arrows (right) whereas STAT3 was confined to the cytoplasm in cells transfected with tTA only (left).

Figure 2-6917

Figure 2-6917

TIMP1 levels in supernatants evaluated by ELISA. TIMP1 levels were measured using an ELISA assay in MCF-7 cells transfected with tTA-TRE-STAT3C and treated with 60 ng/ml of tetracycline. Supernatants from the tissue culture were collected on days 1, 2, and 3. TIMP1 levels were significantly higher in cells treated with 60 ng/ml than in cells with no tetracycline in all 3 days. Increasing levels of TIMP1 (ng/ml) were observed with the duration of experiment, but the rate of increment was significantly higher in cells without tetracycline. Day 1 supernatant collected from MCF-7 transfected with tTA alone was included as a reference. Experiments were performed in triplicates.

Figure 3-6917

Figure 3-6917

TIMP1 expression correlated with invasiveness of MCF-7 cells. A: An increase in Matrigel invasion was observed in MCF-7 cells transfected with tet-off STAT3C and treated with increasing levels of tetracycline for a total of 48 hours. Cells treated with tetracycline displayed a higher degree of invasion in a dose-dependent manner. MCF-7 cells transfected with tTA only were used as controls. Experiments were performed in triplicate. B**:** MCF-7 cells transfected with tetracycline-off STAT3C were treated with 0, 0.5, or 2.0 μg/ml of neutralizing anti-TIMP1 antibody. Invasiveness of Matrigel was increased in cells with higher levels of neutralizing anti-TIMP1. No tetracycline was added to the cell culture. Experiments were performed in triplicate.

Figure 4-6917

Figure 4-6917

pSTAT3 expression correlated with TIMP1 expression in breast tumors. Immunohistochemical staining using pSTAT3 (A and B) and TIMP1 (C and D) applied to breast cancer tissues. Strong nuclear pSTAT3 expression as shown in A correlated with strong cytoplasmic TIMP1 expression as shown in C. Another case was negative for pSTAT3 and TIMP1 (B and D).

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