J Breast Cancer.  2013 Jun;16(2):138-145. 10.4048/jbc.2013.16.2.138.

Leptin as a Potential Target for Estrogen Receptor-Positive Breast Cancer

• Yom CK
• Lee KM
• Han W
• Kim SW
• Kim HS
• Moon BI
• Jeong KY
• Im SA
• Noh DY

• KMID: 2286392
• DOI: http://doi.org/10.4048/jbc.2013.16.2.138

Abstract

PURPOSE
Leptin is a potent adipokine that plays a significant role in tumor development and the progression of breast cancer. The aim of this study was to evaluate whether leptin affects the response to tamoxifen treatment in estrogen receptor (ER)-positive breast cancer cells.
METHODS
Leptin, leptin receptor (Ob-R), and activation of signaling pathways were studied by Western immunoblotting. The effects of leptin on tamoxifen-dependent growth inhibition were studied in MCF-7 and T-47D cells.
RESULTS
Leptin was expressed in MCF-7 and T-47D and had a proliferative effect on MCF-7 cells. Leptin significantly inhibited the antiestrogenic effect of tamoxifen in both cells only under beta-estradiol (E2) (20 nM) conditions. In MCF-7, the inhibitory effect against tamoxifen was a result from the activation of the ERK1/2 and STAT3 signal transduction pathway.
CONCLUSION
Leptin interferes with the effects of tamoxifen under E2 stimulated conditions in ER-positive breast cancer cells. These results imply that inhibition of leptin is expected to enhance the response to tamoxifen in ER-positive breast cancer cells, and, therefore, could be a promising way to overcome endocrine resistance.

Keyword

Breast neoplasms; Hormone resistance; Leptin

MeSH Terms

Adipokines
Blotting, Western
Breast
Breast Neoplasms
Estrogen Receptor Modulators
Estrogens
Leptin
MCF-7 Cells
Receptors, Leptin
Signal Transduction
Tamoxifen
Adipokines
Estrogen Receptor Modulators
Estrogens
Leptin
Receptors, Leptin
Tamoxifen

Figure

• 

  Figure 1 Leptin and leptin receptor (Ob-R) expression in various breast cancer cell lines. (A) Notably, there is increased leptin expression in estrogen receptor (ER)-positive cell lines (MCF-7, T-47D, and HCC-70) compared to other types of breast cancer cells. β-Actin was used for loading control in both cell lines. (B) In breast cancer cell lines, the Ob-R long (Ob-Rb) is the predominat receptor expressed compared to normal breast tissue, whereas there is no difference in Ob-R short (Ob-Ra) expression between breast cancer cell lines and normal breast tissue. Top, expression by Western blot assay; Bottom, quantitative analysis of Western blot results using densitometry.

• 

  Figure 2 Cell viability after single treatment of estrogen, leptin, and tamoxifen in MCF-7 and T-47D cells. MCF-7 cells at 70% confluence were synchronized in serum-free medium for 24 hours and treated. Experiments were performed at least 3 times. Cell viability assays are shown as histograms. The bars represent relative cell number (±standard deviation). Cell viability was assessed by CellTiter-Glo® Luminescent Cell Viability assay. (A) Estrogen and leptin had proliferative effects on MCF-7 cells compared to no treatment. Tamoxifen had a significant inhibitory effect on MCF-7 cell proliferation in a dose-dependent manner. (B) Estrogen had proliferative effects at 20 nM, and leptin had no significant effect on T-47D cell proliferation. *Statistical significance compared to no treatment (p<0.05).

• 

  Figure 3 Leptin inhibition of the antiestrogenic effect of tamoxifen under estradiol stimulated conditions in MCF-7 and T-47D cells. In MCF-7 cells with estradiol 10 nM stimulation, leptin had no effect on the activity of tamoxifen for each combination treatment (A). At estradiol 20 nM stimulation, when leptin 50 ng/mL was added to tamoxifen treatment, leptin significantly inhibited the 10 and 20 µM tamoxifen responses (B). In T-47D cells, leptin 50 ng/mL had an inhibitory effect on tamoxifen 10 µM with estradiol 10 and 20 nM (C and D, respectively). There was no significant difference between leptin 100 ng/mL treatment and no leptin treatment. With tamoxifen 20 µM, cell viability was notably decreased compared to tamoxifen 10 µM treatment, and there was no difference between each combination. All cell viability results are expressed by the ratio of comparison to control (no treatment) group.

• 

  Figure 4 Leptin activates multiple signal transduction pathways in MCF-7 cells compared to T-47D cells. MCF-7 and T-47D cells were synchronized in serum-free medium and then stimulated with estrogen (20 nM), leptin (50 ng/mL), and tamoxifen (10 µM). Stimulation of Ob-Rb was assessed at different time points from 5 minutes to 6 hours. Hsc70 was used as a loading control at each time point for both cell lines. Activation (phospho) and levels of ERK1/2, STAT3, p38 MAPK, and estrogen receptor α (ERα) were assessed by Western blotting in 25 µg of protein using specific antibodies. Figure 4 shows multiple signal transduction pathways being activated at 1 hour. Tamoxifen 10 µM effectively inhibited the activity of total- and phospho-ERK1/2 and STAT3 in both cell lines. (A) In MCF-7 cells, when leptin was added to the combination of estrogen and tamoxifen, leptin induced activation of total- and phospho-ERK1/2, total- and phospho-STAT3 signaling (grey box). There were no significant differences in total- and phospho-p38 MAPK, and ERα signaling when leptin was added to the combination of estrogen and tamoxifen. (B) In contrast to the results obtained in MCF-7 cells, in T-47D cells, there was no activation of the phospho-ERK1/2 and phospho-STAT3 signal transduction pathways. Also, total- and phospho-p38 MAPK, ERα signaling were not activated by addition of leptin (grey box).

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