An Autocrine VEGF/VEGFR2 and p38 Signaling Loop Confers Resistance to 4-Hydroxytamoxifen in MCF-7 Breast Cancer Cells

Reidun Aesoy,Jens Henrik Norum,Rolf Lewensohn,Barbro Linderholm,Kristina Viktorsson,Betzabe Chavez Sanchez

doi:10.1158/1541-7786.mcr-07-2172

Abstract

Tamoxifen, a partial estrogen receptor antagonist, is part of the standard treatment of both primary and advanced breast cancers. However, significant proportions of breast cancers are either de novo resistant or develop tamoxifen resistance during the course of treatment through mechanisms which have been only partly characterized. We have previously found that high vascular endothelial growth factor (VEGF) or VEGF receptor 2 (VEGFR2) expression and concomitant high p38 mitogen-activated protein kinase activity within breast cancers predict a poor outcome for tamoxifen-treated patients. Here, we have molecularly dissected how VEGF/VEGFR2 and p38 are linked, and contribute to tamoxifen resistance within breast cancer using a MCF-7 BC cell model with different 4-hydroxytamoxifen (4-OHT) responsiveness. We report that MCF-7 breast cancer cell lines with tamoxifen resistance have increased secretion of VEGF and increased signaling through VEGFR2 compared with parental MCF-7 cells. 4-OHT treatment caused the ablation of VEGF secretion in parental MCF-7 cells, whereas in the tamoxifen-resistant subline, a VEGF/VEGFR2 signaling loop was still evident upon treatment. Increased basal levels of total and phosphorylated p38 were observed in tamoxifen-resistant cells. Pharmacologic inhibition of p38 reduced the proliferation of both tamoxifen-responsive and tamoxifen-resistant cells and showed an additive growth-inhibitory effect in combination with 4-OHT. A connection between VEGF/VEGFR2 and p38 signaling was identified by VEGF and VEGFR2 knockdown, which equally reduced both the total and the active forms of p38 in tamoxifen-resistant cells. Taken together, our results suggest that decreased sensitivity to 4-OHT is caused by a death-protecting VEGF/VEGFR2 and p38 growth factor loop in breast cancer cells. Inhibition of these signaling pathways may be beneficial to overcome tamoxifen resistance.

Highlights

75% of all patients with breast cancer have a tumor expressing the estrogen receptor (ER) and/or the progesterone receptor
In agreement with Brunner and colleagues, we found that the LCC2 cells showed significantly less decrease in viability upon 4-OHT treatment compared with parental MCF-7 cells using a trypan blue assay over a period of 10 days (Fig. 1)
Decreased ERa expression is known to cause tamoxifen resistance, in line with previous reports using this cell model, as well as findings from clinical studies showing retained expression of functional ER in breast cancer patients with acquired resistance to tamoxifen, we found that both MCF-7 and LCC2 express ERa to a similar extent and its expression was not affected by 4-OHT treatment (Supplementary Fig. S1; refs. 20, 21)

Summary

Introduction

75% of all patients with breast cancer have a tumor expressing the estrogen receptor (ER) and/or the progesterone receptor. These tumors depend on estrogens for their survival and endocrine therapy is used to deprive breast cancer cells of such stimuli either through competitive binding to ER or by estrogen deprivation [1]. Increased ERK, p38, and phosphoinositide-3-kinase/ Akt signaling, as a consequence of either epidermal growth factor receptor or human epidermal growth factor receptor 2 (HER2) signaling within the tumor, have been shown to block the antiproliferative effect of tamoxifen [4,5,6,7]

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