Tamoxifen-Induced Apoptosis of MCF-7 Cells via GPR30/PI3K/MAPKs Interactions: Verification by ODE Modeling and RNA Sequencing

Milad Rouhimoghadam,Nader Sheibani,Gholamreza Bidkhori,Jason S Carroll,Shahrokh Safarian

doi:10.3389/fphys.2018.00907

Abstract

Tamoxifen (Nolvadex) is one of the most widely used and effective therapeutic agent for breast cancer. It benefits nearly 75% of patients with estrogen receptor (ER)-positive breast cancer that receive this drug. Its effectiveness is mainly attributed to its capacity to function as an ER antagonist, blocking estrogen binding sites on the receptor, and inhibiting the proliferative action of the receptor-hormone complex. Although, tamoxifen can induce apoptosis in breast cancer cells via upregulation of pro-apoptotic factors, it can also promote uterine hyperplasia in some women. Thus, tamoxifen as a multi-functional drug could have different effects on cells based on the utilization of effective concentrations or availability of specific co-factors. Evidence that tamoxifen functions as a GPR30 (G-Protein Coupled Receptor 30) agonist activating adenylyl cyclase and EGFR (Epidermal Growth Factor Receptor) intracellular signaling networks, provides yet another means of explaining the multi-functionality of tamoxifen. Here ordinary differential equation (ODE) modeling, RNA sequencing and real time qPCR analysis were utilized to establish the necessary data for gene network mapping of tamoxifen-stimulated MCF-7 cells, which express the endogenous ER and GPR30. The gene set enrichment analysis and pathway analysis approaches were used to categorize transcriptionally upregulated genes in biological processes. Of the 2,713 genes that were significantly upregulated following a 48 h incubation with 250 μM tamoxifen, most were categorized as either growth-related or pro-apoptotic intermediates that fit into the Tp53 and/or MAPK signaling pathways. Collectively, our results display that the effects of tamoxifen on the breast cancer MCF-7 cell line are mediated by the activation of important signaling pathways including Tp53 and MAPKs to induce apoptosis.

Highlights

Breast cancer is the second leading cause of cancer death in the United States in women after lung cancer (Holt, 2010)
The designed signaling network for normal cells is modeled based on the experimental evidences and previous models of the EGFR, PI3K, signal transducer and activator of transcription (STAT) and GPCR signaling pathways (Schoeberl et al, 2002; Yamada et al, 2003, 2004; Sasagawa et al, 2005; Heitzler et al, 2012)
These pathways are activated through two ligands alongside the two axes: 1- through the epidermal growth factor (EGF) binding to EGFR, and 2- via tamoxifen binding to GPR30 (Supplementary Table S1)

Summary

Introduction

Breast cancer is the second leading cause of cancer death in the United States in women after lung cancer (Holt, 2010). Tamoxifen-Induced Apoptosis by GPR30 sequences are located in the 7P22.3 of human genome (Albanito et al, 2008) These receptors are structurally different from estrogen receptors (ER) and are expressed extensively within early mammary tumors and in breast cancer cell lines (Prossnitz et al, 2008; Prossnitz and Barton, 2009). Activation of the GPR30 signaling cascade mediates the non-genomic route of estrogen hormones and stimulates the PI3K/Akt and MAPK (MitogenActivated Protein Kinase) signaling intermediates. This pathway controls different cellular processes that can culminate in cell proliferation, migration, differentiation, and apoptosis (Filardo et al, 2000; Ge et al, 2012; Luo et al, 2012). Besides interacting with estrogenic hormones, the receptor GPR30 has a tendency to bind a number of anti-estrogenic compounds such as tamoxifen and ICI182780 (Thomas et al, 2005)

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