Epidermal growth factor inhibition of c-Myc-mediated apoptosis through Akt and Erk involves Bcl-x_L upregulation in mammary epithelial cells

Abstract

In earlier studies, we and others have established that activation of EGFR can promote survival in association with upregulation of Bcl-x_L. However, the mechanism responsible for upregulation of Bcl-x_L is unknown. For the current studies we have chosen pro-apoptotic, c-Myc-overexpressing murine mammary epithelial cells (MMECs) derived from MMTV-c-Myc transgenic mouse tumors. We now demonstrate that EGFR activation promotes survival through Akt and Erk1/2. Blockade of EGFR kinase activity and the PI3-K/Akt and MEK/Erk pathways with pharmacological inhibitors resulted in a significant induction of cellular apoptosis, paralleled by a downregulation of both Akt and Erk1/2 proteins. Consistent with a survival-promoting role of Akt, we observed that constitutively activated Akt (Myr-Akt) inhibited apoptosis of pro-apoptotic, c-Myc-overexpressing cells following the inhibition of EGFR tyrosine kinase activity. In addressing possible downstream effectors of EGFR through activated Akt, we detected significant upregulation of Bcl-x_L protein, suggesting this pro-survival protein is a target of Akt in MMECs. By using pharmacological inhibitors of PI3-K/Akt and MEK/Erk together with dominant-negative Akt and Erk1 we observed the decrease in Bcl-x_L protein. Our findings may be of importance for understanding the emerging role of Bcl-x_L as a potential marker of poor prognosis in breast cancer.

Introduction

The c-myc gene is thought to play an important role in the onset and progression of breast cancer, where it is commonly amplified and/or overexpressed [1], [2]. Depending on the availability of survival factors, cells that constitutively express c-Myc undergo proliferation, growth arrest, or apoptosis through poorly defined mechanisms. Previously, we demonstrated that an epidermal growth factor receptor (EGFR)-mediated survival signaling pathway(s) inhibited apoptosis in c-Myc-induced transgenic mouse mammary tumors [3]. A comparison of tumors and cell lines derived from bitransgenic tgf a/c-myc mice with those from single transgenic c-myc mice indicated that only the latter model contained a significant fraction of apoptotic cells [3], suggesting that transforming growth factor α (TGFα) protects c-Myc-overexpressing cells from apoptosis in vivo. Further in vitro studies of c-Myc-overexpressing mammary tumor-derived cells confirmed that EGFR ligands, acting through EGFR tyrosine kinase activity, suppressed apoptosis and upregulated the survival molecule Bcl-x_L, at both mRNA and protein levels [4]. However, it was not clear which pathways downstream of EGFR are responsible for these effects.

Presently, there is limited information on the signaling pathways linking EGFR to the regulation of cellular survival in mouse and human mammary epithelial and carcinoma cells. However, studies in some nonmammary epithelial cells (hepatic carcinoma cells and keratinocytes) have identified two survival pathways downstream of EGFR: phosphatidylinositol 3-kinase (PI3-K)/Akt and extracellular signal-regulated kinase (Erk1/2). In most cases, the PI3-K/Akt pathway delivers the most potent survival signal downstream of EGFR [5], [6].

Akt is a serine–threonine kinase, downstream of PI3-K, which delivers strong survival signals in many cell types [7], [8], [9]. Both growth factors and integrins activate Akt through activation of PDK1 and putative PDK2 kinase that subsequently phosphorylate Akt at Thr308 and Ser473 respectively [10]. There are several isoforms of Akt (Akt1, Akt2, Akt3); each has been shown to be expressed at different levels in various tissues [11]. The targets of Akt in epithelial cells, including mouse and human breast cells, include proteins involved in cell growth, metabolism, and apoptosis. The Akt targets involved in apoptosis include Bad, a pro-apoptotic member of the Bcl-2 family of proteins [12], caspase 9 [13], and the forkhead transcription factor [14]. Recently, Akt also has been reported to upregulate the expression of anti-apoptotic proteins in lymphoid cells such as Bcl-x_L [15], Bcl-2 [16], and Mcl-1 [17]. Akt activates NF-κB in both fibroblasts and epithelial cells [18], [19].

In most cell types, both growth factors and integrins are capable of activating the MAPK/Erk pathway. Of particular relevance to our studies, the MAPK/Erk pathway was previously shown to convey survival signals in response to EGF [20], [21]. Recently, it has been shown that the PI3-K/Akt and the MAPK/Erk pathways can cooperate in the inhibition of Bad in some cell types [22]. However, the pro-survival targets and the interactions of EGFR-activated PI3K/Akt and MAPK/Erk pathways have not been established in murine mammary epithelial cells (MMECs), human breast epithelial cells, or human carcinomas. Our preliminary data indicated that Bad is expressed in MMECs; however, the phosphorylation status of endogenous Bad was difficult to determine due to the lack of reliable antibodies. With the emerging role of activated EGFR in breast cancer, we believed it would be important to determine the anti-apoptotic targets of EGFR-stimulated Akt and Erk, in MMECs and human breast cancer cells.

In our investigations to determine which survival molecules downstream of the EGFR are responsible for upregulation of Bcl-x_L and inhibition of c-Myc-mediated apoptosis in MMECs, we show that constitutively activated Akt provides protection from c-Myc-mediated apoptosis in association with upregulation of the Bcl-x_L protein. By using pharmacological inhibitors of both PI3-K/Akt and MEK/Erk1/2, and dominant-negative (DN)-Erk1 and DN-Akt we detected significant decreases in Bcl-x_L protein expression.

In conclusion, our results demonstrate that EGFR-dependent Akt activity provides a major survival signal against c-Myc-mediated apoptosis in MMECs; both Akt and Erk are obligatory for regulation of Bcl-x_L expression in this model. To our knowledge, these studies provide the first comprehensive evaluation of the role of EGFR-dependent survival molecules in inhibition of c-Myc-mediated apoptosis in murine models of breast cancer.