HER2 Upregulates ATF4 to Promote Cell Migration via Activation of ZEB1 and Downregulation of E-Cadherin

Abstract

HER2 (human epidermal growth factor receptor 2) activation is critical in breast cancer development. HER2 promotes cell proliferation, angiogenesis, survival, and metastasis by activation of PI3K/Akt, Ras/MEK/ERK, and JAK/STAT pathways. However, beyond these signaling molecules, the key proteins underlining HER2-mediated metastasis remain elusive. ATF4 (Activating transcription factor 4), a critical regulator in unfolded protein response (UPR), is implicated in cell migration and tumor metastasis. In this study, we demonstrate that HER2 upregulated ATF4 expression at both mRNA and protein levels, resulting in cell migration increased. In addition, ATF4 upregulated ZEB1 (Zinc finger E-box-binding homeobox 1) and suppressed E-cadherin expression resulting in promoting cell migration. Restoration of E-cadherin expression effectively inhibited HER2- or ATF4-mediated cell migration. In addition, upregulated expression of ATF4 was found in HER2-positive breast cancer specimens. Together, this study demonstrates that ATF4-ZEB1 is important for HER2-mediated cell migration and suggests that ATF4-ZEB1 may be potential therapeutic targets for breast cancer metastasis.

Keywords: ATF4; E-cadherin; HER2; ZEB1; cell migration.

Figure 1

HER2 induces scattered cell growth and cell migration through upregulation of ATF4 and downregulation of E-cadherin expression. MCF10A cells stably expressing HER2 (A–C) were infected with a recombinant lentivirus expressing specific shRNA targeting to ATF4 (D–F) or a recombinant lentivirus expressing E-cadherin (H–J). Cells were then subjected to cell morphology observations (A,E,I), transwell assays (B,F,J), and Western blot analyses (C,D,H). MCF10A cells stably expressing HER2 or ATF4 were subjected to cell proliferation assays by Real-Time Cell Analyzer (G). Results are representative of three independent experiments. Data were presented as means ± SD; scale bars: 100 μm; NS: Not significant; **: p < 0.01.

Figure 2

HER2 upregulates ATF4 expression at both transcriptional and translational levels. MCF10A-HER2 cells were subjected to Q-PCR analyses (A), protein half-life assay and the relative ATF4 protein expression levels were quantitated as described in the Materials and Methods (B–C), or were treated with or without rapamycin (100 nM) prior to Western blot analyses (D). The Q-PCR data were derived from three independent experiments; *: p < 0.05.

Figure 3

ATF4 promotes cell migration via suppression of E-cadherin expression. MCF10A-ATF4 cells were subjected to Western blot analyses (A,I), transwell assays (C), and Q-PCR assays (H,J). MCF7-ATF4 cells were subjected to Western blot analyses (B). MCF10A cells stably expressing specific shRNA targeting ATF4 or a control (shC), were subjected to Western blot analyses (D) or transwell assays (E). (F,G) MCF10A-ATF4 cells infected with a lentivirus expressing E-cadherin were subjected to Western blot analyses (F) or transwell assays (G). MCF10A-ATF4 cells infected with a lentivirus expressing shZEB1-#1, shZEB1-#2 or a control (shC), were subjected to Western blot analyses (K). Data were analyzed from three independent experiments and were presented as means ± SD; scale bars: 100 μm; **: p < 0.01.

Figure 4

HER2, ZEB1, and ATF4 expression were analyzed in human breast cancers. mRNA levels of ATF4 or ZEB1 in HER2-positive or -negative breast cancer biopsy samples were analyzed using TCGA databases (A,B). A schematic model for the role of ATF4 in HER2-mediated cell migration (C).