Original ArticleEssential role of Notch4/STAT3 signaling in epithelial–mesenchymal transition of tamoxifen-resistant human breast cancer
Introduction
Despite tamoxifen (TAM) showing clear benefits for the prevention and treatment of estrogen receptor α (ERα)-positive breast cancer, continuous exposure to TAM confers drug resistance [1]. As a consequence, TAM resistance is a considerable limiting factor in the management of advanced breast cancer. Previous studies demonstrated increased cell motility in vitro and morphological distinctions between TAM-resistant human breast cancer (TAMR-MCF-7) cells and their parent MCF-7 cells [1], [2].
Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and gain migratory and invasive properties to become mesenchymal stem cells [3]. As cells undergo EMT, they lose epithelial cell–cell coherence, reorganize their actin cytoskeleton, downregulate the expression of cell adhesion molecules such as E-cadherin and upregulate the expression of mesenchymal markers such as N-cadherin and vimentin [3]. In addition, the activities of matrix metalloproteinases (MMPs) such as MMP2 and MMP9 are elevated during EMT progression [4].
Upregulation of Notch receptors and their ligands has been reported in several types of cancer cells, including breast cancer [5]. The Notch family comprises four receptors, Notch1, 2, 3 and 4 [6]. Notch1 and Notch4 are required for cell proliferation and stimulate matrix invasion in both ERα-negative and -positive breast cancer cells [7]. Moreover, constitutively active forms of Notch1 and Notch4 stimulate the transformation of normal human mammary epithelial cells in vitro [8], [9]. Consistent with this finding, activation of Notch1 [9] or Notch4 [8] can cause mammary carcinogenesis in mice.
A number of stimuli and transcription factors have emerged as potent EMT drivers during normal development and cancer. Several studies have indicated the Notch signaling pathway as a crucial regulator in the induction of EMT and metastasis [8], [10]. Notch ligand binding to an adjacent Notch receptor activates Notch signaling and leads to the morphology and phenotype consistent with mesenchymal transformation [10]. Upon activation, the Notch receptor is cleaved and undergoes conformational changes in which the Notch intracellular domain (ICD) is released through a cascade of proteolytic cleavages by metalloproteases, tumor necrosis factor-α-converting enzyme and the γ-secretase complex [10], [11]. Accordingly, γ-secretase inhibition may prevent Notch-induced EMT in cancer cells. Notch4 activation significantly increases the tumorigenic potential of mammary epithelial cells by changing their morphogenetic properties [12]. Yun et al. revealed that PKCα-overexpressing T47D cells showing TAM-resistant phenotype expressed high levels of Notch4, but not of Notch1 [13]. Notch4 is up-regulated in TAM-resistant MCF-7 breast cancer cells which possessed more invasive and migratory phenotype compared to wild type MCF-7 cells [14].
Endogenous activity of the Notch signaling pathway is critical for activation of signal transducer and activator of transcription 3 (STAT3) in neuroepithelial cells [15]. STAT3 is frequently activated in many human cancer types, including breast cancer [16]. Tumorigenic STAT3 activation is often associated with increased malignant cancer behaviors, including uncontrolled growth, EMT, migration, invasion, metastasis and therapeutic resistance [17], [18].
Here, we observed that basal expression and activity of Notch4 were amplified in TAMR-MCF-7 cells, and we investigated the role of Notch4/STAT3 signaling in EMT of TAM-resistant human breast cancer, and the potential of Notch inhibitors in the suppression of the metastatic tumor burden in vivo.
Section snippets
Antibodies and reagents
Anti-E-cadherin and anti-N-cadherin antibodies were purchased from BD Transduction (San Jose, CA). Anti-Snail antibody was supplied from Abcam (Cambridge Science Park, UK). Anti-Notch1, anti-Notch4 and phospho-STAT3, anti-STAT3, anti-c-Jun, anti-c-Fos, Jun B, Jun D antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-P65, anti-Notch1 (for immunohistochemistry), anti-Notch4 (for immunohistochemistry) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Notch activation in TAM-resistant breast cancer is important for cell migration
Recently, Notch1 and Notch4 have emerged as prognostic markers for breast cancer [21]. Here, we observed that basal expression levels of Notch1 and especially Notch4 were significantly increased in TAMR-MCF-7 cells compared with control MCF-7 cells at both the mRNA and protein levels (Fig. 1A and B). Moreover, the ICDs of Notch1 and Notch4 were highly expressed in TAMR-MCF-7 cells (Fig. 1A). To confirm these findings in human, tumor tissues were obtained from two groups of patients. The group
Discussion
Endocrine therapy is the most effective approach to treat ERα-positive breast cancer, but its benefit is limited by common intrinsic and acquired resistance [1]. There is accumulating evidence that breast cancer progression and recurrence are promoted by cancer stem cells (CSCs) [32]. Previous studies have shown that breast CSCs contribute to metastatic progression, which may require cancer cells with the ability to undergo EMT at the primary site and mesenchymal epithelial transition at sites
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) grant (No. 2014M3A9A9073788) and grant from Ministry of Food and Drug Safety (16173MFDS542) in funded by the Korea government.
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