Cancer Letters

Cancer Letters

Volume 390, 1 April 2017, Pages 115-125
Cancer Letters

Original Article
Essential role of Notch4/STAT3 signaling in epithelial–mesenchymal transition of tamoxifen-resistant human breast cancer

https://doi.org/10.1016/j.canlet.2017.01.014Get rights and content

Highlights

  • Notch4 plays a critical role in EMT signaling in tamoxifen-resistant breast cancer.

  • Notch4/STAT3 crosstalk is important for EMT.

  • Notch inhibition suppresses the micrometastatic tumor burden.

Abstract

We previously demonstrated that tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells showed increased expression of mesenchymal marker proteins compared to the parent MCF-7 cells. Notch is functionally important in the promotion of epithelial–mesenchymal transition (EMT) during both development and tumor progression. Notch1 and Notch4 have been reported as prognostic markers in human breast cancer. Here, we indicated that Notch4, but not Notch1, plays a critical role in the regulation of EMT signaling in TAMR-MCF-7 cells. Notch4 suppression by either Notch inhibitors or Notch4 siRNA attenuated EMT signaling. Tyrosine-phosphorylated STAT3 protein is known as a crucial signaling molecule in the regulation of tumorigenesis and metastasis. We found that TAMR-MCF-7 cells exhibited constitutive STAT3 phosphorylation, and Notch inhibition reduced the level of activated STAT3 in TAMR-MCF-7 cells. An intrasplenic injection model of liver metastases was performed using TAMR-MCF-7 cells. Mice injected with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, 10 mg/kg) formed smaller splenic tumors and showed a reduced micrometastatic tumor burden in their livers compared with the control group treated with vehicle. To conclude, Notch4 could be a potential target to prevent metastasis in TAM-resistant 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.

References (46)

  • G. Moreno-Bueno et al.

    Transcriptional regulation of cell polarity in EMT and cancer

    Oncogene

    (2008)
  • S. Stylianou et al.

    Aberrant activation of notch signaling in human breast cancer

    Cancer Res.

    (2006)
  • G.L. Kevin et al.

    Recent insights into the role of Notch signaling in tumorigenesis

    Blood

    (2006)
  • P. Rizzo et al.

    Crosstalk between Notch and the estrogen receptor in breast cancer suggests novel therapeutic approaches

    Cancer Res.

    (2008)
  • R. Callahan et al.

    Notch signaling in mammary development and oncogenesis

    J. Mammary Gland. Biol. Neoplasia

    (2004)
  • A. Dievart et al.

    Involvement of Notch1 in the development of mouse mammary tumors

    Oncogene

    (1999)
  • L. Miele

    Rational targeting of Notch signaling in breast cancer

    Expert Rev. Anticancer Ther.

    (2008)
  • C. Sahlgren et al.

    Notch signaling mediates hypoxia induced tumor cell migration and invasion

    Proc. Natl. Acad. Sci. U. S. A.

    (2008)
  • J.V. Soriano et al.

    Expression of an activated Notch4(int-3) oncoprotein disrupts morphogenesis and induces an invasive phenotype in mammary epithelial cells in vitro

    Int. J. Cancer

    (2000)
  • J. Yun et al.

    Crosstalk between PKCα and Notch4 in endocrine-resistant breast cancer cells

    Oncogenesis

    (2013)
  • Y. Lombardo et al.

    Nicastrin and Notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in MCF7 breast cancer cells

    Breast Cancer Res.

    (2014)
  • S. Kamakura et al.

    Hes binding to STAT3 mediates crosstalk between Notch and JAK–STAT signaling

    Nat. Cell Biol.

    (2004)
  • H.W. Lo et al.

    Constitutively activated Stat3 frequently coexpresses with epidermal growth factor receptor in high-grade gliomas and targeting STAT3 sensitizes them to iressa and alkylators

    Clin. Cancer Res.

    (2008)
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