Cancer Letters

Cancer Letters

Volume 380, Issue 2, 1 October 2016, Pages 424-433
Cancer Letters

Original Articles
Apigenin inhibits the inducible expression of programmed death ligand 1 by human and mouse mammary carcinoma cells

https://doi.org/10.1016/j.canlet.2016.06.023Get rights and content

Highlights

  • Apigenin inhibits interferon-γ-induced PD-L1 expression by breast cancer cells.

  • The apigenin metabolite luteolin inhibits inducible PD-L1 expression.

  • Interferon-γ mediated STAT1 activation is inhibited by apigenin.

  • T cell proliferation is enhanced in the presence of apigenin-treated breast cancer cells.

Abstract

Programmed death ligand 1 (PD-L1) is expressed by many cancer cell types, as well as by activated T cells and antigen-presenting cells. Constitutive and inducible PD-L1 expression contributes to immune evasion by breast cancer (BC) cells. We show here that the dietary phytochemical apigenin inhibited interferon (IFN)-γ-induced PD-L1 upregulation by triple-negative MDA-MB-468 BC cells, HER2+ SK-BR-3 BC cells, and 4T1 mouse mammary carcinoma cells, as well as human mammary epithelial cells, but did not affect constitutive PD-L1 expression by triple-negative MDA-MB-231 BC cells. IFN-β-induced expression of PD-L1 by MDA-MB-468 cells was also inhibited by apigenin. In addition, luteolin, the major metabolite of apigenin, inhibited IFN-γ-induced PD-L1 expression by MDA-MB-468 cells. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 and 4T1 cells was associated with reduced phosphorylation of STAT1, which was early and transient at Tyr701 and sustained at Ser727. Apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by MDA-MB-468 cells also increased proliferation and interleukin-2 synthesis by PD-1-expressing Jurkat T cells that were co-cultured with MDA-MB-468 cells. Apigenin therefore has the potential to increase the vulnerability of BC cells to T cell-mediated anti-tumor immune responses.

Introduction

Although there have been substantial advances in the screening and treatment of breast cancer (BC), the disease remains the most common cancer in women and has the second highest rate of mortality [1]. The ability to evade detection and elimination by the immune system has been identified as an important factor in cancer progression [2]. In this regard, constitutive or induced expression of programmed death-ligand 1 (PD-L1), also known as B7-H1, is among the immune evasion mechanisms observed in many cancers, including BC, and is typically associated with a poor prognosis [3], [4], [5], [6], [7], [8], [9].

Interferon (IFN)-γ is a potent inducer of PD-L1 on cancer cells [10], acting via MyD88/tumor necrosis factor receptor-associated factor 6/extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (STAT1) [11], the phosphoinositide 3-kinase (PI3K)/ERK signaling pathway [12], and the IFN-γ receptor-associated Jak1/Jak2/STAT1 signaling cascade [13]. Since the PD-1 ligand for PD-L1 is expressed by most tumor-infiltrating T cells [14] and IFN-γ is among the cytokines present in the microenvironment of some breast carcinomas and other tumors [15], [16], it is believed that IFN-γ-induced upregulation of PD-L1 on tumor cells suppresses the cell-mediated anti-tumor immune response [17]. For example, tumor cell expression of PD-L1 is correlated with decreased tumor infiltration by CD8+ cytotoxic T cells, reduced T cell activation and interleukin (IL)-2 production, and T cell anergy or apoptosis [18], [19], [20]. In addition, tumor growth in mouse models of cancer is inhibited by antibody (Ab)-mediated blockade of PD-1 or PD-L1, DNA vaccination with the extracellular region of PD-1, genetic elimination of the PD-1 gene, RNA interference, or expression of recombinant soluble PD-1 and PD-L1 proteins [21], [22], [23]. A number of clinical trials have investigated the effects of various drugs that interrupt the PD-1/PD-L1 pathway on cancer progression [24], [25], [26], [27]. Based on strong evidence that the PD-1/PD-L1 pathway is a suitable target for cancer treatment, MPDL3280A (anti-PD-L1 Ab) has been granted breakthrough therapy status for the treatment of bladder cancer [24]. Patients who respond to PD-1/PD-L1 inhibitors have higher numbers of CD8-, PD-1- and PD-L1 expressing cells at the invasive tumor margin and within the tumor itself [27]. Since immune evasion promotes tumor development and progression, agents that have the potential to interfere with the PD-1/PD-L1 pathway may be the basis for new cancer treatments.

Epidemiological studies have established that a diet rich in fruits and vegetables is associated with a lower risk of certain cancers, including BC [28], [29], [30]. Many of the disease-preventing components of fruits and vegetables belong to the flavonoid class of phytochemicals [31]. Apigenin is a bioavailable flavone found in a variety of fruits, vegetables, and beverages, including parsley, onions, grapefruit, oranges, and chamomile tea [32], [33]. Apigenin protects against carcinogen-induced mammary tumor development in rats [34] and inhibits the proliferation of BC cells and other cancer cell types by causing G2/M cell cycle arrest and, at higher concentrations, inducing apoptosis [35], [36], [37], [38], [39]. Importantly, apigenin does not harm normal, healthy cells [35]. However, the immunomodulatory properties of apigenin are less understood. Since apigenin and its metabolite luteolin reduce microglia-mediated inflammation via inhibition of IFN-γ-induced STAT1 phosphorylation [40], we predicted that apigenin would also inhibit STAT1-dependent PD-L1 expression induced in BC cells following exposure to IFN-γ or other cytokines that trigger STAT-1 phosphorylation. The present investigation focuses on the effect of the phytochemical apigenin on inducible and constitutive PD-L1 expression by mammary carcinoma cells. We provide evidence that apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by BC cells may allow T cells to mount an anti-tumor immune response.

Section snippets

Cell culture

MDA-MB-231 human BC cells were kindly provided by Dr. S. Dover (Memorial University of Newfoundland, St. John's, NL). Dr. P. Lee and Dr. G. Dellaire generously provided MDA-MB-468 and SK-BR-3 human BC cells, respectively (Dalhousie University, Halifax, NS). 4T1 mouse mammary carcinoma cells were provided by Dr. D. Waisman (Dalhousie University). All BC cell lines were authenticated by short tandem repeat profiling performed by ATCC (Manassas, VA). Human mammary epithelial cells (HMEC) were

Apigenin inhibits IFN-γ-induced PD-L1 expression by mammary carcinoma cells

Consistent with findings from earlier studies [8], [10], we observed that IFN-γ-treated MDA-MB-468 BC cells showed a time- and dose-dependent increase in PD-L1 expression (Fig. 1A and Appendix: Supplementary data S1). Optimal induction of PD-L1 was obtained after 24 h treatment with 10 ng/ml IFN-γ, although PD-L1 expression was also increased after 12 h exposure to IFN-γ. As shown in Fig. 1B, pretreatment of MDA-MB-468 cells with 30 µM apigenin, which does not affect the viability of the

Discussion

Apigenin has been extensively studied for its direct inhibitory effects on the growth of cancer cells [35], [36], [37], [38], [39]; however, to our knowledge, the present investigation is the first to provide evidence of a potential role for apigenin in enhancing a T cell-mediated anti-tumor immune response against BC cells by targeting the PD-L1/PD-1 checkpoint, which is considered a promising target for immunotherapy [17]. Many solid tumors use IFN-γ, which is often present within the tumor

Conflict of interest

All authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the Dalhousie Medical Research Foundation and the Canadian Breast Cancer Foundation-Atlantic Region. M. Harrison was the recipient of a Frederick Banting and Charles Best Canada Graduate Scholarship, a Canadian Imperial Bank of Commerce Graduate Scholarship in Medical Research and a Trainee Award from The Beatrice Hunter Cancer Research Institute as part of the Terry Fox Strategic Health Research Training Program in Cancer Research at the Canadian Institutes of Health

References (46)

  • E.J. Choi et al.

    Apigenin causes G2/M arrest associated with the modulation of p21Cip1 and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells

    J. Nutr. Biochem

    (2009)
  • B. Schreiner et al.

    Interferon-β enhances monocyte and dendritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis

    J. Neuroimmunol

    (2004)
  • R.L. Siegel et al.

    Cancer statistics, 2016

    CA Cancer J. Clin

    (2015)
  • Y. Ohigashi et al.

    Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer

    Clin. Cancer Res

    (2005)
  • R.H. Thompson et al.

    Costimulatory B7-H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target

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

    (2004)
  • J. Hamanishi et al.

    Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer

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

    (2007)
  • R.H. Thompson et al.

    Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up

    Cancer Res

    (2006)
  • H. Ghebeh et al.

    Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule

    Breast Cancer Res

    (2010)
  • S. Muenst et al.

    Expression of programmed death ligand 1 (PD-L1) is associated with poor prognosis in human breast cancer

    Breast Cancer Res. Treat

    (2014)
  • H. Dong et al.

    Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion

    Nat. Med

    (2002)
  • R. Bellucci et al.

    Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression

    Oncoimmunology

    (2015)
  • K.B. De Oliveira et al.

    CXCL12, CXCR4 and IFNγ genes expression: implications for proinflammatory microenvironment of breast cancer

    Clin. Exp. Med

    (2013)
  • K. Abiko et al.

    IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer

    Br. J. Cancer

    (2015)
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    These individuals contributed equally to this work.

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