Elsevier

Biochemical Pharmacology

Volume 144, 15 November 2017, Pages 78-89
Biochemical Pharmacology

Antihelminthic drug niclosamide inhibits CIP2A and reactivates tumor suppressor protein phosphatase 2A in non-small cell lung cancer cells

https://doi.org/10.1016/j.bcp.2017.08.009Get rights and content

Abstract

Protein phosphatase 2A (PP2A) is a critical tumor suppressor complex responsible for the inactivation of various oncogenes. Recently, PP2A reactivation has emerged as an anticancer strategy. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A, is upregulated in many cancer cells, including non-small cell lung cancer (NSCLC) cells. We demonstrated that the antihelminthic drug niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A in NSCLC cells. We performed a drug-repurposing screen and identified niclosamide as a CIP2A suppressor in NSCLC cells. Niclosamide inhibited cell proliferation, colony formation, and tumor sphere formation, and induced mitochondrial dysfunction through increased mitochondrial ROS production in NSCLC cells; however, these effects were rescued by CIP2A overexpression, which indicated that the antitumor activity of niclosamide was dependent on CIP2A. We found that niclosamide increased PP2A activity through CIP2A inhibition, which reduced the phosphorylation of several oncogenic proteins. Moreover, we found that a niclosamide analog inhibited CIP2A expression and increased PP2A activity in several types of NSCLC cells. Finally, we showed that other well-known PP2A activators, including forskolin and FTY720, did not inhibit CIP2A and that their activities were not dependent on CIP2A. Collectively, our data suggested that niclosamide effectively suppressed CIP2A expression and subsequently activated PP2A in NSCLC cells. This provided strong evidence for the potential use of niclosamide as a PP2A-activating drug in the clinical treatment of NSCLC.

Introduction

Lung cancer, the leading cause of cancer deaths worldwide, is broadly classified into small cell lung cancer (13% of cases) and non-small cell lung cancer (NSCLC) (87% of cases) [1]. Despite recent advances in the development of more effective therapeutic agents such as targeted drugs, the long-term outcome for many patients with NSCLC remains poor [2]. Chemotherapy is currently the standard treatment for NSCLC [3]. However, the clinical benefits of chemotherapy are limited by the development of resistance. Therefore, there is an unmet need for the understanding of the molecular mechanisms underlying tumor resistance to improve therapeutic treatment in patients with NSCLC.

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases and plays a crucial role in tumor suppression by dephosphorylating and inactivating the numerous kinase-driven intracellular signaling pathways in multiple cancers [4], [5]. Many studies have suggested that PP2A inhibition is closely associated with tumor progression and resistance in multiple cancers, including NSCLC [6], [7], [8], [9], [10], [11]. Moreover, PP2A inactivation is a critical step in the promotion of cellular transformation and tumor progression [4]. The inhibition of PP2A activity leads to the upregulation of many cancer-related signaling pathways, such as Akt, c-Myc, and ERK-1/2 [12], [13], [14]. However, the restoration of PP2A activity reduces tumor progression and enhances chemosensitivity in multiple cancers including NSCLC [6], [7], [8], [9], [10]. Therefore, PP2A reactivation is considered an attractive therapeutic strategy for anticancer treatment, including tumor resistance.

Cancerous inhibitor of PP2A (CIP2A) is an oncogene known to inhibit endogenous PP2A activity and thereby activate multiple oncogenic proteins, including Akt, c-Myc, E2F1, Plk1, and mTORC1, in various cancers [15], [16]. CIP2A is highly expressed in many cancers and its inhibition was shown to reduce tumor formation in various cancer models [16]. Furthermore, CIP2A knockout mice showed no severe defects in development and viability owing to its low expression in most normal tissues [17]. Thus, CIP2A inhibitors have emerged as an attractive anticancer drug without any adverse side effects [16]. Recent studies have shown that numerous anticancer drugs suppress CIP2A through various signaling pathways (Chk1 inhibitor UCN01 [18], EGFR inhibitor afatinib [19], MEK inhibitor U0126 [20], mTOR inhibitor temsirolimus [21], and the proteasome inhibitors bortezomib [22] or celastrol [23]). However, these drugs are thought to affect other signaling pathways in normal cells that are often linked to severe systemic toxicity. Thus, it is critical to identify more specific inhibitors of CIP2A with selective anticancer activity.

As drug discovery is a time-consuming and an expensive process, the repositioning of approved drugs has emerged as an alternative approach for the identification of new treatments for diseases. Drug repositioning has an advantage over de novo drug discovery, as many drugs have known pharmacokinetic and safety profiles; thus, any newly identified uses can be rapidly evaluated in clinical trials. In this study, we performed high-throughput screening using 1771 clinically approved compounds and identified niclosamide as a potent CIP2A inhibitor in NSCLC cells. We found that niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A. Moreover, niclosamide exhibited antitumor activity via the suppression of multiple oncogenic pathways in NSCLC cells. Our findings suggested that niclosamide is a novel suppressor of CIP2A in NSCLC cells.

Section snippets

Cell lines and reagents

H1299, H460, A549, H23, H358, IMR90, and MRC5 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in DMEM (Gibco-BRL, Rockville, MD) supplemented with 10% FBS (Corning, NY) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator. The cells were preserved and passaged in accordance with ATCC protocols for a maximum of 2 months and tested monthly for mycoplasma infection by using Hoechst 33258 staining. Niclosamide, UCN-01, U0126, and

Antihelminthic niclosamide suppresses CIP2A expression in NSCLC cells

To identify the repositioning drug that inhibits CIP2A in NSCLC cells, we conducted high-throughput screening of a clinically approved compound library using H1299-CIP2A-Luc cells that expressed CIP2A promoter linked to luciferase reporter [20]. Celastrol, which is known to inhibit CIP2A in NSCLC cells [23], was used as a positive control. In the primary screen, cell viability was measured by an MTT assay, and then the CIP2A reporter assay was conducted. Any compound that demonstrated at least

Discussion

In this study, we showed that the antihelminthic drug niclosamide was a CIP2A suppressor with the ability to activate tumor-suppressive PP2A activity in NSCLC cell lines. Using a clinically approved library containing 1771 compounds, we screened CIP2A-suppressor compounds with cancer-selective activity and identified niclosamide as a specific inhibitor of CIP2A. In addition, we demonstrated that the antitumor activity of niclosamide was dependent on CIP2A in various models. Notably, we

Acknowledgements

The chemical library used in this study was gifted by Korea Chemical Bank of Korea Research Institute of Chemical Technology. This work was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare (No. HI14C1864).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

References (45)

  • D.H. Johnson et al.

    Recent clinical advances in lung cancer management

    J. Clin. Oncol.

    (2014)
  • J. Sangodkar et al.

    All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase

    FEBS J.

    (2016)
  • I. Cristobal et al.

    PP2A inhibition is a common event in colorectal cancer and its restoration using FTY720 shows promising therapeutic potential

    Mol. Cancer Ther.

    (2014)
  • R. Rincon et al.

    PP2A inhibition determines poor outcome and doxorubicin resistance in early breast cancer and its activation shows promising therapeutic effects

    Oncotarget

    (2015)
  • M.H. Hung et al.

    SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A

    Oncotarget

    (2016)
  • A.S. Farrell et al.

    Targeting inhibitors of the tumor suppressor PP2A for the treatment of pancreatic cancer

    Mol. Cancer Res.

    (2014)
  • K.G. Roberts et al.

    Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers

    Cancer Res.

    (2010)
  • H. Liu et al.

    Overexpression of PP2A inhibitor SET oncoprotein is associated with tumor progression and poor prognosis in human non-small cell lung cancer

    Oncotarget

    (2015)
  • A.A. Sablina et al.

    Identification of PP2A complexes and pathways involved in cell transformation

    Cancer Res.

    (2010)
  • H.K. Arnold et al.

    Protein phosphatase 2A regulatory subunit B56alpha associates with c-myc and negatively regulates c-myc accumulation

    Mol. Cell. Biol.

    (2006)
  • A. Khanna et al.

    Cancerous inhibitor of protein phosphatase 2A, an emerging human oncoprotein and a potential cancer therapy target

    Cancer Res.

    (2013)
  • S. Ventela et al.

    CIP2A promotes proliferation of spermatogonial progenitor cells and spermatogenesis in mice

    PLoS One

    (2012)
  • Cited by (0)

    View full text