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