Hydroxychloroquine suppresses lung tumorigenesis via inducing FoxO3a nuclear translocation through STAT3 inactivation

doi:10.1016/j.lfs.2020.117366

Life Sciences

Volume 246, 1 April 2020, 117366

https://doi.org/10.1016/j.lfs.2020.117366 Get rights and content

Highlights

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HCQ inhibits cell proliferation and induces cell cycle arrest at G1/S transition in LUAD.
•
JAK-STAT and FoxO3a pathways are related to HCQ and LUAD by bioinformatic analysis.
•
HCQ impairs lung tumorigenesis dependent of STAT3/FoxO3a/p27^Kip1 signaling.
•
HCQ alone for LUAD exhibits anticancer activity in vivo without obvious toxicity.

Abstract

Background

Hydroxychloroquine exhibits synergistic anticancer properties as an adjuvant. However, the role and molecular mechanisms underlying of HCQ as monotherapy for lung adenocarcinoma (LUAD) have yet to be elucidated.

Methods

We assessed the antitumor effects of HCQ in LUAD cells through a series of in vitro and in vivo assays. GEO database and R packages were used to predict molecular mechanisms of HCQ in the treatment of lung adenocarcinoma, followed by verification of gene expression and subcellular localization via immunoblotting, immunofluorescent and immunohistochemistry assays.

Results

We showed the phenotypic effects that HCQ inhibited cell growth, induced apoptosis and cell cycle arrest at G1/S transition in A549 and PC-9 cells, which was associated with inhibition of CDK2, CDK4, CyclinD1 and CyclinE, but up-regulation of p21 and p27^Kip1. Bioinformatic analysis predicted that 63 targets related to HCQ and LUAD were mainly enriched in JAK-STAT and FoxO pathways. Then, we observed that HCQ decreased the phosphorylation of STAT3, but increased the expression of FoxO3a and its accumulation in the nucleus. The specific STAT3 inhibitor cryptotanshinon augmented the HCQ-induced upregulation and nuclear translocation of FoxO3a. In addition, HCQ increased the expression of p27^Kip1, which was impaired by FoxO3a blockade with siRNA. Finally, ablation of p27^Kip1 expression abrogated the cytotoxicity of HCQ. More importantly, similar results were further confirmed in vivo.

Conclusions

Taken together, this study suggests that STAT3/FoxO3a/p27^Kip1 signaling pathway is involved in the anticancer effects of HCQ, and provides preliminary evidence for therapeutic prospects of HCQ alone in LUAD.

Graphical abstract

Introduction

Lung cancer is ranked as one of the most common causes of cancer-related morbidity and mortality worldwide, of which nearly half are lung adenocarcinomas [1,2]. Although standardized treatment strategies benefited partial LUAD patients in the past decades, the majority of LUAD patients still have poor prognosis due to drug resistance, adverse effects or economic burden [3]. Thus, it seems to be more imperative to find a safer, cheaper and more effective drug for LUAD patients.

Hydroxychloroquine (HCQ), one of the classic anti-malarial drugs, has been used to treat cancer in several preclinical studies and clinical trials. Previous study suggested that HCQ was active in suppressing cell migration and metastasis, both in vitro and in vivo [4]. When combined with monotherapy, HCQ sensitized resistant non-small cell lung cancer to these anticancer agents [5,6]. For example, HCQ, combined with erlotinib or crizotinib, inhibited xenografted tumor growth and enhanced apoptosis, which was relevant to the inhibition of autophagic flux [7,8]. In clinical trials, more attentions have been put on HCQ in combination with other cytotoxic drugs. The limited public results were positive on the safety and therapeutic efficacy of HCQ as an adjuvant in the treatment of patients with cancer [9,10]. The fact that HCQ has been available as an adjuvant in the treatment of cancers needs a better insight of pharmaceutical mechanisms. Therefore, searching for novel anticancer mechanisms of HCQ awaits further elucidation in lung cancer.

Signal transducer and activator of transcription 3 (STAT3) belongs to a unique protein family STATs and acts as a transcription factor in carcinogenesis. More evidence indicated that STAT3 was aberrantly activated in several malignant tumors. In lung cancer, activated STAT3 was responsible for cell proliferation, metastasis and therapy resistance [[11], [12], [13]]. Accordingly, STAT3 blockade contributed to induce cell apoptosis, abrogate metastasis and treat resistant lung cancer [14,15]. Thus, it seems to be a novel therapeutic strategy for cancer by targeting STAT3.

Transcription factor forkhead box class O 3a (FoxO3a) is recognized as an important member of forkhead family and involved in many cellular processes such as cell proliferation, differentiation and metabolism [16,17]. Unlike phosphorylated STAT3, FoxO3a translocates into the nucleus in a dephosphorylated state, and further modulates the expression of downstream genes related to cell cycle and proliferation, such as p27^Kip1, p21^Cip1 and Bim [18]. Clinically, decreased expression of FoxO3a was significantly correlated with poor prognosis and therapeutic resistance in lung cancer patients [19]. Thus, it is necessary to explore new therapeutic approaches to increase the levels of FoxO3a.

In this study, we attempted to clarify the effects and underlying molecular mechanisms of HCQ for LUAD treatment. With the help of bioinformatics analysis and experimental results, our study discovered a novel role of STAT3/FoxO3a/p27^Kip1 axis in the anticancer activity of HCQ.

Section snippets

Cell culture

Human LUAD cell lines A549 and PC-9 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). PC-9 cells were cultured in Dulbecco's modified Eagle's medium/high glucose medium plus 10% fetal bovine serum (FBS, BI, Israeli); A549 cells were cultured in PRMI-1640 plus 10% FBS. They were grown at 37 °C under a 5% CO₂ humidified atmosphere.

Chemicals and reagents

Hydroxychloroquine sulfate was purchased from MedChemExpress (MCE, USA) and dissolved in saline. Cryptotanshinon (CTN) was obtained from

HCQ induces apoptosis and modulates cell cycle arrest at G1/S transition

In order to identify the effects of HCQ on the proliferation of LUAD cells in vitro, A549 and PC-9 cells were cultured with increasing concentrations (0, 10, 20, 40, 80, 120μmol/l) of HCQ for 24, 48 h and 72 h. We found that HCQ caused the growth attenuation in these cell lines in a dose- and time- dependent manner (Fig. 1A). For A549 cells, the IC50s of distinct time (24, 48, 72 h) were 115.23 ± 5.04, 91.95 ± 9.86 and 71.01 ± 8.38μmol/l, respectively. For PC-9 cells, IC50s were 98.98 ± 8.29,

Discussion

In this study, we elaborated the anticancer action and molecular mechanisms of HCQ in LUAD cells. We observed that HCQ induced LUAD cell apoptosis and cell cycle arrest at G1/S transition. Bioinformatic analysis indicated that HCQ-LUAD-related genes were enriched in JAK-STAT and FoxO3a signalings. Further experiments provided sufficient evidence that HCQ was able to target STAT3/FoxO3a/p27^Kip1 signaling to reduce LUAD growth in vitro and in vivo.

In past decades, HCQ has been used to treat

Conclusions

In conclusion, our study showed that HCQ restrained cell proliferation and induced cell cycle arrest at G1/S transition in LUAD cells via regulating the STAT3/FoxO3a/p27^Kip1 signaling. HCQ reduced the activation of STAT3, increased the expression and nuclear translocation of FoxO3a, and enhanced the expression of p27^Kip1, leading to suppress LUAD growth. The data in vitro and in vivo supported that HCQ alone may be a novel and well tolerated treatment approach for LUAD.

The following are the

Funding

This work was supported by National Natural Science Foundation of China [grant number 81672300] and Science and Technology Funds of Shaanxi Province [grant number 2017JM8159].

CRediT authorship contribution statement

Xin Lyu: Conceptualization, Data curation, Formal analysis, Methodology, Visualization, Writing - original draft. Lizhong Zeng: Investigation. Hua Zhang: Methodology. Yue Ke: Methodology. Xuan Liu: Conceptualization, Visualization. Nannan Zhao: Conceptualization. Jingyan Yuan: Conceptualization. Guoan Chen: Supervision, Writing - review & editing. Shuanying Yang: Conceptualization, Funding acquisition, Project administration, Writing - review & editing.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgement

Not applicable.

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Hydroxychloroquine suppresses lung tumorigenesis via inducing FoxO3a nuclear translocation through STAT3 inactivation

Highlights

Abstract

Background

Methods

Results

Conclusions

Graphical abstract

Introduction

Section snippets

Cell culture

Chemicals and reagents

HCQ induces apoptosis and modulates cell cycle arrest at G1/S transition

Discussion

Conclusions

Funding

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgement

Cell.

Lancet.

J. Control. Release

J. Thorac. Oncol.

J. Thorac. Oncol.

Cancer Treat Res Commun.

Cancer Cell

J. Thorac. Oncol.

Biochim Biophys Acta

J. Thorac. Oncol.

Lancet Infect. Dis.

Semin. Arthritis Rheum.

Biochem. Pharmacol.

Kaohsiung J. Med. Sci.

Cell. Signal.

Trends Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

Cancer treatment and survivorship statistics, 2016

CA Cancer J. Clin.

Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells

J. Exp. Clin. Cancer Res.

Induction of autophagy contributes to crizotinib resistance in ALK-positive lung cancer

Cancer Biol Ther.

Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) pathway in EGFR-Mutant NSCLC

J Natl Cancer Inst.

AKR1C1 activates STAT3 to promote the metastasis of non-small cell lung cancer

Theranostics.