Elsevier

Life Sciences

Volume 281, 15 September 2021, 119763
Life Sciences

Apelin/APJ signaling activates autophagy to promote human lung adenocarcinoma cell migration

https://doi.org/10.1016/j.lfs.2021.119763Get rights and content

Highlights

  • Apelin/APJ signaling pathway activates autophagy in lung adenocarcinoma.

  • Apelin promotes BECN1 transcription via HIF1A.

  • Autophagy promotes lung adenocarcinoma cells migration.

  • Autophagy induces cofilin phosphorylation in lung adenocarcinoma.

  • Autophagy inhibition is a potential strategy in lung adenocarcinoma metastasis therapy.

Abstract

Aims

Beclin1(BECN1) is known as an autophagy-related protein and the expression is promoted by apelin in lung adenocarcinoma cells, suggesting that apelin activates autophagy in lung adenocarcinoma. However, the functions of apelin-induced autophagy in lung adenocarcinoma tumorigenesis and deterioration are still unknown. Thus, this study aims to investigate the effects of apelin-induced autophagy on lung adenocarcinoma tumorigenesis and deterioration.

Main methods

Protein expression of exogenous genes were detected by Western blotting analysis. Lung adenocarcinoma cell migration was assessed with cell migration assays. Autophagy was measured with quantification of GFP-LC3 or RFP-GFP-LC3 puncta using fluorescence microscopy in cells by an observed blinded to experimental condition and by western blot analysis of LC3 and p62 in cell lysates as well as autophagy flux. Immunofluorescence staining was performed in human lung adenocarcinoma A549 cells with p-cofilin antibody. The proteins expression in cancer specimens were examined with immunohistochemistry.

Key findings

Here, we reveal that apelin induces autophagy activation in lung adenocarcinoma. Apelin/APJ regulates BECN1 transcription via HIF1A. Apelin/APJ-activated autophagy promotes lung adenocarcinoma cell migration. Moreover, treatment with autophagy inhibitors significantly decreases apelin/APJ-induced lung adenocarcinoma cell migration. Evaluation of patient samples of lung adenocarcinoma reveals an association between APJ with BECN1 expression and a poor prognosis.

Significance

Our studies demonstrate that apelin-induced autophagy promotes lung adenocarcinoma cell migration which suggests a potential therapeutic target for lung adenocarcinoma.

Introduction

Apelin, the endogenous ligand of APJ [1], was isolated from the bovine stomach in 1998 [2]. Apelin isoforms have at least four bioactive forms including apelin-12, -13, -17, and -36. Researchers have confirmed that apelin and APJ exist in a variety of tissues such as heart, lung, brain, kidney, liver, blood vessels, and apelin/APJ system involved in a wide range of physiological and pathological functions [3], [4]. Apelin is reported as an oncogene in many cancer including lung cancer [5], breast cancer [6], prostate cancer [7], gastric cancer [8], oral cancer [9], colon cancer [10], ovarian cancer [11] and glioblastoma [12]. It plays important roles in carcinogenesis and tumor progress by inducing angiogenesis, metastasis, and therapy resistance [13].

In lung cancer, apelin significantly stimulates non-small cell lung cancer growth and micro-vessel formation [14]. Our previous research reveals that apelin/APJ promotes lung adenocarcinoma cell proliferation [15] and migration [16]. However, the intracellular mechanisms of apelin-regulated lung adenocarcinoma cell migration are not clear.

Autophagy is a conservative intracellular process in different species [17]. Recent studies discover that autophagy played major role in human cancer progression. Numerous studies demonstrate that autophagy promotes cancer cell proliferation and inhibits cancer cell apoptosis [18], [19], [20]. Autophagy is considered to play an important role in cancer cell migration [21], [22], [23], [24]. In lung cancer, researchers have verified that autophagy promotes cell migration [25]. However, it is unclear how autophagy controls migration. What's more, the upstream pathway that controls autophagy activation in lung cancer remains unclear.

Apelin plays inverse roles in autophagy reported in present studies. Apelin inhibited glucose deprivation-induced autophagy in rat cardiomyocytes [26] and hypoxia-induced autophagy in pulmonary arterial smooth muscle cells [27]. Apelin was involved in progression of diabetic nephropathy by inhibiting autophagy [28]. However, we found that the ROS-autophagy pathway mediates monocytes-human umbilical vein endothelial cells adhesion induced by apelin-13 [29]. The autophagy pathway is involved in apelin-13-induced cardiomyocyte hypertrophy [30], [31]. Additionally, over-expressed apelin/APJ activated autophagy in cancer. In lung adenocarcinoma, our previous research has revealed that apelin secretion in plasma and APJ expression in tissue of lung adenocarcinoma patients was increased. We also have proved that both over-expressed apelin and APJ increased BECN1 expression in A549 cells, revealing that apelin and APJ may activate autophagy in lung adenocarcinoma [15]. Therefore, we hypothesize that apelin promotes autophagy to induce migration in lung adenocarcinoma. Given the important role of autophagy, we focus on autophagy in therapeutic targeting.

Section snippets

Cell culture

Lung cancer A549 cells, H1299 cells, Human HEK293T were purchased from American Type Culture Collection (ATCC). A549 and H1299 cell lines that overexpress exogenous APJ or knockdown BECN1 were established as the below description. Cells were cultured in RPMI-1640 (GIBCO), supplemented with 10% FBS (GIBCO). Cells were kept in a 37 °C, 5% CO2-water jacket (Thermo scientific). A549 and H1299 cell lines were also recently authenticated using a STR DNA fingerprinting at Shanghai Biowing Applied

Autophagy-related genes are highly expressed in lung adenocarcinoma

Studies indicate that autophagy plays crucial roles in tumorigenesis and tumor progression including metastasis [35], [36]. To reveal autophagy activation in lung adenocarcinoma, firstly, we investigated the change of autophagy-related gene expression in lung adenocarcinoma. We downloaded a lung adenocarcinoma microarray dataset, GSE 2514 [37], and assessed the expression of autophagy-related genes including ATG5, ATG9, et al. As shown in Fig. 1A, autophagy-related gene expression was entirely

Discussion

Presently researches implied that apelin/APJ was a novel cancer factor [44]. Evidence confirmed that apelin/APJ played key roles in cancer cell migration [45], [46]. However, the underlying mechanisms keep major unclear. What's more, apelin has conflicting roles in autophagy. Here, we demonstrate that apelin induces autophagy activation in lung adenocarcinoma which dependents on its endogenous receptor APJ. Apelin/APJ activates autophagy by promoting Beclin1 expression via HIF1A. In our

Conclusion

In summary, we report a novel link in which the connection between G protein-coupled receptor APJ and autophagy in lung adenocarcinoma which connected to cell metastasis. We also reveal a novel mechanism that apelin increases cofilin phosphorylation in autophagy dependent manner (Fig. 7). Targeting autophagy is a potential strategy for lung adenocarcinoma therapy.

CRediT authorship contribution statement

Conception and design: Deguan Lv, Lanfang Li, Cuiqing Zhong, and Linxi Chen.

Acquisition of data (acquired patients, provided facilities, etc.): Deguan Lv, Jiaqi Liu, Zhe Chen, Xuling Luo, and Li Yang.

Analysis and interpretation of data (e.g. statistical analysis, biostatistics, computational analysis): Deguan Lv, Xuling, Zhe Chen, Meiqing Liu, Yao Li, Shifang Huang, and Feng Xie.

Writing, reviewing, and/or revision of the manuscript: Deguan Lv, Zhe Chen, Cuiqing Zhong, Linxi Chen, Mingzhu. Tang,

Declaration of competing interest

All authors have completed the ICMJE uniform disclosure form. The authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81703515, 81670265, and 81503074). This work was also supported in part by Initiative Postdocs Supporting Program (BX201600108 to Cuiqing Zhong).

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  • Cited by (5)

    The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

    1

    Deguan Lv, Xuling Luo and Zhe Chen all contribute equally to this study.

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