Autocrine Semaphorin3A signaling is essential for the maintenance of stem-like cells in lung cancer

https://doi.org/10.1016/j.bbrc.2016.10.057Get rights and content

Highlights

  • Sema3a enhances tumorigenic capacity of cancer stem-like cells.

  • Sema3a is essential for the maintenance of cancer stem-like cells.

  • Sema3a can be a therapeutic target to eradicate cancer stem-like cells.

Abstract

Cancer stem-like cells (CSCs) exist in tumor tissues composed of heterogeneous cell population and are characterized by their self-renewal capacity and tumorigenicity. Many studies demonstrate that eradication of CSCs prevents development and recurrences of tumor; yet, molecules critical for the maintenance of CSCs have not been completely understood. We previously reported that Semaphorin3A (Sema3a) knockdown suppressed the tumorigenicity and proliferative capacity of Lewis lung carcinoma (LLC) cells. Therefore, we identified Sema3a as an essential factor for the establishment or maintenance of CSCs derived from LLC (LLC-stem cell). shRNA against Sema3a was introduced into LLC cells to establish a LLC-stem cell line and its effects on tumorigenesis, sphere formation, and mTORC1 activity were tested. Sema3a knockdown completely abolished tumorigenicity and the sphere-formation and self-renewal ability of LLC-stem cells. The Sema3a knockdown was also associated with decreased expression of mRNA for stem cell markers. The self-renewal ability abolished by Sema3a knockdown could not be recovered by exogenous addition of recombinant SEMA3A. In addition, the activity of mammalian target of rapamycin complex 1 (mTORC1) and the expression of its substrate p70S6K1 were also decreased. These results demonstrate that Sema3a is a potential therapeutic target in eradication of CSCs.

Introduction

In spite of the development of new treatments, lung cancer is currently the third leading cause of cancer death [1]. Therefore, a detailed understanding of lung cancer biology is essential for improvements in diagnosis and therapeutic strategies. There is increasing evidence that a small population of cells called cancer stem-like cells (CSC), which have stem cell-like properties including self-renewal, multi-potency, and drug-resistance, exist in many types of cancers [2], [3], [4]. CSCs have been shown to be responsible for tumorigenesis, indicating that their eradication could prevent tumor development and recurrence. Therapeutic strategies targeting CSCs have been reported to decrease tumor growth or prolong tumor latency in several tumor models [4], [5], [6], [7], [8]. However, signaling pathways that regulate the maintenance of CSCs remain to be elucidated.

Semaphorin molecules, which were originally discovered as axonal guidance factors, exhibit their activity by binding to plexin receptors and neuropilin co-receptors [9]. Recently, the regulatory role of semaphorin signaling in various diseases including immunological or bone disorders and cancer became clear [10]. Several groups demonstrated that Semaphorin3A (Sema3a) can act as a tumor suppressor by inhibiting angiogenesis, migration, metastasis, and cell proliferation [11], [12], [13]. However, there are contradictory reports that show that Sema3a promotes malignancy by increasing angiogenesis and migration [14], [15]. A recent study revealed that activation of the mutant, but not wild type, PLEXA1 by Sema3a increased the invasive and proliferative capacity of pancreatic cancer cells [16]. These conflicting results make it difficult to understand the role of Sema3a in tumor biology and further studies are needed to reveal the mechanism by which Sema3a exhibits tumor-suppressive or -promoting ability in different tumor microenvironments.

Previously we reported that shRNA mediated knockdown of Sema3a in Lewis lung carcinoma (LLC) cells suppressed its proliferative capacity and tumorigenicity [17], [18]. In this study, we examined the effect of Sema3a knockdown on stem cell-like properties of LLC-derived cancer stem-like cells (LLC-stem) to understand the role of Sema3a signaling in lung cancer.

Section snippets

Mice

Male C57BL/6J mice (5 weeks) used as recipients were provided from SLC (Japan). All animal experiments were approved by the Committee on Animal Experimentation of Niigata University of Pharmacy and Applied Life Sciences and performed in compliance with the University's Guidelines for the Care and Use of Laboratory Animals.

Cell culture

LLC-GFP cells were provided by AntiCancer Japan. LLC-stem cells were maintained in DMEM/F12 (Sigma-Aldrich) containing B27/RA(−) (Life technologies), 10 μg/ml EGF

Loss of tumorigenicity following Sema3a knockdown

By culturing tumor cells under nonadherent conditions, CSCs with sphere-forming and tumorigenic capacity have been established [19], [20], [21]. In this study, we used LLC cells that constitutively express GFP (LLC-GFP). When LLC-GFP cells were cultured on ultra-low attachment plates in DMEM/F12 containing 10 ng/ml FGF2, 10 ng/ml EGF, and B27 without retinoic acid (RA) for 1 week, the cells formed spheroid structures and were designated as LLC-stem cells. Scramble shRNA or Sema3a shRNA

Discussion

Several signaling pathways are associated with the maintenance of CSCs, the cells from which tumors originate [5], [8], [24], [25], [26]. This is the first known study demonstrating that autocrine Sema3a signaling plays a critical role in maintaining the self-renewal capacity of CSCs. Sema3a is a bivalent factor; serving as either tumor-inhibitory or tumor-promoting factor depending on tumor microenvironment. Our studies show that Sema3a promotes malignancy in lung cancer. Recently, Sema3f was

Author contribution

Daisuke Yamada and Takehiko Maeda conceived and designed the experiments. Daisuke Yamada and Kensuke Takahashi performed the experiments. Daisuke Yamada and Kensuke Takahashi analyzed data. Daisuke Yamada and Takehiko Maeda contributed reagents/materials/analysis tools. Daisuke Yamada, Kensuke Takahashi, Kohichi Kawahara and Takehiko Maeda wrote the paper.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number 15K18449 and 15K08682.

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