Elsevier

Cellular Signalling

Volume 27, Issue 6, June 2015, Pages 1208-1213
Cellular Signalling

SEMA4B inhibits growth of non-small cell lung cancer in vitro and in vivo

https://doi.org/10.1016/j.cellsig.2015.02.027Get rights and content

Highlights

  • SEMA4B inhibits the growth of NSCLC in vitro and in vivo.

  • Adaptation of SEMA4B in NSCLC cells altered cell proliferation.

  • SEMA4B induces FoxO1 nuclear retention through suppressing PI3K/Akt signaling.

Abstract

We have recently shown that Semaphorin 4B (SEMA4B) inhibits the invasion of non-small cell lung cancer (NSCLC) through PI3K-dependent suppression of MMP9 activation. In the current study, we evaluated whether SEMA4B may also affect the growth of NSCLC. We thus used two human NSCLC lines, A549 and Calu-3, to examine our hypothesis. We found that overexpression of SEMA4B significantly decreased NSCLC cell growth, while SEMA4B inhibition significantly increased NSCLC cell growth, both in vitro and in vivo in an implanted NSCLC model. Adaptation of SEMA4B in NSCLC cells did not alter cell apoptosis, but changed the cell proliferation. Further analyses show that SEMA4B may induce FoxO1 nuclear retention through suppressing PI3K/Akt signaling pathway, which subsequently inhibited cell growth through the direct nuclear target of FoxO1, p21. Our study thus demonstrate a role of SEMA4B in suppressing NSCLC growth, besides its role in inhibiting cell metastasis, and highlights SEMA4B as a promising therapeutic target for NSCLC.

Introduction

Non-small cell lung cancer (NSCLC) is a lung cancer with a high incidence and mainly consists three subtypes: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma [1], [2]. NSCLCs are generally insensitive to chemotherapy and radiation therapy, and often appear to grow very fast [1], [2], [3]. Thus, understanding of the mechanism controlling the growth of NSCLC is extremely important for its therapy.

The semaphorins are a family of proteins that regulate cell migration, angiogenesis and immune response [4]. The semaphorin family has been divided into 7 subclasses. The class 4 semaphorins has 7 members, among which SEMA4B has recently been shown to play a critical role in the tumorigenesis of NSCLC, by us [5], [6], and by others [7]. Specifically, the downstream signal transduction of semaphorins has been studied in different cell types, and has been found to involve different pathways including the phosphatidylinositol 3-kinase (PI3K) pathway, the extracellular-related kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, and the Jun N-terminal kinase (JNK) pathway [4], [5], [7], [8], [9], [10], [11], [12], [13], [14]. However, we recently reported that only PI3K signaling pathway is affected by SEMA4B in NSCLC [6].

The PI3K pathway is the major signaling pathway to regulate cell proliferation. Activated PI3K generates several phosphoinositols, leading to Akt activation by phosphorylation at Thr308 and Ser473, which further modulate downstream DNA-binding molecules, to trigger DNA synthesis [15], [16], [17], [18], [19]. Forkhead box protein O1 (FoxO1) is a key target of Akt, and the nuclear form of FoxO1 is a DNA binding protein to control the expression of many genes. When the phosphorylation status of FoxO1 protein is altered by phosphorylated Akt, FoxO1 will be excluded from the nuclei to cytoplasm in a process called nuclear exclusion, resulting in the adaptation of expressions of FoxO1-binding genes in the nuclei. On the other hand, FoxO1 nuclear translocation and retention have exactly the adverse effect in this context [3], [20], [21], [22], [23], [24], [25]. In most occasions, nuclear FoxO1 binds to the promoter of cell-cycle-inhibitor p21, which suppresses cell proliferation [3], [20], [21], [22], [23], [24], [25].

In our previous study, we have reported a significant decrease in SEMA4B levels and a significant increase in MMP9 levels in NSCLC from the patients. We have further shown that SEMA4B negatively regulated MMP9 levels, a key factor that induces NSCLC metastasis, exclusively through PI3K signaling pathway [6]. Here we aimed to figure out whether SEMA4B may affect NSCLC growth through its inhibition on PI3K downstream signals.

Section snippets

Cell lines and reagents

A549 and Calu-3 are two human NSCLC lines purchased from ATCC, and were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). The A549 cell line was first developed in 1972 by Dr. Giard through the removal and culturing of cancerous lung tissue in the explanted tumor a of 58-year-old Caucasian male [26]. Calu-3 cell line was developed by Dr. Fogh in 1975 [27].

Transduction of the cell lines

SEMA4B-adapted plasmids have been previously described [6].

SEMA4B inhibited NSCLC growth in vitro

In our previous study, we have reported a significant decrease in SEMA4B levels and a significant increase in the MMP9 levels in NSCLC from the patients. We have further shown that SEMA4B negatively regulated MMP9 levels, a key factor that induces NSCLC metastasis, exclusively through the PI3K signaling pathway [6]. Here we aimed to figure out whether SEMA4B may affect NSCLC growth through its inhibition on the PI3K downstream signals. Thus, we used two human NSCLC lines, A549 and Calu-3, for

Discussion

SEMA4B has been shown to play an important role in lung cancer invasion. However, whether SEMA4B may also regulate the cancer cell growth in lung cancer is not determined yet. Recently, we have shown that hypoxia-inducible factor 1 (HIF-1) could downregulate the expression of SEMA4B in human NSCLC lines, and then abolished its suppressive effect on NSCLC metastasis [5]. Moreover, we also reported a significant decrease in SEMA4B levels and a significant increase in the MMP9 levels in NSCLC from

Conflict of interest

The authors have declared that no competing interests exist.

Acknowledgment

This work is financially supported by Wujieping Medical Foundation No: 320.6750.14060, and Shanghai Chest Hospital Science and Technology Development Fund (2014 YZDC10600).

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