FOXD3 suppresses tumor growth and angiogenesis in non-small cell lung cancer

Highlights

• FOXD3 is downregulated in NSCLC cell lines and tissues.

• FOXD3 overexpression inhibited cell proliferation in NSCLC cells.

• FOXD3 overexpression led to decreased angiogenesis in NSCLC cells in vitro and in vivo.

Abstract

The transcription factor forkhead box D3 (FOXD3), widely studied as a transcriptional repressor in embryogenesis, participates in the carcinogenesis of many cancers. However, the expression pattern and role of FOXD3 in non-small cell lung cancer (NSCLC) have not been well characterized. We report that FOXD3 is significantly downregulated in NSCLC cell lines and clinical tissues. FOXD3 overexpression significantly inhibits cell growth and results in G1 cell cycle arrest in NSCLC A549 and H1299 cells. In a xenograft tumor model, FOXD3 overexpression inhibits tumor growth and angiogenesis. Remarkably, expression of vascular endothelial growth factor (VEGF) was reduced in FOXD3 overexpression models both in vitro and in vivo. These findings suggest that FOXD3 plays a potential tumor suppressor role in NSCLC progression and represents a promising clinical prognostic marker and therapeutic target for this disease.

Introduction

Lung cancer is the second most common cancer, ranked at the top of all cancer-related deaths in the world that is a serious threat to human health and life [1], [2], [3]. Non-small cell lung cancer (NSCLC) includes adenocarcinoma and squamous cell carcinoma, and accounts for approximately 80% of all lung cancer cases [4]. Early diagnosis poses a challenge, as there is a lack of obvious symptoms in the early stages; most symptoms are presented in the late stages, after diagnosis [5]. In recent years, despite great progress in clinical diagnosis and treatment, lung cancer patients have a poor prognosis, with 5-year survival rates of only 9–20% [2], [6]. Therefore, there is a compelling need to develop novel diagnosis and therapeutic strategies. Investigations of the molecular mechanisms underlying the occurrence and progression of NSCLC can help to develop novel prognostic biomarkers and therapeutic targets for malignancy, and thus are clinically important.

Forkhead box (FOX) transcription factors, a family of proteins containing a conserved DNA-binding region known as the forkhead box or winged helix domain, mediate a wide spectrum of biologic processes such as metabolism, differentiation, proliferation, apoptosis, and migration [7], [8], [9]. As one important member of the FOX family, FOXD3 (Genesis/HFH2) was originally identified due to its expression in embryonic stem (ES) cells and serves numerous indispensable roles during development [10], [11]. With the expanding knowledge of FOXD3, its role in the tumorigenesis attracted tremendous attention in oncology research. It has been reported that FOXD3 has abnormal expression in tumor cells and participates in tumor onset and progression. Studies have demonstrated that FOXD3 significantly inhibits gastric cancer and melanoma cell growth and invasion [12], [13], [14], [15]. In line with these findings, FOXD3 was downregulated in neuroblastoma and affected growth, invasion, metastasis, and angiogenesis as a novel tumor suppressor [16]. However, contrary to these findings, FOXD3 was found to be upregulated in renal and endometrial cancers and might exert tumor suppressive and oncogenic roles in cervical and renal cancers, respectively [16]. Therefore, FOXD3 may exhibit tissue-specific expression patterns and functions in human tumors. It is worthy to note that the expression profile and role of FOXD3 in NSCLC has not been well characterized as of yet.

The evidence of FOXD3 function in tumor cells led us to examine the expression and function of FOXD3 in human NSCLC cells, and more importantly in nude mice with NSCLC in vivo. Herein, we demonstrate that FOXD3 expression is reduced in multiple NSCLC cells lines and tissues. Enforced expression of FOXD3 results in potent inhibition of proliferation in NSCLC cells lines by inducing cell cycle arrest in the G1 stage. Furthermore, this phenomenon was also observed in nude mice with NSCLC in vivo. In addition, FOXD3 overexpression led to an obvious inhibition in angiogenesis, as shown by decreased expression of VEGF both in vitro and in vivo. The microvessel density reflected by CD31 staining in xenograft tumor tissues was significantly decreased in the FOXD3 overexpression group. Taken together, these findings uncovered FOXD3 as novel cell-cycle repressor and angiogenesis inhibitor in NSCLC.