Transforming growth factor-β1 increases cell migration and β1 integrin up-regulation in human lung cancer cells

Abstract

Transforming growth factor-β1 (TGF-β1) plays a crucial role in adhesion and migration of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found that TGF-β1 increased the migration and cell surface expression of β1 integrin in human lung cancer cells (A549 cells). TGF-β1 stimulation increased phosphorylation of p85α subunit of phosphatidylinositol 3-kinase (PI3K) and Ser⁴⁷³ of Akt was determined. Besides, we performed that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed the TGF-β1-induced migration activities of A549 cells. Treatment of A549 cells with NF-κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also repressed TGF-β1-induced cells migration and β1 integrins expression. In addition, treatment of A549 cells with TGF-β1 induced IκB kinase α/β (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB-luciferase activity. Furthermore, the TGF-β1-mediated increases in IKKα/β, IκBα phosphorylation and p65 Ser⁵³⁶ phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85α and Akt mutants also reduced the TGF-β1-induced κB-luciferase activity. Taken together, our results suggest that TGF-β1 acts through PI3K/Akt, which in turn activates IKKα/β and NF-κB, resulting in the activations of β1 integrins and contributing the migration of human lung cancer cells.

Introduction

Lung cancer is the leading cause of cancer-related mortality in both men and women [1], with 1.2 million new cases diagnosed every year and 1 million deaths being recorded worldwide [2]. Non-small cell lung cancer (NSCLC) affects approximately 80% of all lung cancer patients. Most patients present with locally advanced (37%) or metastatic (38%) disease at the time of diagnosis [1], and a large percentage of those diagnosed with early-stage disease eventually experience recurrence of metastatic disease. Thus, the high invasiveness of NSCLC to regional lymph nodes, liver, adrenal glands, contralateral lung, brain, and bone marrow, etc. may play a key role in its biological virulence [1].

Decades of scrutiny into the molecular bases of cancer have largely focused on what causes oncogenic transformation and the incipient emergence of tumors [3]. The invasion of tumor cells is a complex, multistage process. To facilitate the cell motility, invading cells need to change the cell–cell adhesion properties, rearrange the extracellular matrix (ECM) environment, suppress anoikis and reorganize their cytoskeletons [4]. Integrins are a family of transmembrane adhesion receptors comprising 19 α and 8 β subunits that interact noncovalently to form up to 24 different heterodimeric receptors. The combination of different integrin subunits on the cell surface allow cells to recognize and respond to a variety of different ECM proteins including fibronectin, laminin, collagen and vitronectin [5]. Because integrins are the primary receptors for cellular adhesion to ECM molecules, they act as crucial transducers of bidirectional cell signaling, regulating cell survival, differentiation, proliferation, migration and tissue remodelling [6]. Integrin has been heavily implicated in tumor development [7], [8], was correlated to reduced patient survival in colon carcinoma and melanoma [9], [10], and has been associated with breast cancer cell metastasis to bone [11]. In addition, in vitro studies have found that integrins facilitated prostate cancer cell adhesion and migration through several ECM substrates [12], [13], and transendothelial migration [14].

In the last 20 years, a large family of secreted polypeptides, known collectively as the transforming growth factor-β (TGF-β) super-family has been revealed [15]. TGF-β, originally called “sarcoma growth factor” was discovered in 1978 [16]. TGF-β is a potent growth inhibitor for a wide variety of cells including epithelial cells, vascular endothelial cells, hematopoietic cells, and immune lymphocytes. Perturbations of the TGF-β signaling pathways result in loss of cell growth regulation which is one of the most crucial steps in oncogenesis [17]. TGF-β family consists of three closely related isoforms (TGF-β1, -β2, and -β3) that are prototypes of the larger TGF-β super-family. TGF-β family members elicit a diverse range of cellular responses including cell proliferation, migration, fibrosis, inflammation, and wound repair [15], [18]. TGF-β1 knockout mice develop diffuse mononuclear cell infiltrates that prove lethal within a few weeks of birth [19]. Recent studies have suggested a fundamental role for TGF-β1 as a critical mediator of the metastasis activity of cancer cells [20]. Evidence for the role of TGF-β signaling in the complex process of cancer metastasis has recently been documented specifically in breast cancer. In mouse models of breast cancer, TGF-β promotes bone metastasis mediated by secreted factors such as parathyroid hormone-related peptide, interleukin-11 and CTGF [21], [22]. Besides, Smad signaling is required for this TGF-β-induced bone metastasis of breast cancer cells [23], [24]. Although the mechanisms underlying TGF-β1-mediated tumor invasion have been studied in some cancers [20], the role of TGF-β1 in the process of human lung cells migration remains large unknown.

Previous studies have shown that TGF-β1 modulates cell migration and invasion in several cancer cells [20]. TGF-β1-mediated invasion may involve activation of integrins receptors [32], [33]. However, the effect of TGF-β1 on integrins expression and migration activity in human non-small cell lung cancer cells is mostly unknown. Here we found a phenomenon that TGF-β1 increased the migration and the expression of β1 integrin of human lung cancer cells. In addition, phosphatidylinositol 3-kinase (PI3K), Akt, IKKα/β and NF-κB signaling pathways were involved it.