GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin-dependent RhoA activation

Abstract

The precise relationship between GCF2 expression and carcinogenesis has not yet been established. To clarify the metastatic potential of GCF2 in colorectal cancer, HT-29 cells stably suppressing GCF2 expression were injected into the spleens of severe combined immunodeficient (SCID) mice. GCF2 suppression reduced the number of metastatic foci in the liver and reduced fibronectin-induced cell adhesion, migration, and invasion. Downstream from the integrin signaling pathways, GCF2 regulates RhoA interaction with the RGS domain of Leukemia associated RhoGEF (LARG). Altogether, our results suggest that GCF2 plays an important role in colorectal cancer metastasis by regulating RhoA-induced cell adhesion, migration, and invasion.

Introduction

The study of metastasis, or the spread of cancer cells from the primary neoplasm to distant sites, is a crucial aspect in cancer research. Liver is the most common target organ in the metastasis of colorectal cancer [1]. Contrary to most digestive organs, the sinusoidal endothelium of the liver is characterized by an incomplete covering of microvessel structures, which leaves extracellular matrix (ECM) components directly accessible to circulating cancer cells [2]. Modifications in tumor metastasis is known to be associated with altered adhesive properties of cancer cells, and highly metastatic tumors show higher rates of adhesion to fibronectin, which is a major component of ECM [3]. The interaction between the fibronectin and integrin receptors is crucial in the progression of metastatic disease [4]. The adhesion of metastatic cells to fibronectin of the ECM activates integrin receptor-dependent signaling pathways [5], which induce their migration into the tissue [6], [7].

Several kinds of integrin superfamilies are expressed on colorectal cancer cells, of which αvβ5 and αvβ6 are the most important ones involved in the formation of metastasis of colorectal cancer, by mediating cellular attachment to the ECM or regulating the motility of cancer cells [5], [8], [9]. Integrin regulates downstream signaling pathways such as the phosphorylation of focal adhesion kinase, the recruitment of adaptor proteins, and the activation of small GTPases. RhoA, one of the small GTPase proteins, regulates signal transduction from cell surface receptors to intracellular target molecules [10]. It plays a pivotal role in remodeling actin stress fibers, inducing firm cell adhesion by exchanging the focal complexes to focal adhesion and cell migration by regulating the actomyosin contractility and rear cell detachment [11], [12].

Rho proteins are activated when they bind to GTP, while they are inactivated when GTP is hydrolyzed to GDP [11]. This regulatory cycle is controlled by various protein families within particular spatiotemporal contexts, and RhoA activation is regulated by guanine nucleotide exchange factors (GEFs) [13], [14]. When metastasis occurs, the fibronectin–integrin interaction activates the downstream signaling pathways, and it has been hypothesized that specific RhoGEFs such as p115RhoGEF, leukemia-associated RhoGEF (LARG), and P190RhoGEF become involved [10], [15]. These RhoGEFs activate RhoA.

The GC-binding factor 2/leucine-rich repeat in Flightless-1 interaction protein 1 (GCF2/LRRFIP1) gene encodes a 752-amino acid protein with a molecular mass of 160 kDa [16]. Previously, a comprehensive analysis of the human genome sequence revealed GCF2 to be one of the cancer-associated genes [17]. However, its role in carcinoma cells remains to be elucidated. Previous reports regarding GCF2 function have indicated that this molecule binds to the promoter region of several genes, consequently repressing transcriptional activity [16], [18], [19], [20]. In addition to the function of GCF2 as a transcriptional regulator, it has been indicated to interact with several molecules in the cytoplasm, such as leucine-rich repeat of flightless 1 (Fli1) or Dishevelled (Dvl) [21], [22], [23]. Dishevelled (Dvl) is a key molecule in the Wnt signaling cascade. When interacting with Dvl, GCF2 positively RhoA activity in the Wnt non-canonical planar cell polarity (PCP) pathway [23]. Since RhoA is involved in the development of metastasis [24], we hypothesized that GCF2 might also play an essential role in cancer metastasis. Therefore, in our study, we aimed to investigate the role of GCF2 in detail. To reveal the impact of GCF2 on the formation of liver metastasis, the metastatic capacity assay was performed by injecting cancer cells into the spleen of immunodeficient mice [25]. The colorectal cancer cell lines that inhibited GCF2 expression stably were established and used for the assay. Our data demonstrated that the inhibition of GCF2 dramatically reduced the number of metastatic foci in the liver. Moreover, a macromolecular analysis demonstrated that the GCF2 expression contributes to fibronectin–integrin-mediated cancer metastasis by regulating cell adhesion and migration. In conclusion, we demonstrated that GCF2 plays an important role in colorectal cancer metastasis. Ours is the first study to reveal the effect of GCF2 in cancer metastasis both in vitro and in vivo.