Cancer-associated fibroblasts promote hepatocellular carcinoma metastasis through chemokine-activated hedgehog and TGF-β pathways

Highlights

• CAFs promote the malignancy of HCC cells in vitro and in vivo.

• CCL2, CCL5, CCL7 and CXCL16 secreted by CAFs promote HCC cell migration and invasion.

• Hh and TGF-β pathways are involved in the effect of CAF-secreted chemokines on HCC.

Abstract

Fibroblasts are rich in the surrounding microenvironment of hepatocellular carcinoma (HCC) because most HCCs occur in fibrotic or cirrhotic livers. However, the role of cancer-associated fibroblasts (CAFs) in HCC metastasis remains obscure. Here, we reported that CAFs promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice. The RayBio human chemokine antibody array revealed that CAFs secret higher levels of CCL2, CCL5, CCL7 and CXCL16 than peri-tumor fibroblasts. CCL2 and CCL5 increase the migration but not the invasion of HCC cells, while CCL7 and CXCL16 promote both migration and invasion of HCC cells. Moreover, CCL2 and CCL5 stimulate the activation of the hedgehog (Hh) pathway, while CCL7 and CXCL16 enhance the activity of the transforming growth factor-β (TGF-β) pathway in HCC cells. The neutralizing antibodies of chemokines notably attenuate the effect of CAFs on HCC metastasis and compromised the activation of Hh and TGF-β pathways in HCC cells. In summary, CAF-secreted CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.

Introduction

The tumor microenvironment is a complicated mixture of tumor cells within the extracellular matrix (ECM), including diffusible growth factors, cytokines, and various types of stromal cells. A specialized group of fibroblasts called cancer-associated fibroblasts (CAFs) is believed to actively participate in the metastasis and invasion of tumor cells [1], [2]. Evidence from clinical and epidemiological studies has shown a strong association between CAFs and poor prognosis in several types of cancer, including HCC [3], [4].

Although CAFs have a high degree of heterogeneity due to their various origins, all of them express certain mesenchymal markers including α-SMA, Vimentin, and FSP-1. However, CAFs do not express the common markers of epithelial and endothelial cells [1], [2]. In addition, CAFs are quite different from PTFs in both their molecular constitution as well as their functional impact on neighboring epithelial cells [5]. It is known that most HCCs occur in fibrotic or cirrhotic livers. The major producers of the fibrotic extracellular matrix in the liver are myofibroblasts derived from quiescent fibroblasts and hepatic stellate cells activated by chronic injury. Consistently, HCC cells are embedded in a surrounding microenvironment that is rich in fibroblasts. Previous studies have successfully isolated CAFs and PTFs from human HCC samples [4], [6]. The myofibroblast marker α-SMA is expressed at higher levels in CAFs than in normal fibroblasts [6], [7]. CAFs in HCCs also proliferated more efficiently than PTFs [6].

Previous studies have revealed that CAFs promote cancer progression and metastasis by secreting a variety of soluble factors, including inflammatory cytokines, growth factors and chemokines [6], [8]. TGF-β and hepatocyte growth factor (HGF) released by CAFs have been shown to induce the malignant transformation of human breast cells [9]. The inhibition of CAF-secreted fibroblast growth factor-2 (FGF-2) reduces angiogenesis [10]. CAF-derived HGF can active the c-Met pathway in pre-invasive DCIS and enhance the transition to invasive carcinoma in breast cancer [11]. It has been reported that CAFs also increase the proliferation and invasive property of HCC cells [12]. However, the cytokine profile of CAFs in HCC remains unclear.

As mediators of inflammation, chemokines have been reported to modulate the recruitment of inflammatory cells and, thus, modify the environment in which cancer eventually develops. Recently, several studies demonstrated that chemokines also directly affect tumor cells. CCL2 produced by CAFs stimulates cancer stem cell self-renewal of breast cancer cells [13]. CCL5 promotes tumor growth and invasion of ovarian cancer [14]. A recent study found the CXCR2/CXCL5 axis contributes to the spread of HCC by inducing the EMT through activation of the PI3K/Akt/GSK-3β/Snail pathway [15]. These findings suggest that chemokines directly promote tumor progression. Nevertheless, the role of CAF-derived chemokines in HCC progression remains elusive.

In this study, we investigated the effect of CAFs on HCC metastasis in vitro and in vivo. The differential chemokines secreted by CAFs and PTFs were also analyzed. We found that CAF-derived CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through inducing the activation of Hh and TGF-β pathways in HCC cells.