Original ArticleVascular mimicry formation is promoted by paracrine TGF-β and SDF1 of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma
Introduction
Development of adequate microcirculation is essential for overgrowth and metastasis of tumors. It has long been believed that microcirculation in cancers was dependent on angiogenesis, the formation of new capillaries from pre-existing vessels [1]. Recent studies have revealed that vascular mimicry (VM) is an important complement for tumor microcirculation [2], [3]. VM refers to the vascular channel-like structure that is consisted of tumor cells but not endothelial cells, which is similar to embryonic vasculogenesis [4], [5]. VM is correlated with short survival of malignant tumors [6], [7], [8]. Studies in the xenografts of breast cancer and melanoma reveal the perfusion ability of the VM channels [9], [10], suggesting that VM provides an alternative pathway to supply oxygen and nutrition for rapidly growing tumors, as well as an escape route for metastasis. A few molecules, including VE-cadherin, matrix metallopeptidases (MMPs) and laminin have been shown to be critical for VM formation [5], [11]. Expression of VE-cadherin in tumor cells can upregulate MMP14 expression and then activates MMP2, in turn cleaves laminin5γ2 (LAMC2) chain and results in the formation of γ2′ and γ2x fragments, which stimulates migration, invasion and VM formation of tumor cells [8], [12]. Moreover, knockdown of VE-cadherin, LAMC2 or neutralizing antibody of MMP2/MMP14 inhibits melanoma cells to form VM structure in vitro, suggesting that activation of VE-cadherin/MMPs/LAMC2 pathway is essential for VM formation [13]. However, the extracellular signals that trigger the VM formation of tumor cells remain largely unknown.
The interaction between stromal cells and tumor cells plays a major role in tumor progression. Cancer-associated fibroblasts (CAFs) constitute the majority of stromal cells in cancer tissues [14]. The presence of CAFs is correlated with tumor development and worse survival of cancer patients [15], [16]. CAFs secrete a wide spectrum of chemokines and cytokines to tumor microenvironment, thus promoting growth, invasion and angiogenesis of cancers [14], [17]. To date, the role of CAFs in VM formation is not reported yet.
In this study, we found that the CAF-secreted TGF-β and SDF1 enhanced the expression of VE-cadherin, MMP2 and LAMC2 in tumor cells, consequently promoted VM formation. We further showed that miR-101, a microRNA (miRNA) that was down-regulated in both tumor cells and CAFs, could suppress the CAF-promoted VM formation by abrogating both TGF-β and SDF1 signaling. These findings disclose a novel regulatory network of VM formation and provide potential targets for anti-cancer therapy.
Section snippets
Human tissue specimens
Normal liver tissues were obtained from patients underwent resection of hepatic hemangiomas. Tumor tissues were obtained from pathologically confirmed HCC patients who underwent tumor resection at the Cancer Center of Sun Yat-sen University. No local or systemic anticancer treatment had been conducted before operation and no post-operative anticancer therapies were administered prior to relapse. Informed consent was obtained from each patient and the study was approved by the Institute Research
CAFs promote VM formation in vitro and in vivo
To elucidate the effect of CAFs on VM formation, CAFs were isolated from primary human HCC tissues and then characterized by positive staining for the activated fibroblast marker (α-SMA) and mesenchymal marker (vimentin) and negative staining for epithelial marker (E-cadherin) and macrophage marker (CD68, Supplementary Figure S1). We first evaluated the effect of CM-CAF on the VM formation of tumor cells, using the in vitro 3-D culture model. As shown, QGY-7703, MHCC-97L and two cell lines
Discussion
CAF is a major constituent of tumor microenvironment [14]. However, the effect of CAFs on VM formation is unknown. In this study, we revealed that CAFs promoted VM formation through paracrine TGF-β and SDF1. On the other hand, miR-101 significantly inhibited VM formation by suppressing the TGF-β and SDF1 signaling pathways in tumor cells and repressing SDF1 expression in CAFs (Fig. 8C). Our findings reveal a novel function of CAFs as the promoter of VM formation and suggest miR-101 as a
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (81230049, 91440205, 81172343, 81401922), Ministry of Health of China (2012ZX10002011) and Natural Science Foundation of Guangdong Province (2014A030311031). We thank Prof. Yunfei Yuan for providing clinical samples.
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These authors contributed equally to this work.