Cancer Letters

Cancer Letters

Volume 383, Issue 1, 1 December 2016, Pages 18-27
Cancer Letters

Original Article
Vascular mimicry formation is promoted by paracrine TGF-β and SDF1 of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma

https://doi.org/10.1016/j.canlet.2016.09.012Get rights and content

Highlights

  • CAFs promoted tumor cells to form VM structure in vitro and in vivo.

  • CAFs secreted TGF-β and SDF1 induced the expression of VE-cadherin, MMP2 and LAMC2.

  • MiR-101 attenuated TGF-β and SDF1 signaling by targeting TGF-βR1, Smad2 and SDF1.

  • MiR-101 suppressed the CAF-promoted VM formation in vitro and in vivo.

Abstract

Vascular mimicry (VM) describes the phenomenon that tumor cells but not endothelial cells form vascular-like channels, which provide blood perfusion for tumor tissues. VM is associated with tumor growth, metastasis and worse survival of different cancers. The mechanisms of VM formation remain largely unknown. We showed that the conditioned medium of cancer-associated fibroblast (CM-CAF) promoted tumor cells to form capillary-like structure in vitro. Consistently, co-implantation of CAFs with tumor cells significantly enhanced VM formation in mouse xenografts, and higher amount of CAFs was found in VM+ human HCC tissues compared to VM ones. However, the CM-CAF-promoted VM formation was attenuated when TGF-β or SDF1 signaling was abrogated. Similar to CM-CAF, recombinant TGF-β1 and SDF1 induced VM formation. We further disclosed that the CAF-secreted TGF-β and SDF1 enhanced the expression of VE-cadherin, MMP2 and laminin5γ2 via TGF-βR1 and CXCR4 in tumor cells, thereby promoted VM formation. Moreover, tumor cells with high activity of self-sustaining TGF-β signaling displayed strong capability of VM formation. Subsequent investigations showed that miR-101, which was down-regulated in both tumor cells and CAFs, suppressed the CAF-promoted VM formation in vitro and in vivo. Gain- and loss-of-function analyses revealed that miR-101 attenuated TGF-β signaling transduction by targeting TGF-βR1 and Smad2 in tumor cells, and simultaneously abrogated SDF1 signaling by suppressing SDF1 expression in CAFs and inhibiting VE-cadherin expression in tumor cells. Our findings suggest that the miR-101-TGF-β/SDF1-VE-cadherin/MMP2/LAMC2 networks regulate VM formation and represent the potential targets for cancer therapy.

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.

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