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Tumor Biology

, Volume 36, Issue 11, pp 8413–8424 | Cite as

Robo1 promotes angiogenesis in hepatocellular carcinoma through the Rho family of guanosine triphosphatases’ signaling pathway

  • Jian-Yang Ao
  • Zong-Tao Chai
  • Yuan-Yuan Zhang
  • Xiao-Dong Zhu
  • Ling-Qun Kong
  • Ning Zhang
  • Bo-Gen Ye
  • Hao Cai
  • Dong-mei Gao
  • Hui-Chuan Sun
Research Article

Abstract

Robo1 is a member of the Robo immunoglobulin superfamily of proteins, and it plays an important role in angiogenesis and cancer. In this study, we investigate the role of roundabout 1 (Robo1) in tumor angiogenesis in hepatocellular carcinoma (HCC). Firstly, the relationship between Robo1 expression on tumors and patient’s survival and endothelial cells in tumor blood vessels and patient’s survival was studied. Secondly, Robo1 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). Cell proliferation, motility, and tube formation were compared in HUVEC with different Robo1 expression. Also, HUVECs with different Robo1 expression were mixed with HCCLM3 and HepG2 hepatoma cells and then implanted in a nude mouse model to examine the effects of Robo1 in endothelial cells on tumor growth and angiogenesis. Cell motility-related molecules were studied to investigate the potential mechanism how Robo1 promoted tumor angiogenesis in HCC. The disease-free survival of the patients with high Robo1 expression in tumoral endothelial cells was significantly shorter than that of those with low expression (P = 0.021). Overexpression of Robo1 in HUVECs resulted in increased proliferation, motility, and tube formation in vitro. In the implanted mixture of tumor cells and HUVECs with an increased Robo1 expression, tumor growth and microvessel density were enhanced compared with controls. Robo1 promoted cell division cycle 42 (Cdc42) expression in HUVECs, and a distorted actin cytoskeleton in HUVECs was observed when Robo1 expression was suppressed. In conclusion, Robo1 promoted angiogenesis in HCC mediated by Cdc42.

Keywords

Hepatocellular carcinoma Angiogenesis Roundabout receptors 1 Rho GTPase 

Abbreviations

HCC

Hepatocellular carcinoma

HUVEC

Human umbilical vein endothelial cell

GTPase

Guanosine triphosphatase

AFP

α-Fetoprotein

TEC

Tumor endothelial cell

Rho

Ras homolog family member

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

Notes

Acknowledgments

This study was jointly supported by the grants from the National Major Science and Technology Project (2012ZX10002012-004, 2013ZX10002007), the National Natural Science Foundation of China (No. 81101805, No. 81472224), and the Shanghai Health Bureau Research Project (No. 2009Y007). We thank Ke Qiao from Key Laboratory of Medical Molecular Virology, Ministry of Education and Public Health, Shanghai Medical School, Fudan University, for technical expertise in confocal microscopy.

Conflicts of interest

None

Authors’ contributions

JYA, ZTC, YYZ, and LQK performed the experimental work. NZ, BGY, HC, and DMG participated in the experiments. HCS participated in its design and coordination. The manuscript was written by HCS, JYA, and CTZ.

Supplementary material

13277_2015_3601_Fig7_ESM.gif (105 kb)
Fig. S1

Representative immunostaining of Robo1 on sections of HCC and survival analysis. The representative immunostaining images of high Robo1 expression (A) and low Robo1 expression (B) in HCC. (C) Cumulative disease-free survival (DFS) curves of patients with high and low Robo1 expression could not discriminate patient from different DFS (P = 0.299). Scale bar: 100.0 μm (GIF 104 kb)

13277_2015_3601_MOESM1_ESM.tif (586 kb)
High Resolution Image (TIFF 586 kb)
13277_2015_3601_Fig8_ESM.gif (354 kb)
Fig. S2

Knockdown of Robo1 resulted in distorted F-actin of human umbilical vein endothelial cells. Confocal laser scanning microscopy images of F-actin expression in different Robo1 expression cells. F-actin was stained by phalloidin in (A) HUVEC-oe-Robo1, (B) HUVEC-nc-Robo1, and (C) HUVEC-kd-Robo1 cells. F-actin in HUVEC-kd-Robo1 cells was found distorted. F-actin expression was compare in HUVEC-oe-Robo1, HUVEC-nc-Robo1 cells and HUVEC-kd-Robo1 cells (D). (^P < 0.05 compared with HUVEC-oe-Robo1; *P < 0.05 compared with HUVEC-nc-Robo1; #P < 0.05 compared with HUVEC-kd-Robo1). Scale bar: 100.0 μm (GIF 353 kb)

13277_2015_3601_MOESM2_ESM.tif (106 kb)
High Resolution Image (TIFF 105 kb)
13277_2015_3601_MOESM3_ESM.docx (16 kb)
Supplementary Table 1 Clinicopathological features of the cohort 1 HCC patients (DOCX 16 kb)
13277_2015_3601_MOESM4_ESM.docx (16 kb)
Supplementary Table 2 Clinicopathological features of the cohort 2 HCC patients (DOCX 16 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Jian-Yang Ao
    • 1
    • 2
    • 4
  • Zong-Tao Chai
    • 1
    • 2
    • 5
  • Yuan-Yuan Zhang
    • 1
    • 2
  • Xiao-Dong Zhu
    • 1
    • 2
  • Ling-Qun Kong
    • 3
  • Ning Zhang
    • 1
    • 2
  • Bo-Gen Ye
    • 1
    • 2
  • Hao Cai
    • 1
    • 2
  • Dong-mei Gao
    • 1
    • 2
  • Hui-Chuan Sun
    • 1
    • 2
  1. 1.Liver Cancer Institute and Zhongshan HospitalFudan UniversityShanghaiChina
  2. 2.Key Laboratory of Carcinogenesis and Cancer InvasionMinistry of EducationShanghaiChina
  3. 3.Department of Hepatobiliary SurgeryBinzhou Medical College Affiliated HospitalBinzhouChina
  4. 4.The First Affiliated Hospital of Wenzhou Medical UniversityWenzhouChina
  5. 5.Changhai HospitalSecond Military Medical UniversityShanghaiChina

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