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. 2015 Jan 1;6(1):243-57.
doi: 10.18632/oncotarget.2801.

CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cells

Shi-Jie Wang  1 , Hong-Yong Cui  1 , Yan-Mei Liu  1 , Pu Zhao  2 , Yang Zhang  1 , Zhi-Guang Fu  1 , Zhi-Nan Chen  1 , Jian-Li Jiang  1
Affiliations

CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cells

Shi-Jie Wang et al. Oncotarget. .

Abstract

Metastasis is considered a dynamic process in tumor development that is related to abnormal migration and invasion. Tumor cells can move as individual cells in two interconvertible modes: mesenchymal-type and amoeboid. Previously, we reported that the interaction between CD147 and Annexin II can inhibit the amoeboid movement in hepatocellular carcinoma (HCC) cells. However, the mechanism of CD147 involved in mesenchymal movement is still unclear. Notably, our results show overexpression of CD147 led to mesenchymal-type movement in HCC cells. Evidence indicated that the mesenchymal-type cell movement induced by CD147 was Src dependent, as observed by confocal microscopy and Rac1 activity assay. The phosphorylation of Src (pY416-Src) can be up-regulated by CD147, and this regulation is mediated by focal adhesion kinase (FAK). Next, we identified DOCK8 as a GEF for Rac1, a key molecule driving mesenchymal-type movement. We also found that Src promotes STAT3 phosphorylation and STAT3 facilitates DOCK8 transcription, thus enhancing DOCK8 expression and Rac1 activation. This study provides a novel mechanism of CD147 regulating mesenchymal-type movement in HCC cells.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Fig.1
Fig.1. CD147 regulates cell morphology and motility via coordinating Rac and Rho signalings
(A) CD147 was examined in total lysates of 7721, K7721, R7721 and K7721-pcDNA3.1 cells (K7721 cells transfected with pcDNA3.1 vector as a mock control for R7721) using western blotting. (B) Wound healing assay of the 7721, K7721 and R7721 cell lines. (C) Migration assay of the 7721, K7721 and R7721 cell lines. (D) In vitro invasion assay of the 7721, K7721 and R7721 cell lines. (E) Confocal microscopy images of 7721, K7721 and R7721 cells. Red: CD147; Green: actin; Blue: DAPI. Scale bar = 20μm. (F) Rac1 activity, WAVE2 expression, and RhoA and MLC2 activities were examined in total lysates of 7721, K7721, R7721 and K7721-pcDNA3.1 cells using western blotting. (G) Src and Rac1 activities, WAVE2 expression, and RhoA and MLC2 activities were examined in total lysates of Huh-7 cells and Huh-7 cells transfected with si-147 or sncRNA using western blotting. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P < 0.01, * P < 0.05, by one-way ANOVA (B-D).
Fig.2
Fig.2. Src activity alteration leads to morphological and migratory activity changes in HCC cells
(A) Src activity level (pY416-Src/total Src) was assessed using western blotting in pcDNA3.1 or Src-pc3.1 transfected 7721 cells. (B) Images (Scale bar = 500μm) and confocal microscopy images (Scale bar = 20μm) demonstrating the effect of Src overexpression on morphological changes in 7721 cells. Green: actin; Blue: DAPI. Left panel: representative image. Right panel: quantification. (C) Src activity was assessed using western blotting after treatment of 7721 cells with Src kinase inhibitor (Src I-1). (D) Images (Scale bar = 500μm) and confocal microscopy images (Scale bar = 20μm) demonstrating the effect of Src I-1 treatment on morphological changes in 7721 cells. Green: actin; Blue: DAPI. Left panel: representative image. Right panel: quantification. (E) Effects of Src I-1 treatment on cell motility of 7721 cells. (F) Effects of Src I-1 treatment on cell migration of 7721 cells. (G) Effects of Src I-1 treatment on cell invasion of 7721 cells. (H) Effects of Src I-1 treatment on cell proliferation of 7721 cells. (I) Src and Rac1 activities, WAVE2 expression, and RhoA and MLC2 activities were examined in 7721 and HepG2 cells treated with 300 nM of Src I-1. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P < 0.01, * P < 0.05, # P > 0.05, by t-test (B, D-G) and one-way ANOVA (H).
Fig.3
Fig.3. Src is required for CD147 regulated cell movement in HCC cells
(A) Src activity in CD147-pcDNA3.1-expressing K7721 cells treated with Src I-1. (B) Effects of CD147 overexpression and/or Src I-1 treatment on cell motility in 7721 cells. (C) Confocal microscopy images (Scale bar = 10μm) of CD147 overexpression and/or Src I-1 treatment on cytoskeleton rearrangement in 7721 cells. (D) Rac1 activity in CD147-pcDNA3.1-transfected 7721 cells. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P < 0.01, by one-way ANOVA (B).
Fig.4
Fig.4. CD147 promotes Src activation through FAK
(A) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with Src I-1. Left panel: representative image. Right panel: quantification. (B) Src activity in 7721 or CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK inhibitor (PF573,228). Left panel: representative image. Right panel: quantification. (C) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK inhibitor-14 (Y-15). Left panel: representative image. (D) Src activity in CD147-pcDNA3.1-transfected 7721 cells was blocked with FAK siRNA (si-FAK). Left panel: representative image. Right panel: quantification. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P < 0.01, * P < 0.05, # P > 0.05, by one-way ANOVA.
Fig.5
Fig.5. DOCK8 is a Rac1 GEF involved in the Src-regulated Rac/WAVE signaling pathway
(A) Images (Scale bar = 500μm) and confocal microscopy images (Scale bar = 20μm) of the DOCK8 siRNA (si-D8)-induced morphological changes in 7721 cells. Green: actin; Blue: DAPI. Left panel: representative image. Right panel: quantification. (B) DOCK8 was detected using western blotting after the transfection of 7721 cells with DOCK8 siRNA. (C) The DOCK8 mRNA level was detected using real-time PCR after the transfection of 7721 cells with si-D8. (D) Effects of si-D8 on cell motility of 7721 cells. Cells were transfected with si-D8 prior to use. (E) Rac1 immunoprecipitated with DOCK8. (F) Rac1 activity, WAVE2 expression and MLC2 activity in 7721 and 7721-siD8 cells. (G) The mRNA level of DOCK8 was detected using real-time PCR after the transfection of 7721 cells with ANXA2 siRNA (si-A2). (H) DOCK8 expression was examined in total lysates of SMMC-7721, K7721, R7721 and K7721-pcDNA3.1 cells using western blotting. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. ** P < 0.01, * P < 0.05, by t-test (A, C, D, G).
Fig.6
Fig.6. Src promotes DOCK8 expression via enhancing STAT3 phosphorylation
(A) DOCK8 expression was detected using western blotting after treatment of 7721 cells with Src I-1. (B) Confocal microscopy images of 7721, 7721 treated with Src I-1 and K7721 cells. Red: CD147; Green: DOCK8; Blue: DAPI. Scale bar = 20μm. (C) DOCK8 expression and Src activity were examined in total lysates of 7721, K7721, K7721 transfected with SrcY530F and K7721-pcDNA3.1 cells (K7721 cells were transfected with pcDNA3.1 vector as a mock control for R7721) using western blotting. Phosphorylation of Src at Tyr416 can also be seen in the overexposed panel (right).(D) Phosphorylation level of STAT3 and DOCK8 expression were detected using western blotting after treatment of 7721 cells with WP1066. (E) The DOCK8 mRNA level was detected using real-time PCR after treatment of 7721 cells with WP1066. (F) Phosphorylation level of STAT3 was detected using western blotting after treatment of 7721 cells with Src I-1. (G) Phosphorylation level of STAT3 and CD147 expression were determined in 7721 cells overexpressing CD147 and/or transfected with STAT3 siRNA. (H) The effects of CD147 overexpression and/or STAT3 silencing on cell motility of 7721 cells. (I) CD147 expression using normalized microarray gene expression data. The bars represent each sample performed in triplicate, and the error bars indicate ± SD. * P < 0.05, ** P < 0.01, # P > 0.05, by unpaired t-test (E), one-way ANOVA (H) and paired t-test (I).
Fig.7
Fig.7. Schematic representation of the major molecular mechanisms of CD147 in regulating hepatocellular carcinoma cells motility
FAK, focal adhesion kinase; DOCK8, dedicator of cytokinesis 8; ROCK, Rho-kinase; MLC2, myosin light chain 2; STAT3, signal transducer and activator of transcription 3.
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