Figure 1.
Expression levels of CAV1 in various HCC samples.
1A, 1B: CAV1 mRNA and protein levels of HCC cell lines detected by Q-PCR and Western Blotting. 1C, Membranous and strong cytoplasmic CAV1 expression in a metastatic HCC; 1D, Weak CAV1 expression in hepatocytes of a non-metastatic HCC and strong CAV1 expression in the endothelium of the blood vessels. Original magnification, ×200; 1E and 1F, Q-PCR and Western blot analysis indicated the expressed CAV1 in HepG2 cell lines. The CAV1 expression levels upon CAV1 adenovirus expression are comparable to the endogenous CAV1 level in MHCC97-L cell lines; 1G, Cell anoikis assays using poly-HEMA. Data are presented as mean values (n = 3); error bars represent ± SD; 4H, Stable CAV1 overexpression pools from HepG2 cells were injected subcutaneously into mice; each group contained 6 mice. A Kaplan-Meier survival plot 4 weeks after injection indicates that the mice injected with the CAV1 cells survived for a significantly longer period of time than the controls (P<0.01); 4I, all xenograft tumors were removed from the experimental mice.
Table 1.
Relationship between CAV1 expression and clinicopathological variables in the 96 HCC patients
Figure 2.
CAV1 modulates HCC cell migration, growth, and metastasis.
A, CAV1 overexpression promotes cell migration in HepG2 cells that express GFP and CAV1 using a wound-healing assay. B, Transwell migration assay of HepG2 cell, viewed at ×200 and stained with purple for three independent experiments. C, Comparison analysis with data as mean ± SD, and the statistically significant differences with Student's t-test. D and E, Effect of CAV1 overexpression on the HepG2 cell metastasis using letivirus–CAV1 via orthotopic implantation. Locoregional metastases, classified as abdominal wall metastases and intrahepatic metastases (black arrow) were observed by day 40. F, representative images show intrahepatic metastases (black arrow) and H&E-stained sections. G, The quantification of average weight of xenograft tumors at 6 weeks is shown. * P<0.05.
Figure 3.
CAV1 knockdown inhibited HCC tumor growth and metastasis.
A and B, CAV1 knockdown in MHCCLM3 cells by shRNA-CAV1-1, as demonstrated by qPCR and western blot analysis, with noshRNA as the control. C and D, The growth of subcutaneous tumors was recorded for 30 days, monitored in terms of tumor diameter after 3 days (mean ± SD), and photographed as mice killed. Cells (5×106) in 0.2 mL of PBS were injected s.c. into right upper flank region of each of the six nude mice. E, After orthotopic implantation, tumor growth was assayed using small piece of subcutaneous tumor. The mice were killed after 6 weeks, and autopsied for photo (left panel) and weight (right panel, mean ± SD). F, Abdominal metastases to mesenteric lymph nodes (black arrow) in noshRNA tumors. G, Representative images of liver cancer metastasis (black arrow) on the mesenteric fold (red arrow, intestine) (H&E-stain, original magnification, ×40). H, No abdominal metastasis was observed in shRNA-CAV1-1 tumors. I and J, Representative images of metastases formed in lungs of each nude mouse (n = 6) at 6 weeks after orthotopic implantation with MHCCLM3 no-shRNA or shRNA-CAV1-1 tumors (black arrow, metastatic tumors).
Figure 4.
CAV1 changes the subcellular distribution and signaling of β-catenin in EMT.
A, CAV1 induce EMT in HCC cells. Whole cell lysates from the control, CAV1-expressing, and sh-CAV1 HCC cells were separated and probed with antibodies for epithelial and mesenchymal markers as indicated. B, CAV1 decreases E-cadherin mRNA level, increases Vimentin and Twist mRNA level. C, The β-catenin distribution in CAV1-expressing and sh-CAV1 HCC cells was determined. The purity of nuclear and cytoplasmic fractions was confirmed by immunoblotting. D, The control and CAV1-RNAi MHCC97-H cells were transiently transfected with TOP-tk to determine the transcriptional activity of β-catenin–mediated signaling. The activity levels are represented as fold activation compared with the control in triplicate wells. E, Immunohistochemistry staining of E-cadherin and Vimentin in the tumors from control and CAV1-overexpression HepG2 cells. F, mRNA level of MMP-7 was determined by Q-PCR. G, Protein levels of phospho-ERK 1/2, and total ERK1/2 were analyzed by western blot. Actin is a loading control.