Correlation of CTHRC1 expression and clinicopathological factors.

<p>Correlation of CTHRC1 expression and clinicopathological factors.</p

CTHRC1 promotes cell adhesion through activation of integrin β1/FAK pathway.

<p>(A) Adhesion assay. Approximately 1×10⁴ cells were seeded into fibronectin-coated 96-well plates and incubated at 37°C for 30 min, washed, fixed, and stained with crystal violet. The remaining cells were counted. Knockdown of CTHRC1 markedly reduced the number of cells adherent to fibronectin. (B) Treating Huh7 cells with CTHRC1 (100 ng/ml) enhanced adhesion to fibronectin at 15 min and 30 min. (C) Knockdown of CTHRC1 decreased the formation of focal adhesion structures. Cells were trypsinized and replated onto fibronectin-coated slides and incubated at 37°C for 60 min, fixed, and stained. Mouse anti-vinculin was used to visualize focal adhesions (upper panel), and rhodamine-phalloidin was used to visualize actin filaments (lower panel). (D) CTHRC1 treatment enhanced phosphorylation of FAK at residues 576/577. (E) Knockdown of CTHRC1 inhibited phosphorylation of FAK. (F) CTHRC1 enhanced expression of integrin β1. (G) The adhesion-promoting effect of CTHRC1 was abolished by integrin β1 blocking antibody.</p

Overexpression of CTHRC1 in Hepatocellular Carcinoma Promotes Tumor Invasion and Predicts Poor Prognosis

<div><p>Collagen triple helix repeat containing-1 (CTHRC1) is a secreted glycoprotein that activates the planar cell polarity pathway of Wnt signaling. Using microarray analysis, we found that the <i>CTHRC1</i> gene is overexpressed in hepatocellular carcinoma (HCC). The level of <i>CTHRC1</i> mRNA was measured in 201 surgically resected HCCs using real time reverse transcription-polymerase chain reaction. Overexpression of CTHRC1 in HCC was associated with large tumor size and advanced tumor stage. Furthermore, expression of CTHRC1 as was identified as an independent prognostic factors in the multivariate analysis. Suppression of CTHRC1 expression inhibited tumor migration and invasion whereas overexpression of CTHRC1 promoted tumor invasion. Activation of RhoA, but not Rac1 or Cdc42, was found to play a crucial role in CTHRC1-induced cell migration. CTHRC1 promoted adhesion of cancer cells to extracellular matrix through induction of integrin β1 expression and activation of focal adhesion kinase. These results suggest CTHRC1 promotes tumor invasion and metastasis by enhancing the adhesion and migratory abilities of tumor cells. It is also a promising biomarker for predicting the prognosis of patients with HCC.</p></div

CTHRC1 activates RhoA activity.

<p>(A) Pull-down assay of Rho family proteins showed that knockdown of CTHRC1 inhibited the activation of RhoA, but not Rac1 or Cdc42. (B) Treating Huh7 cells with recombinant CTHRC1 (100 ng/ml) activated the RhoA pathway. (C) CTHRC1 enhanced phosphorylation of MLC2, a target of ROCK. (D) Pretreatment with a ROCK inhibitor Y2732 (5 µmol/L) abolished the invasion-promoting activity of CTHRC1.</p

p15^PAF expression in normal adult tissues.

<p>Nuclear expression of p15^PAF was detected in the lymphocytes in the germinal center (arrow) of lymph node (B), the epithelial cells in the lower half of crypt (arrow) in the colonic mucosa (D), and the suprabasal keratinocytes (arrow) of epidermis (F), but not in the neurons and glial cells of brain (D), hepatocytes of liver (E), and (F) pneumocytes of lung.</p

CTHRC1 promotes tumor migration and invasion.

<p>(A) shRNA#1 and #5 markedly reduced CTHRC1 mRNA and protein in HA22T cells. (B) Knockdown of CTHRC1 markedly decreased the amount of CTHRC1 in the culture medium. (C) Depletion of CTHRC1 did not affect proliferation, as measured by MTT assay. (D–E) Knockdown of CTHRC1 in HA22T cells inhibited invasion (D) through Matrigel and motility (E). (F) Scratch wound assay. Knockdown of CTHRC1 in HA22T cells delayed closure of scratch wounds Photographs were taken at 30 h. (G) Overexpression of CTHRC1 in Huh7 increased the amount of CTHRC1 in the culture medium. (H) Overexpression of CTHRC1 in Huh7 cells enhanced tumor invasion through Matrigel.</p

p15^PAF is expressed in S phase.

<p>(A) HeLa cells were synchronized at G1/S boundary by thymidine/aphidicholin double block. Immunofluorescence staining of p15^PAF was performed at the indicated time point after release. Flow cytometry was also performed to determine the cell cycle distribution. Expression of p15^PAF peaked at 6 h (mid-to-late S phase) after release. (Scale bar = 30 µm). (B) Quantification of the percentage of p15^PAF-positive cells at the indicated time point. (C) HeLa cells were synchronized at the G1/S boundary thymidine/aphidicholin double block and were collected for Western blotting following release into the cell cycle. Cyclin D1 and B1 were used as markers of S and G2 phase, respectively. (D) After pulse labeling with BrdU, HeLa cells were stained with anti- p15^PAF and anti-BrdU antibodies. The cells with positive BrdU staining were also positive for p15^PAF. (Scale bar = 30 µm). (E) Quantification of the result of immunofluorescence staining. 90% of the cells showed concordant staining of p15^PAF and BrdU.</p

Univariate analysis of prognostic factors in patients with HCCs.

*<p><i>P</i><0.05.</p

Overexpression of CTHRC1 in HCC.

<p>(A) RT-PCR analysis showed overexpression of CTHRC1 in 4 of 8 HCC specimens (T), but in none of the 8 specimens of nontumorous liver parenchyma (N). S26 (ribosomal protein S26) served as internal control. (B) Western blotting showed CTHRC1 was expressed in 5 of 8 HCC specimens (T) but not in nontumourous liver parenchyma (N). (C, D) Cumulative overall (C) and disease-free (D) survival curves for 201 patients with resected, unifocal, primary HCC stratified by <i>CTHRC1</i> mRNA expression level as determined by real time RT-PCR. Patients with HCCs overexpressing the <i>CTHRC1</i> gene had significantly lower 10-year overall and disease-free survival rates than those with HCCs without <i>CTHRC1</i> overexpression.</p

Univariate analysis of S100P protein expression with various clinicopathological features and aberrant gene expression in 305 patients with surgically removed unifocal primary hepatocellular carcinoma.

<p>Univariate analysis of S100P protein expression with various clinicopathological features and aberrant gene expression in 305 patients with surgically removed unifocal primary hepatocellular carcinoma.</p