Clinicopathologic correlation of LRP6 transcript in HCC patients.

<p>IHC, immunohistochemical;</p>*<p>Nuclear stain or cytoplasmic overexpression.</p

Ectopic expression of LRP6 activated the Wnt/β-catenin pathway.

<p>(A) Schematic diagram showing the structural domains of Myc-tagged full length form of LRP6 (myc-FL LRP6) and the constitutively active form (myc-CA LRP6). (B) Western blotting. Myc-FL LRP6 and myc-CA LRP6 were transiently overexpressed in BEL-7402 HCC cell line and human embryonic kidney cell HEK293T cells. The protein level of β-catenin was increased in both BEL-7402 HCC cell line and HEK293T cells. (C) TOP/FOP luciferase reporter assay. Expression of myc-FL LRP6 led to an activation of TCF/β-catenin reporter up to ∼40-fold (without Wnt3a treatment) and ∼120-fold (with Wnt3a treatment), respectively, as compared with the vector control. The fold of induction in myc-CA LRP6 cells reached ∼150-fold even without Wnt3a treatment. (D) Similar results were also observed in HEK293T cells.</p

Constitutively active LRP6 promoted both cell migration and invasion.

<p>Cell migration and invasion assays were performed using LRP6-stably expressing BEL-7402 cells. (A) Overexpression of myc-CA LRP6 enhanced cell migration in BEL-7402 cells. The numbers of migrated cells in CA LRP6 Clones #3 and #8 were significantly higher than the vector control (P<0.001 for both). (B) Overexpression of myc-CA LRP6 promoted cell invasion in BEL-7402 cells. The numbers of invaded cells in CA LRP6 Clones #3 and #8 were significantly higher than the vector control cells (P<0.001 and  = 0.009, respectively).</p

Constitutively active LRP6 enhanced tumor cell growth <i>in vivo</i>.

<p>(A) <i>In vivo</i> nude mice injection assay was performed by injecting myc-CA LRP6 stably expressing and vector control BEL-7402 cells subcutaneously into the flank of the nude mice. (B) The tumor sizes of two myc-CA LRP6 stably expressing tumors were significantly higher as compared with the tumor of vector control (P<0.001). (C) The tumor weight of Clone #3 was higher in myc-CA LRP6 than the control vector (P = 0.002). Another clone (Clone #8) showed a trend of higher tumor weight but the difference did not reach statistical significance (P = 0.165). Error bar = SEM.</p

Overexpression of LRP6 in HCC cell lines and human HCCs.

<p>(A) Quantitative real-time PCR and Western blotting. Both transcripts and protein of LRP6 were expressed in all seven HCC cell lines. (B) In human HCCs, the transcript level of LRP6 was frequently (45%) up-regulated as compared with their corresponding non-tumorous livers (P = 0.003). (C) Western blot analysis. LRP6 protein level was determined in 28 HCC pairs and was found to be overexpressed in 9 (32%) cases. The 9 cases with LRP6 protein overexpression in the tumors are shown. (D) Immunohistochemical analysis. LRP6 protein was found to be overexpressed in the HCC as compared with the corresponding non-tumorous liver. High power magnification of the tumor showed strong positive staining of LRP6 protein in the cytoplasm (white arrows) and also the membranes (black short arrows) of the tumor cells.</p

Constitutively active LRP6 enhanced cell proliferation <i>in vitro.</i>

<p>(A) LRP6 expressing BEL-7402 stable cells were established using myc-CA LRP6 construct. The protein level of β-catenin was upregulated as compared with the parental BEL-7402 cells. (B) Cell proliferation assay. The numbers of cells of CA LRP6 Clones #1, #3, #8 and #11 on Day 7 were significantly higher than the vector control BEL-7402 cells (P = 0.032, <0.001,  = 0.002 and 0.005, respectively).</p

ILK knockdown inhibited cell migration and invasion.

<p>(<b>A</b>) Cells from BEL7402 and HLE ILK knockdown stable clones were grown to confluence and a wound was created. Closure of the wound was monitored and captured 16 hours after the wound was made. (<b>B</b>) BEL7402 and HLE ILK knockdown stable clones were seeded onto migration chambers in triplicate and were allowed to migrate for 24 hours. Cells migrated through the membrane were fixed and visualized by crystal violet staining. (<b>C</b>) ILK knockdown stable clones were seeded onto matrigel-coated invasion chamber. BEL7402 was allowed to migrate for 72 hours while HLE required only 24 hours to invade. Cells were fixed, stained and scored. *<i>P</i><0.05 and **<i>P</i><0.001 were regarded as statistically significant.</p

ILK overexpression enhanced PLC cell growth and motility.

<p>(<b>A</b>) FLAG-tagged ILK was transduced into PLC cells and stable clone of ILK was established. Western blot analysis confirmed stable FLAG-ILK expression in PLC cells but not in vector control clone. (<b>B</b>) PLC/vector and PLC/FLAG-ILK cells were counted in triplicates for 8 consecutive days. (<b>C</b>) PLC ILK overexpressing cells were subjected to migration assay. Cells were seeded in triplicates and allowed to migrate for 16 hours. Migrated cells were fixed and stained by crystal violet. (<b>D</b>) PLC and HEK293T cells overexpressing ILK were collected for western blot analysis to study the phosphorylation of Akt and GSK3β. Expression of β-actin was included as an internal loading control. *<i>P</i><0.05 and **<i>P</i><0.001 were regarded as statistically significant.</p

ILK expression was elevated in HCC clinical samples and cell lines.

<p>(<b>A</b>) ILK mRNA expression was examined by qPCR. ILK was overexpressed in 36.9% of the HCC samples. Cases with T/NT ratio >2 were classified as overexpression, while those with T/NT ratio <0.5 were regarded as underexpression. The remaining cases with 0.5< ratio <2 were regarded as no change in ILK expression. Higher ILK expression (<i>P</i> = 0.004) was observed in tumor samples when compared to their non-tumor counterparts. (<b>B</b>) A stepwise increase of ILK expression along HCC tumor stage was observed. (<b>C</b>) ILK expression in a panel of HCC cell lines was analyzed by western blot analysis. MIHA is an immortalized non-tumorigenic liver cell line. Relative ILK expression normalized with corresponding β-actin expression was shown below.</p

ILK knockdown suppressed phosphorylation of Akt and GSK3β.

<p>Insulin was added to cells to stimulate the PKB/Akt signaling pathway. Cell lysates were then collected for western blotting analysis. Expression levels of pAkt, total Akt, pGSK3β, total GSK3β and ILK were shown. Expression of β-actin was included as an internal loading control. The band intensities were determined by densitometry, and the amount of pAkt and pGSK3β was normalized with that of total Akt and GSK3β respectively.</p