Src Induces Urokinase Receptor Gene Expression and Invasion/Intravasation via Activator Protein-1/p-c-Jun in Colorectal Cancer

Nuclear extracts from c-src–expressing SW480 cells show increased binding of AP-1 transcription factors (c-Jun, JunD, and Fra-1) to an oligonucleotide spanning region −190/−171. Equal amounts of nuclear extract from vector control SW480 cells or Src-transfected SW480 cells were incubated with an end-labeled oligonucleotide (spanning −190/−171 bp region of the u-PAR promoter) in the presence or absence, of a 100-fold excess of the unlabeled competitor sequence and the indicated antibodies. EMSA was carried out using 10 μg of nuclear extract, 1 μg of poly(deoxyinosinic-deoxycytidylic acid), 1 × 10⁶ cpm of ³³P oligonucleotide, and 1 μg of antibody. Complexes were subsequently analyzed by gel electrophoresis. Top arrow, AP-1 complexes. *, nonspecific binding, Bottom arrow, free probes.

Recruitment of p-c-Jun, JunD, and Fra-1, to the AP-1 responsive cis-element within the endogenous region −190/−171 of the uPAR promoter as indicated by quantitative ChIP. A. Schematic presentation of the region of the u-PAR promoter and the primers/probe used for amplification. B. To calculate the amount of immunoprecipitated chromatin, serial dilutions (1 to 10⁻⁷ ng) of pGL3-398 plasmid containing the specific AP-1 binding region were used as a standard curve. The fluorescent signal is shown as the mean of quadruplicate experiments. C. Quantitative measurement of immunoprecipitated chromatin. Equal amounts of chromatin from vector- or c-src–transiently transfected SW480 cells were incubated with the indicated antibodies and quantified by real-time PCR.

Src induces JNK activity and phosphorylation of c-Jun at Ser⁶³ and, especially, Ser⁷³. Cellular extracts (equal protein) were assayed for Src activity by immune complex kinase assay using MBP as a substrate. The corresponding blots were reprobed after decay with Src and MBP antibodies. For all other proteins, Western blotting was done on cell lysates with phospho-JNK, phospho–c-Jun (Ser⁶³), and phospho–c-Jun (Ser⁷³) antibodies. The corresponding blots were stripped and reprobed with JNK, c-Jun, and actin antibodies. After normalization, the fold increase of the Src-transfected cells compared with the vector control was 2.5-fold for phospho-Src, 1.6-fold for phospho-MBP, 2.4-fold for phospho-JNK(p54), 1.2-fold for phospho–c-Jun(Ser⁶³) and 2.8-fold for phospho-c-Jun(Ser⁷³).

Cells expressing constitutively active c-Src show an increased invasion/intravasation, which can be reduced by treatment with Src-kinase inhibitors, u-PAR knockdown, and TAM67. A. A Matrigel assay was used to assess the invasive capacity of Src-transfected SW480 colon cancer cells versus vector-transfected control cells. The transwell filters were coated with 25 μg of Matrigel. Cells (0.5 × 10⁶) were added to the upper chambers. Conditioned NIH-3T3 medium was added to the bottom chambers. After 24 h, the cells that had migrated to the lower chamber of the filter were trypsinized and quantified. B. For si-RNA–mediated u-PAR knockdown, SW480 cells were transiently transfected with an siRNA specific for u-PAR (ID289), or with unspecific (scrambled) siRNA control, at a final concentration of 40 nmol/L. After 24 h, cells were additionally transfected with Src-expressing plasmid or vector control and subjected to Matrigel after an additional 48 h. For PP2-induced inhibition, SW480 cells were transiently Src-transfected, preincubated 12 h with 30 μmol/L PP2, and after 72 h, subjected to matrigel. During the whole assay, the same concentration of PP2 was added to the upper chamber. C. Down-regulation of the invasive capacity (Matrigel) of Src-transfected SW480 cells by dominant negative c-Jun (TAM67). Cells were transiently transfected using the Src expression plasmid either in combination with empty vector (pCMVβ) or the TAM-67 expression plasmid (pCMVβTAM67) and compared with vector-transfected control (pcDNA3.1(−)). *, P < 0.01, differences between vector-transfected and Src-expressing SW480 cells were statistically significant. D. A CAM assay was done to assess the intravasation capacity of Src-transfected SW480 cells versus vector-transfected control cells. 0.33 × 10⁵ cells were inoculated onto the upper CAM of a 9-day-old chicken embryo. After 48 h, the lower CAM was removed, and the genomic DNA was isolated. Human Alu sequences were amplified and quantified by quantitative PCR. To back-calculate the number of intravasated cells, human genomic DNA (equivalent to 600, 200, and 60 cells of SW480) was diluted in 0.5 μg CAM genomic DNA and quantified in parallel. The fluorescent signal is shown as the mean of triplicate experiments. The R² value is at least 92% for the standard curve. Conversion of cycle threshold (CT) to cell numbers (Cell nr.) is shown. Differences between vector-transfected and Src-transfected cells were significant (P < 0.01).