Figure 1.
SSX2 is frequently expressed in melanoma lesions and derived cell lines but not in normal cells.
SSX expression was analyzed by a nested RT-PCR method previously described using primers recognizing SSX1 to SSX 9 cDNA. Fresh biopsies were obtained from metastatic lesions of melanoma patients. The DFW melanoma cell line expressing high levels of SSX1 to SSX5 was used for RNAi studies. NHEM: normal human epithelial melanocytes, HDF: human diploid fibroblasts.
Figure 2.
The conditional knock-down of SSX inhibits the proliferation, survival and cell cycle progression of the melanoma cell line DFW in vitro.
A) Graphic representation of the shRNA sequence (complementary to SSX1 to SSX9) ligated into shRNA vectors for stable and doxycycline regulated shRNA expression (see material and methods). B) Western blot showing SSX expression in control-shRNA and SSX-shRNA transfected cells 24 hours after doxycycline addition to the culture medium. C) Cell colony quantification in control and SSX-shRNA transfected DFW cells grown in the presence of doxycycline for 8 days. D) Cell proliferation curves determined by counting the number of alive cells in control and SSX silence cultures using trypan blue staining. E) S-phase cell cycle progression determined by BrdU incorporation in a fluorescence activated cell sorter (FACS). F) Percentage of cells at G1, S and G2 phases of the cell cycle in control and SSX shRNA knocked down DFW cells over 96 hours period.
Figure 3.
Loss of SSX expression arrest tumor cells in G1/S phase and abrogates -cyclin E expression.
Control (SSX+) and silenced (SSX−) DFW melanoma cells were synchronized in G1/S phase by double thymidine blockade and released in normal medium as described in material and methods. A) Entry of the cells to the S phase (S) was analyzed by quantifying DNA content using propidium iodide (PI) incorporation and a fluorescent activated cell sorter FACS. B) Western blot showing time dependent expression of SSX and cyclin E in control and SSX silenced (+Dox) DFW cells. Cells were syncronized in G1/S (0 h), released into normal medium containing FBS and harvested at the indicated time points.
Figure 4.
SSX is required for Erk mediated signalling.
Western blot showing the expression of Erk1–2 and Akt-1 and their activated (phosphorylated) forms pAkt (Ser 473) and pErk (Thr202/Tyr204) in control shRNA and SSX-shRNA DFW cells. Cells were starved in serum free media for 36 hrs (0 h) and cell signalling was activated by the addition of serum into the media. Samples were collected at 10 and 30 minutes (10′ and 30′) and at 6 and 24 hours (6 h, 24 h) following serum stimulation. SSX expression was determined by immunoprecipitation (fl188 antibody) and western blot (N18 antibody) the later recognozing bands of approximately 22 and 14 kDa.
Figure 5.
SSX interacts with β-catenin and transactivates TCF/β-catenin target genes.
A) DFW and Saos-2 cell lines were synchronized in G1/S by double thymidine blockade as indicated in material and methods (0 hrs), and released into normal medium containing FBS for 6 and 24 hrs. SSX was immunoprecipitated from protein extracts collected at the indicated time points using the rabbit anti SSX antibody (FL188, detecting SSX1–9). An equivalent amount of protein from G1/S blocked Saos-2 cells was immunoprecipitated with an irrelevant anti mouse (m) or ant-rabbit (r) antibody. The protein complex were electrophoresed in reducing conditions and blotted ether with goat anti SSX (N18) or mouse anti β-catenin antibodies. The total input levels of β-catenin are shown in the upper gel image. B) Activity of a TCF/Lef luciferase reporter in SSX silenced and control DFW and Saos-2 cells, 48 hours after transfection of siRNA molecules (n = 5). The activity of the TCF/Lef reporter in SSX silenced cells is relative to that of control cells ( = 1). C) Gene transcription associated with SSX expression in both Saos-2 and DFW cells, determinded by PCR arrays containing 84 genes associated with epithelial to mesenchymal transition (n = 5) and confirmed by Q-RT PCR in SSX silenced and control DFW and Saos-2 cells as described in material and methods. SSX was knocked down in Saous-2 or DFW cells using siRNA molecules or shRNA vectors as indicated. Cells were collected and RNA was isolated 6 ours after siRNA transfection or 6 hours after addition of doxycycline into the medium (conditionally shRNA). The loss of SSX expression following RNAi silencing was confirmed by western blot before each Q-RT-PCR array, as shown in the figure. Fold-Change [2∧(−Delta Delta Ct)] is the normalized gene expression [2∧(−Delta Ct)] in SSX silenced cells divided by the normalized gene expression in the control (SSX+) cells. Values less than one indicate a negative or down-regulation.
Figure 6.
shRNA targeting of SSX in inhibit the growth of xenografts in SCID mice.
A) DFW melanoma cells stably transfected with SSX-shRNA (SSXi) or with a control shRNA (SSXm) vector, expanded and xenografted in SCID mice. A) Tumor volume determined at the indicated times. B) Tumor volume at the end point of the experiment (21 days). C) Growth curves of xenografts from conditionally SSX-shRNA silenced DFW cells (SSX−) and control shRNA (SSX+) cells. Doxycycline was administered to all mice by subcutaneous insertion of a slow release pellet to mantain steady concentration of doxycycline (10 µM) for 21 days. D) Tumor volume at the end point of the experiment. E) Immunohistochemistry of the tumors visualized in the light microscope using 10x objective, inserts 63x magnification: hematoxillin (HTX), the proliferation marker (ki-67) and β-catenin (β-cat).