Oncogenic Functions of the Cancer-Testis Antigen SSX on the Proliferation, Survival, and Signaling Pathways of Cancer Cells

<div><p>SSX is a transcription factor with elusive oncogenic functions expressed in a variety of human tumors of epithelial and mesenchymal origin. It has raised substantial interest as a target for cancer therapy since it elicits humoral responses and displays restricted expression to cancer, spermatogonia and mesenchymal stem cells. Here, we investigated the oncogenic properties of SSX by employing a RNA interference to knock-down the endogenous expression of SSX in melanoma and osteosarcoma cell lines. Depletion of SSX expression resulted in reduced proliferation with cells accumulating in the G1 phase of the cell cycle. We found that the growth promoting and survival properties of SSX are mediated in part though modulation of MAPK/Erk and Wnt signaling pathways, since SSX silencing inhibited Erk-mediated signaling and transcription of cMYC and Akt-1. We also found that SSX forms a transient complex with β-catenin at the G1-S phase boundary resulting in the altered expression of β-catenin target genes such as E-cadherin, snail-2 and vimentin, involved in epithelial-mesenchymal transitions. Importantly the silencing of SSX expression in <i>in vivo</i> significantly impaired the growth of melanoma tumor xenografts. Tumor biopsies from SSX silenced tumors displayed reduced cyclin A staining, indicative of low proliferation and predominantly cycloplasmic β-catenin compared to SSX expressing tumors. The present study demonstrates a previously unknown function of SSX, that as an oncogene and as a tumor target for the development of novel anti-cancer drugs.</p></div

SSX is required for Erk mediated signalling.

<p>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.</p

shRNA targeting of SSX in inhibit the growth of xenografts in SCID mice.

<p>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).</p

SSX interacts with β-catenin and transactivates TCF/β-catenin target genes.

<p><b>A</b>) 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.</p

The conditional knock-down of SSX inhibits the proliferation, survival and cell cycle progression of the melanoma cell line DFW in vitro.

<p>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.</p

SSX2 is frequently expressed in melanoma lesions and derived cell lines but not in normal cells.

<p>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.</p