Auto-stimulatory action of secreted caveolin-1 on the proliferation of Ewing's sarcoma cells

Abstract

Caveolin-1 (CAV1) is highly expressed in Ewing's sarcoma (EWS). We previously showed that increased cellular CAV1 is associated with the regulation of the tumorigenicity, drug resistance and metastatic ability of EWS cells. Because several studies reported that melanoma and prostate cancer cells, which express relatively high CAV1 levels, secrete CAV1, and that secreted CAV1 is associated with tumor progression, our study explored the possibility that EWS cells also secreted CAV1 and that secreted CAV1 may contribute to EWS pathobiology. Results from experiments involving the ectopic expression of a Myc-tagged CAV1 protein in EWS cells as well as the supplementation of culture media with purified CAV1 protein followed by its intracellular localization using immunofluorescence demonstrated that EWS cells secrete CAV1, that they are able to take up the secreted protein, and that extracellular CAV1 enhances EWS cell proliferation. These findings strongly support the notion that secreted CAV1 may also contribute to the malignant properties of EWS.

Figure 1. Secretion of CAV1 by EWS cells

(A) Detection of CAV1 with anti-CAV1 antibodies through dot-blot analysis of the indicated volumes of un-concentrated conditioned media from cultures of A4573, SK-ES-1, TC-71 and PC-3 cells (upper panel); fresh culture medium was used as control (lower panel). (B) Immunodetection of CAV1 in TCA precipitates obtained from conditioned media prepared from cultures of the same cell lines, and resolved by SDS-PAGE (upper panel); Ponceau staining of the region of the membrane corresponding to the range of the CAV1 molecular mass (lower panel) is shown as loading reference. (C) Western blot analysis for CAV1 in the protein fraction purified from conditioned media from EWS and PC-3 cells by 30–70% ammonium sulfate precipitation, and resolved on SDS-PAGE (upper panel); Ponceau staining of the blot is shown as control for equal loading (lower panel). A short exposure of the immunoblots is shown in panels (B) and (C) to better illustrate expression differences.

Figure 2. Secreted CAV1 is taken up by EWS cells

(A) Detection of cellular CAV1 protein levels in CAV1 knocked-down A4573/shCav1 cells, grown in the presence and absence of conditioned media prepared from the EWS cell lines and passages indicated (upper panel); α-tubulin (lower panel) was used as the loading control. (B) Quantification of the relative differences in cellular CAV1 content under the experimental conditions indicated.

Figure 3. Ectopically expressed human CAV1 is secreted and taken up by EWS cells

(A) The Myc-DDK-tagged (MD-CAV1) protein was detected using anti-Myc antibodies in cell lysates (upper panel) and conditioned media (lower panel) prepared from the indicated EWS cells stably transfected with Myc-DDK-CAV1. (B) The MD-CAV1 protein was purified from A4573 (upper panel) and SK-ES-1 cells (lower panel) using anti-FLAG magnetic beads; samples of the cell lysates, flow through and the eluted fractions were resolved by SDS-PAGE and subjected to immunodetection with anti-Myc antibodies. (C) Purification of MD-CAV1. MD-CAV1 was purified using anti-FLAG magnetic beads as described under “Materials and methods”. Samples from the various steps of the purification process were resolved by SDS-PAGE, and the purity of the protein was assessed using silver staining. M, denotes marker, while lanes correspond to the following: (1) cell lysate – 3 µl; (2) flow through following incubation with anti-FLAG beads – 3 µl; (3) TBS washing of the beads – 3 µl; (4) bound protein eluted with glycine HCl – 10 µl ; (5) magnetic beads alone treated with glycine HCl – 10 µl; and (6) bound protein eluted with Laemmli buffer – 10 µl.

Figure 4. Uptake of human CAV1 by EWS cells

Immunofluorescent detection of CAV1 taken up by A4573 or SK-ES-1 cells either from conditioned medium (CM MD-CAV1) or from culture medium supplemented with the purified protein (PP MD-CAV1). MD-CAV1 was visualized using an Alexa Fluor 488 (green)-conjugated secondary antibody following immunodetection of the Myc tag, while the nuclei were visualized using DAPI (blue). The panels show individual channels detecting the protein (Anti-Myc) and nuclei (DAPI) along with fluorescent merged channels (Myc + DAPI) and fluorescent channels merged with phase contrast image (Phase + Myc + DAPI). The size bars corresponds to 50µm.

Figure 5. Exogenously added CAV1 stimulates cell proliferation in EWS cells

(A) Proliferation of A4573 cells was measured using a luminescence-based technique in untreated cells (CT), and in cells incubated with conditioned medium containing CAV1 (CM); with exogenously added purified MD-CAV1; with exogenously added purified MD-CAV1 along with anti-CAV1 antibody (MD-CAV1 + Ab); and with the anti-CAV1 antibody alone (Ab). (B) Comparison of proliferation of EWS cell lines in the presence of either exogenously added MD-CAV1 (gray bar) or exogenously added MD-CAV1 plus the anti-CAV1 polyclonal antibody (black bar), with the control cells (white bar). Cell viability values in both panels are plotted relative to those in untreated control cultures, which were arbitrarily set as 1.00.