Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas

Abstract

Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.

Figure 1. Sox2 is highly expressed in human and murine osteosarcomas

Sox2 mRNA (left) and protein expression (right) in human (A) and murine (B) osteosarcoma cell lines. Quantitative RT-PCR for mRNA levels and Western blot of Sox2 protein in the indicated cell lines. hFOB, Normal Fetal Human Osteoblast; WT osteoblasts, wild type murine primary calvarial osteoblasts. (C) Sox2 expression in human osteosarcoma tissue. Immunohistochemistry on paraffin-embedded human osteosarcoma tissue samples using a polyclonal antibody against Sox2 (Magnification 10×). Representative images of osteosarcoma samples are shown. Higher magnification (40×) at which nuclear Sox2 staining is evident is shown. Secondary antibody alone was used as a negative control. We examined a total of 18 samples. Tumors were identified as Sox2-positive when nuclear staining was present in more than 50% cells per field. All samples were scored positive by these criteria.

Figure 2. Sox2 depletion decreases transformation properties and tumorigenesis

(A) Sox2 protein expression in osteosarcoma cells stably expressing Sox2 shRNA. Western blot of mOS-482 osteosarcoma cells expressing scrambled (sc) shRNA or shRNAs against Sox2 were analyzed by immunoblotting. Tubulin was used as a loading control. (B) Colony formation. Osteosarcoma cells infected with either scrambled (sc) or Sox2 shRNA viruses were selected in Hygromycin B for 7-10 days. Colonies were counted following staining with Crystal Violet. Representative plates of mOS-482 cells are shown. Table shows genotypes and colony counts from four independent cell lines. (C) Soft Agar Colony Formation; (D), Migration and (E) Invasion. mOS-482 cells expressing sc or Sox2 shRNA were analyzed in the indicated assays as described in Materials and Methods. All experiments were performed in at least two independent cell lines. Results shown are an average of triplicate plates. * = p<0.05 (F) Tumorigenicity assay: Tumor-forming ability of osteosarcoma cell lines (mOS-482) expressing either scrambled or Sox2 shRNAs, was measured in NOD/SCID mice. Injection of 1 × 10⁶ cells gave similar results (not shown). Tumor volume was monitored and measured bi-weekly. Difference between groups was measured by ANOVA. * = p<0.05 The inset shows a western blot of Sox2 protein expression in the injected cells and isolated tumors from either scrambled or long latency tumors arising from cells expressing Sox2 shRNAs.

Figure 3. Sox2 marks a population of Sca-1 positive, multipotent osteosarcoma stem cells

(A) Flow cytometric analysis of Sox2 and Sca-1 doubly-labeled osteosarcoma cells. Osteosarcoma cells (mOS-482) were labeled to detect membrane Sca-1 (PE- Sca-1, Phycoerythrin-Sca-1 conjugated antibody) and intracellular Sox2 (Sox2 polyclonal antibody followed by Alexa Fluor 488 conjugated secondary antibody). Forward scatter-Side scatter (FSC-SSC) profile shows that 90% cells were viable (Fig. S4) and was used for anaysis. Sca-1^HISox2^HI population is indicated in top right quadrant. The percentage of Sca-1^HISox2^HI population from three independent cell lines is shown in the adjoining Table. (B) Sox2 protein expression in Sca-1^HI and Sca-1^LO fraction. mOS-482 cells were separated into Sca-1^HI and Sca-1^LO cells using Sca-1 magnetic bead separation. Western blot of Sox2 expression in the total adherent population and the two fractions is shown. (C) Osteosphere Assay. Equal numbers of cells from Sca-1^HI and Sca-1^LO fractions from mOS-482 cells were plated in suspension culture in triplicate. Spheres were counted after 10 days. Each experiment was repeated at least twice. Results from a representative experiment are shown. * = p<0.05 (D) Osteogenic and Adipogenic Differentiation Assay. Sca-1^HI and Sca-1^LO fractions from mOS-482 cells were plated in either osteogenic or adipogenic medium. Osteogenic differentiation was detected using alkaline phosphatase staining. Results from representative plates at the indicated time period are shown. Adipogenesis was detected by Oil Red O staining of cells induced for 15 days (magnification 100×). (E) Serial propagation of Sca-1^HI and Sca-1^LO fractions. Sca-1^HI and Sca-1^LO fractions from mOS-482 cells were doubly purified by magnetic bead and FACS. Each fraction was plated separately and serially passaged. After labeling for both Sox2 and Sca-1, the proportion of cells that were doubly positive (Sca-1^HI Sox2^HI or Sca-1^LOSox2^LO) at each passage was determined by flow cytometry.

Figure 4. Sox2 down-regulation decreases the stem cell population in murine and human osteosarcomas

(A) Surface Sca-1 expression is reduced in Sox 2-depleted cells. mOS-379 cells expressing either scrambled or Sox2 shRNA were stained with a PE-conjugated anti-Sca-1 antibody and analyzed by flow cytometry. Mean fluorescence intensity (MFI) of the indicated cells is plotted. Y axis = % of Max (Percentage of Maximum MFI). X axis = rIgG-PE (rat-PE-conjugated-anti-Sca-1) (B) Osteosphere Assay. Equal numbers of cells expressing scrambled or Sox2 shRNA were plated in suspension culture in triplicate. Spheres were counted after 10 days. Each experiment was repeated at least twice. Results from a representative experiment are shown. * = p<0.05. Similar results were obtained with the mOS-682 and mOS-202M cell lines. (C) and (D) Western Analysis ad Osteosphere Assay in human osteosarcoma samples. Saos-2-LM7 (Figure 4C) or primary osteosarcoma cells from fresh biopsy (OS-NYU1) (Figure 4D) cells expressing either scrambled or human Sox2 shRNA were analyzed for Sox2 expression by Western analysis. For osteosphere assay, equal numbers of cells expressing scrambled or human Sox2 shRNA were plated in suspension culture in triplicate. Spheres were counted after 10 days. Each experiment was repeated at least twice. Results from a representative experiment are shown. * = p<0.05.

Figure 5. Sox2 down-regulation increases osteoblastic differentiation of murine osteosarcoma cells

(A) Osteoblastic differentiation. mOS-379 cells expressing scrambled or Sox2 shRNA were plated in osteogenic for the indicated time periods. Differentiation was assessed using alkaline phosphatase staining. Results from representative plates at the indicated time period are shown. For gene expression analysis, real-time quantitative PCR analysis using primers for osteocalcin at the indicated times is shown. All values are normalized to actin and are expressed relative to expression in uninduced cells at Day 0. (B) Adipogenic differentiation. mOS-379 cells expressing scrambled or Sox2 shRNA were plated in adipogenic medium for 15 days. Differentiation was measured using Oil Red O staining (magnification 100×). For gene expression analysis, real-time quantitative PCR analysis using primers for PPARγ and aP2 at the indicated days. All values are normalized to actin and are expressed relative to uninduced cells at Day 0.

Figure 6. Sox2 depletion leads to activation of Wnt signaling in osteosarcoma cells

(A) Activated β-catenin (ABC) protein expression. Immunoblot of mOS-379 cells expressing either scrambled or Sox2 shRNA with anti-ABC antibody. γ- tubulin was used as a loading control. (B) Wnt reporter activity. Relative TOPFLASH luciferase reporter activity in mOS-379 cells expressing scrambled or Sox2 shRNA. Basal (black bars) or Wnt 3A stimulated (gray bars) activity at 48 hours was measured and normalized to total protein.* = p<0.05 (C) Wnt target gene expression. Real-time quantitative PCR analysis of CTGF, Axin and Timp3 expression in mOS-379 cells expressing scrambled or Sox2 shRNAs as indicated. All values are normalized to actin and expressed relative to expression in cells expressing scrambled shRNA. (D) Wnt reporter activity in osteospheres. Relative luciferase activity in total (adherent) cells and osteospheres from mOS-379 cells expressing TOPFLASH luciferase reporter plasmid. (E) Expression of Osterix and Runx2 in osteospheres. Real-time quantitative PCR analysis of Runx2 and Osterix expression in osteospheres and adherent cells, as in (D). (F) Expression of Sox2 in osteospheres. Real-time quantitative PCR analysis of Sox2 expression in osteospheres and adherent cells, as in (D).

Figure 7. Activation of Wnt signaling decreases Sox2 expression in osteoblasts and osteosarcoma

(A) Sox2 Expression in Wnt-treated cells. Western blot of Sox2 in osteosarcoma cells (mOS-482), immortalized osteoblasts (OB1). and primary osteoblasts transduced with empty vector (-) or Sox2 (+) lentivirus, after treatment with control or Wnt 3A conditioned medium for 48 hours. γ- tubulin was used as a loading control. (B) Model for Sox2-Wnt antagonism in osteosarcoma. Undifferentiated osteosarcoma cells with high Sox2 expression and low Wnt pathway activity are in a stem-like, multipotent, self-renewing state.suggesting that Sox2 and Wnt pathway are mutually antagonistic in osteosarcoma development. Sox2 depletion or activation of Wnt signaling leads to a more differentiated, osteoblast-like state.