Together, these data highlight an additional mechanism in TZD-induced adipogenesis where the transcriptional activity of YAP is decreased upon cytoplasmic sequestration. This finding is corroborated by a decrease in expression of canonical YAP target genes and suggests that TZDs affect YAP-dependent transcription and activation of Hippo signaling in osteosarcoma cells. Importantly Rosi shows synergistic growth inhibitory effects when combined with verteporfin, an inhibitor of YAP-TEAD mediated transcription [ 44 ] in mouse human and dog osteosarcoma cells Figure 7E.

Images were taken using a Leica DM immunofluorescence microscope at 63x magnification. Graph shows growth relative to DMSO control averaged from three replicates in each condition. From the results presented it is apparent that a TZD mediated differentiation therapy would target specifically the OS stem cell population. Our previous analysis had revealed that Wnt signaling is low in CSCs that express Sox2 and form osteospheres. Sox2-depleted cells have higher Wnt signaling, as evidenced by an increase in canonical Wnt targets, such as TIMP3 [ 15 , 37 ].

Thus high Wnt signaling and low YAP target genes high Hippo signaling mark a population of differentiated, less stem-like osteosarcoma cells. Distribution of patients for this data set has been previously published. Survival probability and P values calculated using Kaplan-Meier and Cox proportional hazards methods. Osteosarcomas are genetically highly heterogeneous with multiple oncogenic drivers which has been a major obstacle in developing targeted therapy for this tumor type [ 46 - 49 ].

In this study, we present proof of principle for differentiation therapy for osteosarcoma which relies on the use of TZDs to stimulate adipogenesis in these cells. Across species, osteosarcoma cells demonstrate growth inhibition and enhanced adipogenesis when treated with the TZDs, Pio or Rosi. TZDs also decreased tumorigenicity of osteosarcoma cells in xenotransplantation assays. The effects of TZDs are independent of the origins or driver mutations, suggesting the broad therapeutic potential of these agents for the treatment of osteosarcoma. Gene expression analysis of TZD -treated cells led to the identification of a signature that is predictive of patient outcomes.

This study provides pre-clinical rationale for an osteosarcoma differentiation therapy with a targeted agent. DT represents an attractive alternative to conventional treatment for osteosarcomas by inducing terminal differentiation of CSCs. This two-pronged strategy would ensure tumor debulking through surgery or chemotherapy and CSC depletion, such that relapse through persistence of osteosarcoma stem cells is minimized. FGF21 also acts on bone marrow mesenchymal cells to promote differentiation into adipocytes rather than osteoblasts.

Hippo signaling has been identified as a tumor suppressive pathway in tumors of both epithelial and mesenchymal origin cancers. Loss of YAP decreases the stem cell fraction and restores osteogenic differentiation [ 17 ]. Treatment of osteosarcoma cells with TZDs leads to YAP phosphorylation and nuclear exclusion, with a decrease in canonical YAP target genes, suggesting that TZDs exert their tumor suppressive activity in part through a mechanism akin to activation of the Hippo pathway.

While the exact mechanism of this re-activation remains to be deciphered, experiments in 3T3-L1 cells suggest that TZD treatment activates Lats2, one of the upstream kinases that phosphorylate YAP [ 51 ]. Hippo signaling is probably not the only mechanism of action of TZDs. This points to a novel combination of drugs to target osteosarcomas.

Like the TZDs, verterporfin is also in clinical use thus providing a rationale for drug repurposing. Even more striking was the down regulation of Hippo targets in the osteosarcoma cells. This could be due to the fact that osteosarcoma cells are addicted to YAP expression that needs to be down-regulated for adipogenic differentiation to occur, unlike normal mesenchymal and pre-adipocytic cells. We therefore believe that the survival analyses reflect the fact that the least differentiated, most aggressive tumors contain a larger proportion of CSC than the more benign tumors, a conclusion previously reached by an analysis of lung and breast cancers [ 52 ].

In this view the most aggressive osteosarcomas would be the ones that could mostly benefit from a CSC targeted differentiation therapy.

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Of particular interest is the fact that this signature is predictive of survival in two different data sets and is independent of the tumor heterogeneity of osteosarcomas. Despite the therapeutic potential of TZDs for DT, their clinical use has been challenged because of their safety profile with long-term use as in diabetes. However, a more recent and larger study has determined that pioglitazone use was not associated with an increased risk to bladder cancer [ 53 ].

The relationship between exposure duration and adverse effects makes it important to modulate treatment to maximize the benefit-risk ratio. Unlike the use of TZDs for the treatment of T2D that is typically low-dose and long-term, we anticipate that TZD use in DT would be high-dose, short-term in turning aggressive tumor cells to less proliferative fat cells. This along with the emergence of second-generation TZDs maintains its promising outlook [ 19 ]. Thus DT via TZD treatment may be a potential adjuvant therapy for osteosarcoma and other cancers of the mesenchymal lineage. DT would also be applicable to other cancers such as gliomas where CSCs have been identified.

The mouse osteosarcoma cell line mOS was attained from a spontaneous osteosarcoma and previously described [ 7 ]. Gordon, and Dr. Kleinerman respectively, MD. Survival analysis was carried out for a proposed subset of genes using the Cox-proportional hazards models as implemented survival package in R. For a gene signatures and a patient cohort, using a previously proposed methodology [ 54 ]: by computing an activity score for each patient in the cohort as follows: all genes are z-score transformed, then for each sample we add the z-score for up-regulated genes and subtract it for down-regulated genes.

Specimens were sorted by activity score, then survival association was evaluated using R. Cells were subsequently serum-starved and a scratch was made on the monolayer using a pipette tip. Animals were monitored and weighed twice weekly. Tumor volumes were measured by Vernier calipers. Two treatment groups were established with ten mice per group.

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Tissues were fixed and embedded in paraffin. Paraffin-embedded sections were deparaffinized in Citrosolv and then rehydrated in an ethanol series. Antigen retrieval was performed at pH 6, 10mM sodium citrate buffer and slides were blocked in goat serum.

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Control IgG was used as a negative control. Adipogenesis was detected by staining with Oil-Red-O Sigma. The medium was changed every three days and lysed in radio-immunoprecipitation assay RIPA buffer containing protease inhibitors. Samples were kept on ice for thirty minutes and after centrifugation at 13, rpm for twenty minutes, the supernatants were collected. Anti-tubulin antibodies were used as a normalization control.

Following secondary probing with monoclonal anti-mouse or polyclonal anti-rabbit antibody probes, protein blots were visualized with an enhanced chemiluminesence detection reagent Amersham , exposed to an X-ray film, and developed.

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Actin was used as a normalization control. Gene expression was quantified with Cufflinks 2. Unsupervised clustering visualization was generated using R. Changes in localized bone microstructure were assessed using a microCT system SkyScan ; Bruker microCT, Belgium where projections x pixels of the distal femur were acquired at a nominal isotropic resolution of 9 um. To reduce the variability in the beam intensity profiles across the image, the approximate centerline of each bone was aligned to the axis of rotation of the system.

A 10W power energy setting kV and mA and a 0. An alignment procedure and flat-field detector calibration were performed to minimize ring artifacts and increase signal-to-noise ratio.

These scanning parameters, were chosen in accordance with the guidelines for mCT analysis of rodent bone structure [ 35 ]. A modified back-projection reconstruction algorithm v. Images were optimized and corrected for ring artifacts and further beam hardening correction was achieved using the NRecon software to check that the X-ray intensity profiles across the bone cross-section remained linear. Using manufacturer software CtAn, Bruker microCT, Belgium the tumor regions were re-oriented such that regions of interest ROIs could be defined and compared in the transverse plane.

The standard trabecular bone microstructural parameters are not reported here since this is a pathological bone formation system, and those measurements are not easily interpretable in this setting. Curr Opin Pediatr. Biology and therapeutic advances for pediatric osteosarcoma. Outcomes for children and adolescents with cancer: challenges for the twenty-first century.

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J Clin Oncol. The adolescent and young adult with cancer: state of the art — bone tumors. Curr Oncol Rep. Canine osteosarcoma: a naturally occurring disease to inform pediatric oncology. ILAR J. Helman LJ and Meltzer P. Mechanisms of sarcoma development. Nature reviews Cancer. Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease. Genes Dev. Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage. The transcription factor Sox2 is required for osteoblast self-renewal.

Cell Death Differ. Stem-like cells in bone sarcomas: implications for tumorigenesis. Cancer letters. SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nature genetics.

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Anti-diabetic rosiglitazone remodels the adipocyte transcriptome by redistributing transcription to PPARgamma-driven enhancers.