SOX2 Expression and Transcriptional Activity Identifies a Subpopulation of Cancer Stem Cells in Sarcoma with Prognostic Implications

Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cells (CSC). The role of SOX2 in tumor initiation and progression has been well characterized in osteosarcoma. However, the pro-tumorigenic features of SOX2 have been scarcely investigated in other sarcoma subtypes. Here, we show that SOX2 depletion dramatically reduced the ability of undifferentiated pleomorphic sarcoma (UPS) cells to form tumorspheres and to initiate tumor growth. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. Moreover, using a reporter system (SORE6) which allows to monitor viable cells expressing SOX2 and/or OCT4, we found that SORE6+ cells were significantly more tumorigenic than the SORE6- subpopulation. In agreement with this findings, SOX2 expression in sarcoma patients was associated to tumor grade, differentiation, invasive potential and lower patient survival. Finally, we studied the effect of a panel of anti-tumor drugs on the SORE6+ cells of the UPS model and patient-derived chondrosarcoma lines. We found that the mithramycin analogue EC-8042 was the most efficient in reducing SORE6+ cells in vitro and in vivo. Overall, this study demonstrates that SOX2 is a pro-tumorigenic factor with prognostic potential in sarcoma. Moreover, SORE6 transcriptional activity is a bona fide CSC marker in sarcoma and constitutes an excellent biomarker for evaluating the efficacy of anti-tumor treatments on CSC subpopulations.This work was supported by the Agencia Estatal de Investigación (AEI) [MINECO/Fondo Europeo de Desarrollo Regional (FEDER) (SAF-2016-75286-R to R.R.), ISC III/FEDER (Miguel Servet Program CPII16/00049 to R.R., Sara Borrell Program CD16/00103 to S.T.M. and PI16/00280 and PI19/00560 to J.M.G-P) and Consorcio CIBERONC CB16/12/00390)] and the Plan de Ciencia Tecnología e Innovación del Principado de Asturias/FEDER (IDI/2018/155) to J.P.R and Predoctoral Fellowship Severo Ochoa (BP-17-108) to O.E.S

Trabectedin and Campthotecin Synergistically Eliminate Cancer Stem Cells in Cell-of-Origin Sarcoma Models

Trabectedin has been approved for second-line treatment of soft tissue sarcomas. However, its efficacy to target sarcoma initiating cells has not been addressed yet. Here, we used pioneer models of myxoid/round cell liposarcoma (MRCLS) and undifferentiated pleomorphic sarcoma (UPS) developed from transformed human mesenchymal stromal/stem cells (MSCs) to evaluate the effect of trabectedin in the cell type responsible for initiating sarcomagenesis and their derived cancer stem cells (CSC) subpopulations. We found that low nanomolar concentrations of trabectedin efficiently inhibited the growth of sarcoma-initiating cells, induced cell cycle arrest, DNA damage and apoptosis. Interestingly, trabectedin treatment repressed the expression of multiple genes responsible for the development of the CSC phenotype, including pluripotency factors, CSC markers and related signaling pathways. Accordingly, trabectedin induced apoptosis and reduced the survival of CSC-enriched tumorsphere cultures with the same efficiency that inhibits the growth of bulk tumor population. In vivo, trabectedin significantly reduced the mitotic index of MRCLS xenografts and inhibited tumor growth at a similar extent to that observed in doxorubicin-treated tumors. Combination of trabectedin with campthotecin (CPT), a chemotherapeutic drug that shows a robust anti-tumor activity when combined with alkylating agents, resulted in a very strong synergistic inhibition of tumor cell growth and highly increased DNA damage and apoptosis induction. Importantly, the enhanced anti-tumor activity of this combination was also observed in CSC subpopulations. These data suggest that trabectedin and CPT combination may constitute a novel strategy to effectively target both the cell-of-origin and CSC subpopulations in sarcoma

FUS-CHOP Promotes Invasion in Myxoid Liposarcoma through a SRC/FAK/RHO/ROCK-Dependent Pathway

Deregulated SRC/FAK signaling leads to enhanced migration and invasion in many types of tumors. In myxoid and round cell liposarcoma (MRCLS), an adipocytic tumor characterized by the expression of the fusion oncogene FUS-CHOP, SRC have been found as one of the most activated kinases. Here we used a cell-of-origin model of MRCLS and an MRCLS cell line to thoroughly characterize the mechanisms of cell invasion induced by FUS-CHOP using in vitro (3D spheroid invasion assays) and in vivo (chicken chorioallantoic membrane model) approaches. FUS-CHOP expression activated SRC-FAK signaling and increased the invasive ability of MRCLS cells. In addition, FAK expression was found to significantly correlate with tumor aggressiveness in sarcoma patient samples. The involvement of SRC/FAK activation in FUS-CHOP–mediated invasion was further confirmed using the SRC inhibitor dasatinib, the specific FAK inhibitor PF-573228, and FAK siRNA. Notably, dasatinib and PF573228 could also efficiently block the invasion of cancer stem cell subpopulations. Downstream of SRC/FAK signaling, we found that FUS-CHOP expression increases the levels of the RHO/ROCK downstream effector phospho-MLC2 (T18/S19) and that this activation was prevented by dasatinib or PF573228. Moreover, the ROCK inhibitor RKI-1447 was able to completely abolish invasion in FUS-CHOP–expressing cells. These data uncover the involvement of SRC/FAK/RHO/ROCK signaling axis in FUS-CHOP–mediated invasion, thus providing a rationale for testing inhibitors of this pathway as potential novel antimetastatic agents for MRCLS treatment