For the cancer genomics era, there is a need for clinically annotated close-to-patient cell lines suitable to investigate altered pathways and serve as high-throughput drug-screening platforms. This is particularly important for drug-resistant tumors like chondrosarcoma which has few models available. Here we established and characterized new cell lines derived from two secondary (CDS06 and CDS11) and one dedifferentiated (CDS-17) chondrosarcomas as well as another line derived from a CDS-17-generated xenograft (T-CDS17). These lines displayed cancer stem cell-related and invasive features and were able to initiate subcutaneous and/or orthotopic animal models. Different mutations in Isocitrate Dehydrogenase-1 (IDH1), Isocitrate Dehydrogenase-2 (IDH2), and Tumor Supressor P53 (TP53) and deletion of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) were detected both in cell lines and tumor samples. In addition, other mutations in TP53 and the amplification of Mouse Double Minute 2 homolog (MDM2) arose during cell culture in CDS17 cells. Whole exome sequencing analysis of CDS17, T-CDS17, and matched patient samples confirmed that cell lines kept the most relevant mutations of the tumor, uncovered new mutations and revealed structural variants that emerged during in vitro/in vivo growth. Altogether, this work expanded the panel of clinically and genetically-annotated chondrosarcoma lines amenable for in vivo studies and cancer stem cell (CSC) characterization. Moreover, it provided clues of the genetic drift of chondrosarcoma cells during the adaptation to grow conditions.

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.

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.

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.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.