Alveolar Soft Part Sarcoma Cell Research Articles

Establishment and characterization of NCC-ASPS1-C1: a novel patient-derived cell line of alveolar soft-part sarcoma.

Alveolar soft-part sarcoma is a mesenchymal malignancy characterized by the rearrangement of ASPSCR1 and TFE3 and a histologically distinctive pseudoalveolar pattern. Although alveolar soft-part sarcoma takes an indolent course, its long-term prognosis is poor because of late distant metastases. Currently, curative treatments have not been found for alveolar soft-part sarcoma, and hence, a novel therapeutic strategy has long been required. Patient-derived cell lines comprise an important tool for basic and preclinical research. However, few cell lines from alveolar soft-part sarcoma have been reported in the literature because it is an extremely rare malignancy, accounting for less than 1% of all soft-tissue sarcomas. This study aimed to establish a novel alveolar soft-part sarcoma cell line. Using surgically-resected tumor tissue of alveolar soft-part sarcoma, we successfully established a cell line and named it NCC-ASPS1-C1. The NCC-ASPS1-C1 cells harbored an ASPSCR1-TFE3 fusion gene and exhibited slow growth, and spheroid formation. On the other hand, NCC-ASPS1-C1 did not show the capability of invasion. We screened the antiproliferative effects of 195 anticancer agents, including Food and Drug Administration-approved anticancer drugs. We found that the MET inhibitor tivantinib and multi-kinase inhibitor orantinib inhibited the proliferation of NCC-ASPS1-C1 cells. The clinical utility and molecular mechanisms of antitumor effects of these drugs are worth investigating in the further studies, and NCC-ASPS1-C1 cells will be a useful tool for the in vitro study of alveolar soft-part sarcoma.

Abstract IA20: Autophagy in alveolar soft part sarcomagenesis

Abstract Altered metabolism is considered to be one of the new hallmarks of cancer. Autophagy is one major avenue of altered cancer metabolism, enabling cell survival under metabolic stress and promoting chemoresistance. The nuclear localization of MiTF/TFE3 family transcription factors has associated with upregulated transcription of autophagy genes in pancreatic cancer. Alveolar soft part sarcoma is a rare but deadly soft-tissue sarcoma, with a predilection for adolescent and young adult victims. Alveolar soft part sarcoma is noteworthy for its resistance to traditional cytotoxic chemotherapies. It consistently associates with a t(X;17) chromosomal translocation that produces the ASPSCR1-TFE3 target gene, bearing the DNA-binding domain from TFE3 and protein interaction domains from ASPSCR1. We have demonstrated that conditional expression of ASPSCR1-TFE3 is sufficient to drive alveolar soft part sarcomagenesis in the mouse with complete penetrance. Mouse tumors recapitulate human alveolar soft part sarcoma histology and transcriptomes faithfully. While direct targets of ASPSCR1-TFE3 have been studied in a renal cell carcinoma cell line, they have not been studied in alveolar soft part sarcoma. We report not only the first genome-wide localization of the ASPSCR1-TFE3 oncoprotein on chromatin from alveolar soft part sarcoma cell lines and mouse tumors, but also its association with actively transcribed genes. Among these are found many genes related to autophagy, especially those related specifically to the nutrient responsive pathways that drive autophagy. We demonstrate high expression of autophagy-related lysosomes and proteins at baseline conditions in human tumors and cell lines and mouse tumors. We also demonstrate active autophagic flux even in the absence of stress conditions. Inhibition of autophagy has no apparent impact on the survival of alveolar soft part sarcoma cells alone, but profoundly impacts their protein degradation pathways and the availability of amino acids for protein assembly in stress. Inhibition of autophagy strongly synergizes with chemotherapy to kill alveolar soft part sarcoma cells, suggesting it was a source mechanism for resistance. We have therefore demonstrated the direct targets of ASPSCR1-TFE3 in alveolar soft part sarcomas, including a number of autophagy genes that are expressed in these tumors, independently from nutrient deprivation or stress, rendering cells particularly resistant to many therapy-induced stresses. Citation Format: Kevin B. Jones. Autophagy in alveolar soft part sarcomagenesis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA20.

Abstract 274: Assessing combinations of chemotherapy in alveolar soft part sarcoma cell lines

Abstract Background: Alveolar Soft Part Sarcoma (ASPS) is a rare soft tissue sarcoma subtype occurring mainly in children and young adults. Malignant tumors develop in the connective tissues, typically in the lower extremities of the legs and thighs, but can also be found in the head and neck regions. ASPS is slow growing, highly angiogenic, and may have unique metabolic properties. ASPS is characterized by an unbalanced translocation between the ASPSCR1 gene (whose product tethers GLUT4 transporters) and the TFE3 gene (encoding a transcription factor). The resulting ASPSCR1-TFE3 fusion gene is responsible for an aberrant transcription factor that is thought to be involved in the pathogenesis of the disease. Methods to assess a number of different agents in combination are needed for practical use in clinical trials. Methods: Here, we seek to examine the therapeutic activity of single agent and combinations of epigenetic, anti-angiogenic and a diverse mix of other mechanisms of action agents using high-throughput screening platforms and well-established cell line models for ASPS (ASPS-1 and ASPS-KY). We designed stringent screening conditions that assess the compounds at clinically-relevant concentrations and time of exposures that mimic the in vivo pharmacokinetics in an effort to maximize the translational potential of these results to the clinical setting. 54 agents were screened across the 2 cell lines with attention to existing clinical data and angiogenesis inhibitors. The cell titer glo assay was used to determine cell viability at three clinically achievable concentrations for all agents. The most promising 8 agents were then studied in all two-drug combinations to evaluate for synergy. Results: The results of our screening showed that several agents significantly reduced cell viability at clinically relevant concentrations and exposure times with belinostat (an HDAC inhibitor) and dinaciclib (a CDK 1, 2, 5 and 9 inhibitor) in particular having good activity. Several combinations with doxorubicin showed good efficacy and synergy. Belinostat and doxorubicin affected both ASPS-KY and ASPS-1 cell lines similarly, with an average fraction affected (FA) of 0.87. The combination index (CI) indicated strong synergy in the ASPS-KY (CI = 0.09) and some synergy in ASPS-1 (CI = 0.74). Similarly, dinaciclib and doxorubicin produced a FA of 0.85 with good synergy (CI of 0.51-0.66). In addition, the combination of belinostat and tivantinib showed additive effects in both cell lines (CI of 0.88-1.2) with a FA of ∼0.8. These findings suggest potentially promising opportunities for developing combinational approach to treating childhood soft tissue sarcomas. Citation Format: Justine Clark, Elliot Kahen, Diana X. Yu, Christopher L. Cubitt, Daniel M. Sullivan, Damon R. Reed. Assessing combinations of chemotherapy in alveolar soft part sarcoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 274.

Abstract C197: Fluorescent in situ hybridization confirmation of circulating alveolar soft part sarcoma cells (CTCs) isolated from peripheral blood specimens using ApoStream™ instrumentation.

Abstract Enumeration and characterization of circulating tumor cells (CTCs) by the CellSearch™ instrument platform, which uses antibody capture of fixed cells from blood, provides prognostic information and assists in monitoring therapy for patients with certain epithelial cancers. Our laboratory is clinically evaluating an antibody-independent CTC enrichment technology, ApoStream™, which was co-developed by NCI and SAIC-F with ApoCell Inc. The ApoStream isolates living CTCs from a wide variety of malignancies including sarcomas and lymphomas as well as epithelial cancers, enabled by critical differences in morphology and dielectric properties rather than surface marker expression. In contrast to the CellSearch™ platform, CTCs isolated with the ApoStream™ instrument are amenable to tests that unambiguously identify isolated cells as malignant cells. To validate the performance of the ApoStream™ device for clinical specimens, we focused on isolation of CTCs from patients with advanced alveolar soft part sarcoma (ASPS). Patient samples from an ongoing clinical study at the NCI provided the opportunity to demonstrate the utility of ApoStream™ technology for the isolation of CTCs from patients with this rare non-epithelial malignancy. ASPS is characterized by a specific, unbalanced genetic translocation of der(17)t(X;17)(p11;q25) involving the ASPL and TFE3 genes. The detection of the fusion product has been reported in primary tumors and peripheral blood based on RT-PCR but not in CTCs. Here we show that the ASPL-TFE3 gene translocation can be unambiguously confirmed in CTCs by break-apart fluorescent in situ hybridization (FISH), and the presence of the fusion protein in CTCs was confirmed with specific monoclonal antibodies. Efficient recovery was demonstrated using specified numbers of cancer cells spiked into buffer and normal donor control PBMCs; clinical utility was demonstrated with blood samples from ASPS patients. This study provides not only initial results demonstrating isolation of CTCs from metastatic ASPS patients using the ApoStream™ technology without the need for antibody capture but also a rationale for exploring ApoStream™ technology in additional tumor histologies that cannot be evaluated with the CellSearch™ platform. Our current efforts are focused on evaluating the utility of ApoStream™-isolated CTCs for assessing pharmacodynamic effects of new targeted agents during early stage clinical trials. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C197. Citation Format: Priya Balasubramanian, Lihua Wang, Shivaani Kummar, Melinda Hollingshead, Francis Owusu, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Fluorescent in situ hybridization confirmation of circulating alveolar soft part sarcoma cells (CTCs) isolated from peripheral blood specimens using ApoStream™ instrumentation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C197.