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Let-7a suppress Ewing sarcoma CSCs’ malignant phenotype through forms a positive feedback regulation loop with lin28 via STAT3
abstracts
1720P
Let-7a suppress Ewing sarcoma CSCs’ malignant phenotype through forms a positive feedback regulation loop with lin28 via STAT3
C. Kai, X. jiang Spine Center, 2nd Affiliated Hospital of Nanchang University, Nanchang, China Background: Ewing’s sarcoma (ES) is a highly malignant primary bone tumor, and ewing’s sarcoma stem cells (CSCs) are the main cause of tumor recurrence and metastasis. This paper will further discuss the mechanism of let-7a in the development and progression of ewing’s sarcoma CSCs, laying an experimental basis and theoretical basis for the biological treatment of ewing’s sarcoma. Methods: In this study, we intend to isolate CSCs from ES cells cultured in vitro by side group cell sorting and flow cytometry, and then detect the effect of let-7a on the malignant phenotype of ewing’s sarcoma CSCs, and further verify the regulatory role of let7a in ewing’s sarcoma CSCs in vitro and in vivo experiments. Results: We successfully isolated Ewing sarcoma cancer stem cells(CSCs) by side population cells (SP) sorting method. The immunohistochemical results showed that STAT3 was highly expressed in ES tissues and negatively correlated with let7a.Overexpression of the let - 7a can suppress the malignant phenotype of Ewing sarcoma CSCs, and inhibited the growth of subcutaneous xenograft tumor of ewing’s sarcoma CSCs.Bioinformatics analysis found that singal transducer and activator of transcription 3 (STAT3) is a direct target gene of let - 7a, STAT3 and its downstream factor lin28 form a positive feedback loop with let-7a, participate in Ewing sarcoma CSCs function regulation. Conclusions: let-7a suppress Ewing sarcoma CSCs’ malignant phenotype through forms a positive feedback regulation loop with lin28 via STAT3. Legal entity responsible for the study: The second affiliated Hospital of Nanchang University. Funding: National Natural Science Foundation of China. Disclosure: All authors have declared no conflicts of interest.
1721P
Myoepithelial tumours of soft tissues and extraskeletal myxoid chondrosarcomas feature a distinct transcriptional pattern
D. Racanelli1, S. Stacchiotti2, M. Brenca1, M. Sbaraglia3, K. Fassetta1, D. Baldazzi1, S. Piccinin1, S. Brich4, P.G. Casali2–11, P. Collini5, G.P. Dagrada1–6, M. Fiore7, A. Gronchi7, A. Astolfi8, M.A. Pantaleo8, A. Righi9, S. Pilotti5, A.P. Dei Tos3–10, R. Maestro1 1 Unit of Oncogenetics and Functional Oncogenomics, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, National Cancer Institute, Aviano, Italy, 2Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 3 Department of Pathology, Treviso Regional Hospital, Treviso, Italy, 4Unit of Experimental Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 5Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 6Laboratory of Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 7Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 8"Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy, 9Anatomy and Pathological Histology, IRCCS, Istituto Ortopedico Rizzoli, Bologna, Italy, 10Department of Medicine, University of Padua School of Medicine, Padua, Italy, 11Oncology and Haemato-Oncology Department, University of Milan, Milan, Italy Background: Myoepithelial tumors of soft tissues (MT) and Extraskeletal Myxoid Chondrosarcoma (EMC) are closely related pathological entities whose overlapping features, in terms of morphology and immunoprofile, make differential diagnosis challenging. Different fusion genes have been described in MT. Instead, the rearrangement of NR4A3 is conventionally considered an exquisite feature of EMC. Nevertheless, whether there is a biological overlap between MT and EMC is still controversial. In order to shed light on this issue we compared the transcriptional profiles of the two entities. Methods: A series of EMC (12 cases) and MT (7 cases), retrieved from the pathology files, was selected for the study. The diagnosis was made according to the WHO classification. FISH analyses confirmed that all EMC harbored NR4A3 rearrangement (7 EWSR1-NR4A3 and 5 TAF15-NR4A3); 4 EWSR1 and 1 FUS rearrangement were detected in MT. No rearrangement was detected in 2 cases. RNA was extracted from FFPE specimens with tumor cellularity >70%. RNA-sequencing was carried out on an Illumina Hiseq1000 platform (average 70 million reads/sample). Diverse algorithms and bioinformatic suites were employed to identify fusion transcripts and functional annotation analysis. Results: RNA-seq analysis confirmed the rearrangements detected by FISH and identified one PTCH1-GLI1 fusion in a MT. Principal component analysis and unsupervised hierarchical clustering indicated that MT and EMC feature a distinct transcriptional profile. Functional annotation of the genes differentially expressed highlighted Hedgehog (HH) and WNT signaling as significantly enriched pathways in MT compared to EMC, with canonical GLI1 and WNT target genes upregulated in MT. Ectopic expression in cell models of the PTCH1-GLI1 chimeric transcript identified in the MT sample correlated with the induction of GLI1 target genes. Conclusions: This study corroborates the notion that MT and EMC represent two distinct biological entities, with MT featuring a distinctive activation of HH and WNT pathways. The PTCH1-GLI1 fusion represents one possible mechanism of HH pathway activation in MT. Legal entity responsible for the study: The authors. Funding: Fondazione AIRC per la Ricerca sul Cancro, CRO Intramural Grant, Italian Ministry of Health. Disclosure: P.G. Casali: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Deciphera; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eisai; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eli Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Nektar Therapeutics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Advenchen Laboratories; Research grant / Funding (institution): Amgen Dompe´; Research grant / Funding (institution): AROG Pharmaceuticals; Research grant / Funding (institution): Blueprint Medicines; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Epizyme Inc.; Research grant / Funding (institution): Glaxo; Research grant / Funding (institution): Karyopharm Pharmaceuticals; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): PharmaMar. A.P. Dei Tos: Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer. All other authors have declared no conflicts of interest.
1722P
In vivo efficacy and enhanced tumour accumulation of liposomal vinorelbine (TLC178) in human sarcoma xenograft mice model
W-N. Yu1, J-H. Tang1, Y-C. Tsai2, H-T. Wang2 Non-Clinical Department, Taiwan Liposome Company, Taipei, Taiwan, 2 Pharmacokinetic Department, Taiwan Liposome Company, Taipei, Taiwan
1
Background: Vinorelbine (VNB) is a member of vinca alkaloid medications and approved for the treatment of non-small cell lung cancer as well as off-label use for metastatic breast cancer over 20 years based upon FDA-approved indication. Several studies have suggested the potential of VNB in treating sarcomas. Liposomal VNB (TLC178) is a novel sustained release liposomal formulation. In the presented studies, TLC178 resulted in enhanced tumor accumulation and greater anti-tumor efficacy than non-liposomal VNB.
v704 | Sarcoma
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz283.053/5577429 by guest on 24 October 2019
is still unknown. Cell fusion is a physiological mechanism involved in several processes including myoblast differentiation and already described to be involved in aneuploidy generation and genomic instability. We thus hypothesized that cell fusion could participate to sarcoma oncogenesis by generating genomic instability. Methods: We established cell line of hybrids from spontaneous fusion of immortalized myoblasts. Tumor and metastasis development were evaluated upon grafting and whole genome and transcriptome sequencing with in-vitro mechanistic studies were performed to decipher phenotypes acquired by hybrids upon fusion. Results: In vitro hybrid cell lines show a more aggressive phenotype than parental cell lines by increasing proliferation and clonogenic capacities. Only hybrids form tumors upon grafting and may develop also metastasis. Aneuploidy triggered by cell fusion induces genome reorganization and in vivo tumor genome show alterations corresponding to those observed in human rhabdomyosarcoma. Indeed, 85% of hybrid tumors harbor DMD deletions as observed in sarcoma with myogenic origin. The study of this protein reveals that tumors lose the expression of Dp427 isoform which is the one reported as a tumor suppressor. This malignant transformation of hybrids is associated to transcriptomic remodeling toward a decrease of the expression of genes involved in muscle differentiation. Conclusions: To our knowledge, this is the first model able to reproduce the complex genomic of sarcoma. The detection of those hybrid cells in sarcoma tumor allowed us to push forward they are involved in sarcoma development and progression and their quantification in tumors or in circulating cells could thus establish an early prognostic marker. Finally, specific therapeutic targeting of this hybrids could represent new approach. Legal entity responsible for the study: Frederic Chibon. Funding: Association pour la Recherche contre le Cancer (ARC) Fondation pour la Recherche Me´dicale (FRM). Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology
1720P
Let-7a suppress Ewing sarcoma CSCs’ malignant phenotype through forms a positive feedback regulation loop with lin28 via STAT3
C. Kai, X. jiang Spine Center, 2nd Affiliated Hospital of Nanchang University, Nanchang, China Background: Ewing’s sarcoma (ES) is a highly malignant primary bone tumor, and ewing’s sarcoma stem cells (CSCs) are the main cause of tumor recurrence and metastasis. This paper will further discuss the mechanism of let-7a in the development and progression of ewing’s sarcoma CSCs, laying an experimental basis and theoretical basis for the biological treatment of ewing’s sarcoma. Methods: In this study, we intend to isolate CSCs from ES cells cultured in vitro by side group cell sorting and flow cytometry, and then detect the effect of let-7a on the malignant phenotype of ewing’s sarcoma CSCs, and further verify the regulatory role of let7a in ewing’s sarcoma CSCs in vitro and in vivo experiments. Results: We successfully isolated Ewing sarcoma cancer stem cells(CSCs) by side population cells (SP) sorting method. The immunohistochemical results showed that STAT3 was highly expressed in ES tissues and negatively correlated with let7a.Overexpression of the let - 7a can suppress the malignant phenotype of Ewing sarcoma CSCs, and inhibited the growth of subcutaneous xenograft tumor of ewing’s sarcoma CSCs.Bioinformatics analysis found that singal transducer and activator of transcription 3 (STAT3) is a direct target gene of let - 7a, STAT3 and its downstream factor lin28 form a positive feedback loop with let-7a, participate in Ewing sarcoma CSCs function regulation. Conclusions: let-7a suppress Ewing sarcoma CSCs’ malignant phenotype through forms a positive feedback regulation loop with lin28 via STAT3. Legal entity responsible for the study: The second affiliated Hospital of Nanchang University. Funding: National Natural Science Foundation of China. Disclosure: All authors have declared no conflicts of interest.
1721P
Myoepithelial tumours of soft tissues and extraskeletal myxoid chondrosarcomas feature a distinct transcriptional pattern
D. Racanelli1, S. Stacchiotti2, M. Brenca1, M. Sbaraglia3, K. Fassetta1, D. Baldazzi1, S. Piccinin1, S. Brich4, P.G. Casali2–11, P. Collini5, G.P. Dagrada1–6, M. Fiore7, A. Gronchi7, A. Astolfi8, M.A. Pantaleo8, A. Righi9, S. Pilotti5, A.P. Dei Tos3–10, R. Maestro1 1 Unit of Oncogenetics and Functional Oncogenomics, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, National Cancer Institute, Aviano, Italy, 2Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 3 Department of Pathology, Treviso Regional Hospital, Treviso, Italy, 4Unit of Experimental Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 5Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 6Laboratory of Molecular Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 7Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, 8"Giorgio Prodi" Cancer Research Center, University of Bologna, Bologna, Italy, 9Anatomy and Pathological Histology, IRCCS, Istituto Ortopedico Rizzoli, Bologna, Italy, 10Department of Medicine, University of Padua School of Medicine, Padua, Italy, 11Oncology and Haemato-Oncology Department, University of Milan, Milan, Italy Background: Myoepithelial tumors of soft tissues (MT) and Extraskeletal Myxoid Chondrosarcoma (EMC) are closely related pathological entities whose overlapping features, in terms of morphology and immunoprofile, make differential diagnosis challenging. Different fusion genes have been described in MT. Instead, the rearrangement of NR4A3 is conventionally considered an exquisite feature of EMC. Nevertheless, whether there is a biological overlap between MT and EMC is still controversial. In order to shed light on this issue we compared the transcriptional profiles of the two entities. Methods: A series of EMC (12 cases) and MT (7 cases), retrieved from the pathology files, was selected for the study. The diagnosis was made according to the WHO classification. FISH analyses confirmed that all EMC harbored NR4A3 rearrangement (7 EWSR1-NR4A3 and 5 TAF15-NR4A3); 4 EWSR1 and 1 FUS rearrangement were detected in MT. No rearrangement was detected in 2 cases. RNA was extracted from FFPE specimens with tumor cellularity >70%. RNA-sequencing was carried out on an Illumina Hiseq1000 platform (average 70 million reads/sample). Diverse algorithms and bioinformatic suites were employed to identify fusion transcripts and functional annotation analysis. Results: RNA-seq analysis confirmed the rearrangements detected by FISH and identified one PTCH1-GLI1 fusion in a MT. Principal component analysis and unsupervised hierarchical clustering indicated that MT and EMC feature a distinct transcriptional profile. Functional annotation of the genes differentially expressed highlighted Hedgehog (HH) and WNT signaling as significantly enriched pathways in MT compared to EMC, with canonical GLI1 and WNT target genes upregulated in MT. Ectopic expression in cell models of the PTCH1-GLI1 chimeric transcript identified in the MT sample correlated with the induction of GLI1 target genes. Conclusions: This study corroborates the notion that MT and EMC represent two distinct biological entities, with MT featuring a distinctive activation of HH and WNT pathways. The PTCH1-GLI1 fusion represents one possible mechanism of HH pathway activation in MT. Legal entity responsible for the study: The authors. Funding: Fondazione AIRC per la Ricerca sul Cancro, CRO Intramural Grant, Italian Ministry of Health. Disclosure: P.G. Casali: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Deciphera; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eisai; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eli Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Nektar Therapeutics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Advenchen Laboratories; Research grant / Funding (institution): Amgen Dompe´; Research grant / Funding (institution): AROG Pharmaceuticals; Research grant / Funding (institution): Blueprint Medicines; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Epizyme Inc.; Research grant / Funding (institution): Glaxo; Research grant / Funding (institution): Karyopharm Pharmaceuticals; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): PharmaMar. A.P. Dei Tos: Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer. All other authors have declared no conflicts of interest.
1722P
In vivo efficacy and enhanced tumour accumulation of liposomal vinorelbine (TLC178) in human sarcoma xenograft mice model
W-N. Yu1, J-H. Tang1, Y-C. Tsai2, H-T. Wang2 Non-Clinical Department, Taiwan Liposome Company, Taipei, Taiwan, 2 Pharmacokinetic Department, Taiwan Liposome Company, Taipei, Taiwan
1
Background: Vinorelbine (VNB) is a member of vinca alkaloid medications and approved for the treatment of non-small cell lung cancer as well as off-label use for metastatic breast cancer over 20 years based upon FDA-approved indication. Several studies have suggested the potential of VNB in treating sarcomas. Liposomal VNB (TLC178) is a novel sustained release liposomal formulation. In the presented studies, TLC178 resulted in enhanced tumor accumulation and greater anti-tumor efficacy than non-liposomal VNB.
v704 | Sarcoma
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz283.053/5577429 by guest on 24 October 2019
is still unknown. Cell fusion is a physiological mechanism involved in several processes including myoblast differentiation and already described to be involved in aneuploidy generation and genomic instability. We thus hypothesized that cell fusion could participate to sarcoma oncogenesis by generating genomic instability. Methods: We established cell line of hybrids from spontaneous fusion of immortalized myoblasts. Tumor and metastasis development were evaluated upon grafting and whole genome and transcriptome sequencing with in-vitro mechanistic studies were performed to decipher phenotypes acquired by hybrids upon fusion. Results: In vitro hybrid cell lines show a more aggressive phenotype than parental cell lines by increasing proliferation and clonogenic capacities. Only hybrids form tumors upon grafting and may develop also metastasis. Aneuploidy triggered by cell fusion induces genome reorganization and in vivo tumor genome show alterations corresponding to those observed in human rhabdomyosarcoma. Indeed, 85% of hybrid tumors harbor DMD deletions as observed in sarcoma with myogenic origin. The study of this protein reveals that tumors lose the expression of Dp427 isoform which is the one reported as a tumor suppressor. This malignant transformation of hybrids is associated to transcriptomic remodeling toward a decrease of the expression of genes involved in muscle differentiation. Conclusions: To our knowledge, this is the first model able to reproduce the complex genomic of sarcoma. The detection of those hybrid cells in sarcoma tumor allowed us to push forward they are involved in sarcoma development and progression and their quantification in tumors or in circulating cells could thus establish an early prognostic marker. Finally, specific therapeutic targeting of this hybrids could represent new approach. Legal entity responsible for the study: Frederic Chibon. Funding: Association pour la Recherche contre le Cancer (ARC) Fondation pour la Recherche Me´dicale (FRM). Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology