Abstract 5893: Expression and role of autophagy-associated p62 (SQSTM1) in multidrug-resistant ovarian cancer

Abstract Multidrug resistance is the major cause of treatment failure in ovarian cancer. p62 (SQSTM1) is a multifunctional protein involved in multiple cellular processes including proliferation, drug sensitivity and autophagy-associated cancer cell growth. p62 is a critical indicator of autophagic flux, which is inversely associated with autophagy activity. However, the role of p62 remains controversial in drug resistance in human ovarian cancer. In this study, we examined p62 expression by immunohistochemistry in a unique ovarian cancer tissue microarray (TMA), which was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. Results showed that both the metastatic and recurrent tumor tissues expressed less p62 than the patient-matched primary tumor. A significant inverse correlation has been found between p62 expression and both the disease free survival and overall survival. In addition, multidrug resistant cancer cell lines expressed lower levels of p62 as compared with their parental drug sensitive cell lines. Importantly, cell viabilities determined by MTT assay after exposure to different concentrations of paclitaxel showed inhibition of autophagy or accumulation of p62 enhances paclitaxel sensitivity in ovarian cancer drug resistant cells. Furthermore, the wound healing assay exhibited that inhibition of autophagy significantly decreased multidrug resistant ovarian cancer cell migration in vitro. Collectively, these data highlight that autophagy pathway may be a promising therapeutic target to prevent metastasis, recurrence and to reverse drug resistance in ovarian cancer. Citation Format: Jinglu Wang, Cassandra Garbutt, Francis J Hornicek, Zhenfeng Duan. Expression and role of autophagy-associated p62 (SQSTM1) in multidrug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5893

Abstract 2315: Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma

Abstract Synovial sarcoma is a rare and aggressive form of soft tissue cancer that affects the extremities of the arms or legs, for which current chemotherapeutic agents have not been proven to be very effective. The cyclin-dependent kinase 4/6-retinoblastoma protein (CDK4/6-Rb) pathway is aberrant in a large proportion of cancer. Recent evidences on pre-clinical application of CDK4 inhibitors have been implicated in many types of human cancers, and the FDA has approved the CDK4 selective inhibitor for the treatment of breast cancer. However, the expression and therapeutic potential of CDK4 in synovial sarcoma remain unclear. In the present study, we examined the expression of CDK4 in synovial sarcoma cell lines by western blot and immunofluorescence assay, and in synovial sarcoma tissue microassays by immunohistochemical analysis. Cell viabilities were determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor. Flow cytometry analysis and wound healing assay were conducted to determine the mechanisms underlying the cytotoxic effects of the selective CDK4 inhibitor. CDK4 specific small interference RNA was used to validate the effect of targeting CDK4 by the selective CDK4 inhibitor in synovial sarcoma cells. We found that CDK4 was highly expressed in human synovial sarcoma, and was related to clinical stage and TNM grade in synovial sarcoma patients and poor prognosis in sarcoma patients. Cell viabilities determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor showed that this selective CDK4 inhibitor repressed synovial sarcoma cell proliferation and growth in a dose- and time- dependent manner. The selective CDK4 inhibitor inhibited the CDK4/6-Rb signaling pathway and promoted cell apoptosis without influence on the expression of CDK4/6, suggesting that the selective CDK4 inhibitor only repressed the hyperactivation, not the production of CDK4/6. The inhibition effect of the selective CDK4 inhibitor was confirmed by knockdown of CDK4 with specific small interference RNA. Flow cytometry analysis revealed that the selective CDK4 inhibitor induced G1 cell-cycle arrest by targeting CDK4/6-Rb pathway in synovial sarcoma cells. Furthermore, the wound healing assay exhibited that inhibition of CDK4/6-Rb pathway with the use of the selective CDK4 inhibitor significantly decreased synovial sarcoma cell migration in vitro. Our data highlight the role of dysregulated CDK4/6-Rb pathway and current selective CDK4/6 inhibitor may be a potential promising therapeutic agent in the targeted treatment of human synovial sarcoma. Citation Format: Xiaoyang Li, Cassandra Garbutt, Francis John Hornicek, Zhenfeng Duan. Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2315

Potentials of Long Noncoding RNAs (LncRNAs) in Sarcoma: From Biomarkers to Therapeutic Targets

Sarcoma includes some of the most heterogeneous tumors, which make the diagnosis, prognosis and treatment of these rare yet diverse neoplasms especially challenging. Long noncoding RNAs (lncRNAs) are important regulators of cancer initiation and progression, which implies their potential as neoteric prognostic and diagnostic markers in cancer, including sarcoma. A relationship between lncRNAs and sarcoma pathogenesis and progression is emerging. Recent studies demonstrate that lncRNAs influence sarcoma cell proliferation, metastasis, and drug resistance. Additionally, lncRNA expression profiles are predictive of sarcoma prognosis. In this review, we summarize contemporary advances in the research of lncRNA biogenesis and functions in sarcoma. We also highlight the potential for lncRNAs to become innovative diagnostic and prognostic biomarkers as well as therapeutic targets in sarcoma

Advances in chromosomal translocations and fusion genes in sarcomas and potential therapeutic applications

•Chromosomal translocations and fusion genes are especially common in sarcomas.•Novel chromosomal translocations and fusion genes have been discovered in sarcomas.•Fusion genes and affected downstream signaling pathway provide therapeutic targets.•Novel therapeutic targets show promise in future treatment of sarcomas. Chromosomal translocations and fusion genes are very common in human cancer especially in subtypes of sarcomas, such as rhabdomyosarcoma, Ewing's sarcoma, synovial sarcoma and liposarcoma. The discovery of novel chromosomal translocations and fusion genes in different tumors are due to the advancement of next-generation sequencing (NGS) technologies such as whole genome sequencing. Recently, many novel chromosomal translocations and gene fusions have been identified in different types of sarcoma through NGS approaches. In addition to previously known sarcoma fusion genes, these novel specific fusion genes and associated molecular events represent important targets for novel therapeutic approaches in the treatment of sarcomas. This review focuses on recent advances in chromosomal translocations and fusion genes in sarcomas and their potential therapeutic applications in the treatment of sarcomas

Aberration of p73 Promoter Methylation in Chondrosarcoma

p73 is a tumor-suppressor gene with significant homology to p53. Abnormal promoter methylation of p73 is present in different types of cancer. However, the promoter methylation status of p73 in chondrosarcoma (CS) is unknown. p73 promoter methylation status was evaluated by quantitative polymerase chain reaction (PCR), p73 protein expression by western blot, and the relationship between p73 methylation and clinical data was analyzed. In 42 tumor tissues with CS, we found that three cases (7%) maintained methylation levels between 51% and 75%, and 39 cases (93%) had levels between 76% and 100%. p73 methylation level was significantly (p<0.05) positively associated with histological grade. Loss of p73 protein expression was correlated with high methylation of the p73 promoter; p73 expression was restored after exposure to a demethylating drug. p73 is epigenetically silenced in CS due to promoter methylation, which suggests the utility of p73 methylation as a biomarker

Expression and Therapeutic Potential of SOX9 in Chordoma

Purpose: Conventional chemotherapeutic agents are ineffective in the treatment of chordoma. We investigated the functional roles and therapeutic relevance of the sex-determining region Y (SRY)-box 9 (SOX9) in chordoma.Experimental Design: SOX9 expression was examined by immunohistochemistry (IHC) using 50 chordoma tissue samples. SOX9 expression in chordoma cell lines was examined by Western blot and immunofluorescent assays. We used synthetic human SOX9 siRNA to inhibit the expression of SOX9. Cell proliferation ability and cytotoxicity of inhibiting SOX9 were assessed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) and clonogenic assays. The effect of SOX9 knockdown on chordoma cell motility was evaluated by a wound-healing assay and a Transwell invasion chamber assay. Knockdown of SOX9 induced apoptosis, cell-cycle arrest, as well as decreased expression of cancer stem cell markers were determined by Western blot and flow cytometric assays. The effect of the combination of SOX9 siRNA and the chemotherapeutic drug doxorubicin/cisplatin on chordoma cells was assessed by an MTT assay.Results: Tissue microarray and IHC analysis showed that SOX9 is broadly expressed in chordomas and that higher expression levels of SOX9 correlated with a poor prognosis. RNA interference (RNAi)-mediated knockdown of SOX9 inhibited chordoma cell growth, decreased cell motility, and induced apoptosis as well as cell-cycle arrest. Moreover, the combination of SOX9 inhibition and chemotherapeutic drugs had an enhanced anti-cancer effect on chordoma cells.Conclusions: Our results demonstrate that SOX9 plays a crucial role in chordoma. Targeting SOX9 provides a new rationale for treatment of chordoma. Clin Cancer Res; 23(17); 5176-86. ©2017 AACR

Expression and Clinical Implication of Autophagy-Associated Protein p62 in Osteosarcoma

Purpose: Recent studies highlight the role of autophagy in cancer tumorigenesis, recurrence, metastasis, and chemoresistance. p62 is an adapter protein that is crucial for the autophagy pathway. In this study, we will describe the expression of p62 and its correlation with clinic prognosis in osteosarcoma. Methods: Western blot was used to test the expression of p62 in osteosarcoma cell lines (U2OS, KHOS, MG63, Saos-2, U2OSR2, KHOSR2, and 1438). A tissue microarray (TMA) was analyzed by immunohistochemistry to determine the expression levels of p62 in osteosarcoma patients and evaluate any correlation between p62 and clinical characteristics in osteosarcoma patients. Results: p62 was expressed differently in all cell lines. The TMA also showed differential expression in osteosarcoma tissues. Seventy-five of 79 (94.9%) patient tissues exhibited p62 immunostaining, ranging from no staining (4 of 97, 5.1%) to 1+ staining (40 of 79, 50.6%), 2+ staining (17 of 79, 21.5%), and 3+ staining (18 of 79, 22.8%). The low staining (1+) was classified as the p62 weak group (50.6%), the medium staining (2+) and intense staining (3+) were classified as the p62 strong group (44.3%). Analyzing the clinical data of the osteosarcoma TMA, we found that the 5-year survival rate of patients with weak p62 expression was significantly lower than that of the patients with strong p62 expression (p = 0.0165). Furthermore, the decreased p62 expression may be associated with higher metastatic and chemoresistant rates in osteosarcoma patients. Conclusion: Our results suggest that p62 may be an effective predictor of prognosis and a potential target for therapy in osteosarcoma. (C) 2018 S. Karger AG, Base