Supplementary Material for: 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 143B). 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.

研究目的：近期研究表明，细胞自噬（autophagy）在肿瘤发生、复发、转移及化疗耐药中发挥关键作用。p62是参与细胞自噬通路的重要衔接蛋白（adapter protein）。本研究旨在分析p62在骨肉瘤（osteosarcoma）中的表达情况，及其与骨肉瘤患者临床预后的相关性。

研究方法：采用蛋白质印迹法（Western blot）检测骨肉瘤细胞系（U2OS、KHOS、MG63、Saos-2、U2OSR2、KHOSR2及143B）中p62的表达水平。通过免疫组织化学法（immunohistochemistry）分析组织微阵列（tissue microarray, TMA），以检测骨肉瘤患者组织中p62的表达水平，并评估p62表达与骨肉瘤患者临床特征的相关性。

研究结果：所有受试细胞系中p62的表达均存在显著差异。组织微阵列检测结果亦显示，骨肉瘤组织中p62的表达存在异质性。79例患者组织中有75例（94.9%）可检测到p62免疫染色，染色强度范围从无染色（4/97，5.1%）至1+染色（40/79，50.6%）、2+染色（17/79，21.5%）及3+染色（18/79，22.8%）。其中，1+染色被归为p62低表达组（50.6%），2+染色与3+染色则被归为p62高表达组（44.3%）。对骨肉瘤组织微阵列的临床数据进行分析后发现，p62低表达患者的5年生存率显著低于p62高表达患者（p=0.0165）。此外，p62表达下调可能与骨肉瘤患者更高的转移率及化疗耐药率相关。

研究结论：本研究结果表明，p62可作为骨肉瘤患者预后的有效预测指标及潜在治疗靶点。