Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
Laboratory of Biochemistry, Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Polyclinic, Palermo, Italy.
Bone. 2014 Mar;60:198-212. doi: 10.1016/j.bone.2013.12.021. Epub 2013 Dec 27.
Osteosarcoma is a highly metastatic tumor affecting adolescents, for which there is no second-line chemotherapy. As suggested for most tumors, its capability to overgrow is probably driven by cancer stem cells (CSCs), and finding new targets to kill CSCs may be critical for improving patient survival. TP53 is the most frequently mutated tumor suppressor gene in cancers and mutant p53 protein (mutp53) can acquire gain of function (GOF) strongly contributing to malignancy. Studies thus far have not shown p53-GOF in osteosarcoma. Here, we investigated TP53 gene status/role in 3AB-OS cells-a highly aggressive CSC line previously selected from human osteosarcoma MG63 cells-to evaluate its involvement in promoting proliferation, invasiveness, resistance to apoptosis and stemness. By RT-PCR, methylation-specific PCR, fluorescent in situ hybridization, DNA sequence, western blot and immunofluorescence analyses, we have shown that-in comparison with parental MG63 cells where TP53 gene is hypermethylated, rearranged and in single copy-in 3AB-OS cells, TP53 is unmethylated, rearranged and in multiple copies, and mutp53 (p53-R248W/P72R) is post-translationally modified and with nuclear localization. p53-R248W/P72R-knockdown by short-interfering RNA reduced the growth and replication rate of 3AB-OS cells, markedly increasing cell cycle inhibitor levels and sensitized 3AB-OS cells to TRAIL-induced apoptosis by DR5 up-regulation; moreover, it strongly decreased the levels of stemness and invasiveness genes. We have also found that the ectopic expression of p53-R248W/P72R in MG63 cells promoted cancer stem-like features, as high proliferation rate, sphere formation, clonogenic growth, high migration and invasive ability; furthermore, it strongly increased the levels of stemness proteins. Overall, the findings suggest the involvement of p53-R248W/P72R at the origin of the aberrant characters of the 3AB-OS cells with the hypothesis that its GOF can be at the root of the dedifferentiation of MG63 cells into CSCs.
骨肉瘤是一种影响青少年的高度转移性肿瘤，目前尚无二线化疗药物。正如大多数肿瘤所建议的那样，其过度生长的能力可能是由癌症干细胞（CSC）驱动的，寻找新的靶标来杀死 CSC 可能对提高患者生存率至关重要。TP53 是癌症中最常突变的肿瘤抑制基因，突变型 p53 蛋白（mutp53）可以获得强烈促进恶性肿瘤发生的功能获得（GOF）。迄今为止的研究并未显示骨肉瘤中存在 p53-GOF。在这里，我们研究了 3AB-OS 细胞中 TP53 基因的状态/作用，这是一种先前从人骨肉瘤 MG63 细胞中选择的高度侵袭性 CSC 系，以评估其在促进增殖、侵袭、抗凋亡和干性方面的作用。通过 RT-PCR、甲基化特异性 PCR、荧光原位杂交、DNA 序列、western blot 和免疫荧光分析，我们已经表明，与 TP53 基因超甲基化、重排和单拷贝的亲本 MG63 细胞相比，3AB-OS 细胞中的 TP53 基因无甲基化、重排和多个拷贝，mutp53（p53-R248W/P72R）发生了翻译后修饰并定位于核内。通过短干扰 RNA 敲低 p53-R248W/P72R 可降低 3AB-OS 细胞的生长和复制率，显著增加细胞周期抑制剂水平，并通过 DR5 上调增加 TRAIL 诱导的凋亡敏感性；此外，它还强烈降低了干性和侵袭性基因的水平。我们还发现，p53-R248W/P72R 在 MG63 细胞中的异位表达促进了癌症干细胞样特征，如高增殖率、球体形成、克隆形成、高迁移和侵袭能力；此外，它还强烈增加了干性蛋白的水平。总的来说，这些发现表明 p53-R248W/P72R 参与了 3AB-OS 细胞异常特征的起源，并假设其 GOF 可能是 MG63 细胞向 CSC 分化的根源。
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