Supplementary Material for: Resistance to Fas-Mediated Apoptosis Does Not Correlate to Structural Alterations or Expression Changes of the Death Receptor in Papillary Thyroid Carcinomas

<b><i>Background:</i></b> Malignant cells exhibit significant resistance to FAS-mediated cell death, through different processes, including <i>FAS</i> mutations, soluble <i>FAS</i> expression, or FAS transcriptional dysregulation by P53, eventually escaping from immune surveillance. Since thyroid carcinomas were shown to be resistant to FAS-mediated apoptosis, we investigated the above mechanisms in thyroid carcinoma samples. <b><i>Methods:</i></b> Thirty-seven thyroid carcinoma samples were analyzed for mutations in <i>FAS</i> exon 9 and <i>TP53</i> exons 5–8 and protein expression by means of immunohistochemistry. Moreover, thyroid carcinoma mRNA samples were subjected to reverse transcription – PCR, to evaluate the relative expression of transmembrane FAS versus its soluble form. <b><i>Results:</i></b> Analysis revealed indications for <i>TP53</i> mutations in the anaplastic carcinomas, but not in the other thyroid specimens examined for <i>TP53</i> or <i>FAS</i> exon 9 mutations. FAS receptor expression was observed in almost all thyroid specimens (97%) with significant up-regulation in papillary carcinomas. P53 nuclear staining was observed only in anaplastic carcinomas. Full-length <i>FAS</i> mRNA was detected in all specimens examined, with soluble <i>FAS</i> mRNA being either absent or present in very low amounts. <b><i>Conclusions:</i></b> Our results denote that <i>FAS</i> death domain or <i>TP53</i> DNA-binding domain mutations, down-regulation of FAS receptor expression, or expression of <i>FAS</i> soluble isoform are not responsible for the seeming inhibition of FAS-mediated apoptosis in papillary thyroid carcinoma cells.

<b><i>背景：</i></b> 恶性细胞可通过多种途径对FAS（FAS）介导的细胞死亡产生显著抗性，具体机制包括FAS基因突变、可溶性FAS表达，或P53介导的FAS转录失调，最终使肿瘤细胞逃避免疫监视。鉴于已有研究证实甲状腺癌对FAS介导的细胞凋亡具有抗性，本研究针对甲状腺癌样本展开了上述相关机制的探究。<b><i>方法：</i></b> 本研究采用免疫组织化学法（immunohistochemistry），对37例甲状腺癌样本的FAS第9外显子、TP53第5至8外显子的突变情况，以及蛋白表达水平进行检测。此外，针对甲状腺癌mRNA样本实施逆转录-聚合酶链式反应（reverse transcription-PCR），以评估跨膜型FAS与其可溶性亚型的相对表达量。<b><i>结果：</i></b> 分析结果显示，间变性甲状腺癌中存在TP53突变的相关迹象，但在其余接受TP53或FAS第9外显子突变检测的甲状腺样本中未发现此类突变。几乎所有受试甲状腺样本（97%）均检测到FAS受体表达，其中乳头状甲状腺癌的FAS受体表达显著上调。仅在间变性甲状腺癌中观察到P53核染色。所有受试样本均检出全长FAS mRNA，而可溶性FAS mRNA则几乎未检出或仅呈极低水平表达。<b><i>结论：</i></b> 本研究结果表明，FAS死亡结构域或TP53 DNA结合结构域突变、FAS受体表达下调，或可溶性FAS亚型的表达，均并非乳头状甲状腺癌细胞中FAS介导的细胞凋亡看似受到抑制的原因。