p53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.肾上腺皮质癌（Adrenocortical carcinoma, ACC）是一类极为罕见的内分泌肿瘤，预后异质性较强，具体取决于肿瘤分期与诊断时机。总体而言该病恶性程度极高，确诊后总生存期仅约5年。放疗在ACC治疗中的应用价值长期存在广泛争议，且其疗效似乎与肿瘤分子改变密切相关，此类改变会进一步诱导肿瘤产生放射抵抗。多项研究表明，p53缺失是恶性肾上腺皮质肿瘤发生的重要风险因素，且有报道称，27%~70%的成人散发性ACC病例存在TP53基因体细胞突变。 本研究探讨了TP53基因体细胞突变在肾上腺皮质癌应对电离辐射（ionizing radiation, IR）过程中的作用。我们针对两种肾上腺皮质细胞系H295R与SW-13开展了p53状态分析：二者分别因第8、9号外显子缺失及第6号外显子存在点突变，导致p53蛋白呈现无功能形式；此外，这两种细胞系均高表达磷酸化Akt（p-Akt）与胰岛素样生长因子2（IGF2），其中H295R的表达水平尤为突出。 我们观察到，通过向细胞中转染野生型p53以恢复p53活性后，可显著抑制细胞增殖。我们通过细胞计数与TUNEL检测法，评估了两种细胞系对电离辐射的细胞增殖与活力响应，结果显示，野生型p53过表达还可增强两种细胞系经辐射诱导的细胞凋亡死亡。此外，对BCL2、IGF2及Akt等p53靶基因进行逆转录聚合酶链反应（RT-PCR）与蛋白质印迹法（Western blotting）分析后发现，电离辐射诱导的p53激活可下调IGF2的表达，这一变化同时伴随Akt活性形式水平的降低。 综上，本研究结果阐明了p53在肾上腺皮质癌细胞系应对电离辐射过程中的作用，提示其可作为恶性肾上腺皮质肿瘤患者放疗疗效的潜在预测因子。