Supplementary Material for: Modulation of Pancreatic Neuroendocrine Neoplastic Cell Fate by Autophagy-Mediated Death

Introduction: Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. Methods: 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence, and Western blot. Autophagosomes were detected by electron microscopy and their maturation by real-time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. Results: We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. However, the binding activity of the cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosome synthesis, maturation, and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol and the nucleus, leading to disruption of the organelles, cellular digestion, and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN, and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. Discussion/Conclusion: Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.

引言：癌细胞的自噬性细胞死亡可通过抑制脱乙酰酶（deacetylases）介导。尽管学界已针对癌症中的自噬过程开展了大量研究，但对于自噬在降解胰腺神经内分泌肿瘤（pancreatic neuroendocrine neoplastic, pNEN）细胞的胞质与核组分并进而引发细胞死亡、从而改善pNEN患者治疗效果的作用，目前仍知之甚少。
方法：采用帕比司他（panobinostat）与巴弗洛霉素（bafilomycin）处理二维（2D）及三维（3D）培养的人源pNEN细胞与胰腺星状细胞。通过实时定量聚合酶链式反应（RT-qPCR）、免疫荧光法与蛋白质印迹法（Western blot）检测自噬标志物；通过电子显微镜观测自噬体，并借助稳定转染LC3B的细胞的实时荧光监测自噬体成熟进程。在环腺苷酸应答元件（cAMP responsive element）位点开展染色质免疫沉淀（ChIP）实验；在小鼠胰腺组织中进行免疫荧光检测。
结果：本研究观察到，泛脱乙酰酶抑制剂帕比司他可诱导pNEN细胞发生自噬性细胞死亡。同时发现，尽管AMPK-α的磷酸化可被磷酸化AKT所抵消，但这一抵消作用并不能抑制自噬性细胞死亡。此外，帕比司他可增强环腺苷酸应答元件的结合活性。虽然自噬抑制可阻断自噬体的合成、成熟与细胞死亡过程，但帕比司他处理可诱导成熟自噬体在胞质与细胞核内聚集，进而引发细胞器破坏、细胞消化与降解。对小鼠胰岛中自噬体膜蛋白Beclin1与LC3B聚集情况的观测表明，自噬功能恢复也可诱导小鼠胰岛素瘤细胞中的自噬体聚集。在pNEN患者体内可检测到自噬标志物的基础低表达；值得注意的是，转移性pNEN患者的此类标志物表达水平显著更低。
讨论/结论：本研究证实，通过抑制脱乙酰酶介导的自噬功能恢复与该分解代谢过程的延长，可实现pNEN细胞数量的减少。诱导自噬性细胞死亡有望成为pNEN治疗的潜在有效策略。