Supplementary Material for: Upregulation of SSTR2 expression and radioligand binding of [18F]SiTATE in neuroendocrine tumour cells with combined inhibition of class I HDACs and LSD1.

Introduction: Peptide receptor radionuclide therapy (PRRT) is a highly effective, targeted treatment option in advanced neuroendocrine tumours (NETs). However, NET patients expressing low levels of Somatostatin receptor type (SSTR)2 do not benefit from this powerful tool. Recently, several preclinical studies have revealed that histone deacetylase (HDAC) inhibitors can upregulate the expression of SSTR2 and enhance somatostatin ligand binding to tumour cells. In this preclinical study, we explored the effects of single and combined treatment of NET cells with the class I HDAC inhibitor entinostat and the lysine-specific demethylase 1 (LSD1) inhibitor CC-90011, on cell viability, SSTR2 expression and radioligand binding. Methods: The human NET cell lines BON1, NCI-H727, and QGP1 were treated with entinostat, CC-90011 or a combination of both. Cell viability was measured with a cell viability assay. SSTR2 expression was assessed by quantitative PCR, Western blot analysis, and immunohistochemistry. [18F]SiTATE uptake was investigated by a radioligand binding assay. Results: Treatment of NET cells with entinostat, CC-90011 and especially with the combination of both reduced tumour cell viability and strongly induced SSTR2 expression resulting in potently enhanced radioligand binding of [18F]SiTATE. Conclusion: Combined inhibition of class I HDACs and LSD1 potently increases SSTR2 expression and consequently radioligand binding and might thus be a putative strategy to improve the outcome of PRRT therapy in patients with NETs.

引言：肽受体放射性核素治疗（PRRT）是晚期神经内分泌瘤（NETs）的高效靶向治疗方案。然而，生长抑素受体2型（SSTR2）低表达的NET患者无法从该治疗手段中获益。近期多项临床前研究表明，组蛋白去乙酰化酶（HDAC）抑制剂可上调SSTR2的表达，并增强生长抑素配体与肿瘤细胞的结合能力。本项临床前研究探究了I类HDAC抑制剂恩替诺特（entinostat）与赖氨酸特异性去甲基化酶1（LSD1）抑制剂CC-90011单药及联合给药，对NET细胞的细胞活力、SSTR2表达及放射性配体结合的影响。
方法：将人NET细胞系BON1、NCI-H727及QGP1分别用恩替诺特、CC-90011或二者联合进行处理。采用细胞活力检测实验测定细胞活力；通过定量PCR、蛋白质免疫印迹分析及免疫组织化学评估SSTR2的表达水平；利用放射性配体结合实验研究[18F]SiTATE的摄取情况。
结果：恩替诺特、CC-90011单药处理，尤其是二者联合处理，可降低NET肿瘤细胞的活力，并显著诱导SSTR2的表达，进而有效增强[18F]SiTATE的放射性配体结合能力。
结论：联合抑制I类HDAC与LSD1可强效上调SSTR2的表达，继而增强放射性配体结合能力，或可作为改善NET患者PRRT治疗预后的潜在策略。