Supplementary Material for: Statins Directly Regulate Pituitary Cell Function and Exert Antitumor Effects in Pituitary Tumors

<b><i>Introduction:</i></b> Pituitary neuroendocrine tumors (PitNETs), the most abundant of all intracranial tumors, entail severe comorbidities. First-line therapy is transsphenoidal surgery, but subsequent pharmacological therapy is often required. Unfortunately, many patients are/become unresponsive to available drugs (somatostatin analogues [SSAs]/dopamine agonists), underscoring the need for new therapies. Statins are well-known drugs commonly prescribed to treat hyperlipidemia/cardiovascular diseases, but can convey additional beneficial effects, including antitumor actions. The direct effects of statins on normal human pituitary or PitNETs are poorly known. Thus, we aimed to explore the direct effects of statins, especially simvastatin, on key functional parameters in normal and tumoral pituitary cells, and to evaluate the combined effects of simvastatin with metformin (MF) or SSAs. <b><i>Methods:</i></b> Effects of statins in cell proliferation/viability, hormone secretion, and signaling pathways were evaluated in normal pituitary cells from a primate model (<i>Papio anubis</i>), tumor cells from corticotropinomas, somatotropinomas, nonfunctioning pituitary tumors, and PitNET cell-lines (AtT20/GH3-cells). <b><i>Results:</i></b> All statins decreased AtT20-cell proliferation, simvastatin showing stronger effects. Indeed, simvastatin reduced cell viability and/or hormone secretion in all PitNETs subtypes and cell-lines, and ACTH/GH/PRL/FSH/LH secretion (but not expression), in primate cell cultures, by modulating MAPK/PI3K/mTOR pathways and expression of key receptors (GH-releasing hormone-receptor/ghrelin-R/Kiss1-R) regulating pituitary function. Addition of MF or SSAs did not enhance simvastatin antitumor effects. <b><i>Conclusion:</i></b> Our data reveal direct antitumor effects of simvastatin on PitNET-cells, paving the way to explore these compounds as a possible tool to treat PitNETs.

<b><i>引言：</i></b> 垂体神经内分泌瘤（Pituitary neuroendocrine tumors, PitNETs）是颅内最为常见的肿瘤类型，可引发严重的合并症。其一线治疗方案为经蝶窦手术，但后续往往需要辅以药物治疗。遗憾的是，多数患者对现有治疗药物——生长抑素类似物（somatostatin analogues, SSAs）/多巴胺受体激动剂——产生耐药性，凸显了开发新型治疗手段的迫切需求。他汀类药物（statins）是临床广泛用于治疗高脂血症与心血管疾病的经典药物，同时还具备包括抗肿瘤在内的多种额外获益。目前学界对他汀类药物在正常人体垂体或PitNETs中的直接作用尚缺乏深入认知。因此，本研究旨在探究他汀类药物，尤其是辛伐他汀（simvastatin），对正常及肿瘤性垂体细胞关键功能参数的直接调控作用，并评估辛伐他汀与二甲双胍（metformin, MF）或SSAs联合使用的效果。
<b><i>方法：</i></b> 本研究以橄榄狒狒（*Papio anubis*）正常垂体细胞、促肾上腺皮质激素瘤、生长激素瘤、无功能垂体瘤细胞以及PitNET细胞系（AtT20/GH3细胞）为实验模型，评估他汀类药物对细胞增殖活力、激素分泌及信号通路的影响。
<b><i>结果：</i></b> 所有受试他汀类药物均可抑制AtT20细胞增殖，其中辛伐他汀的抗肿瘤活性最为显著。具体而言，辛伐他汀可降低所有PitNET亚型及细胞系的细胞活力与/或激素分泌水平；同时，通过调控MAPK/PI3K/mTOR信号通路以及调节垂体功能关键受体——生长激素释放激素受体（GH-releasing hormone-receptor, GHRH-R）/饥饿素受体（ghrelin-R）/Kiss1受体（Kiss1-R）——的表达，辛伐他汀可在橄榄狒狒原代细胞培养体系中抑制促肾上腺皮质激素（ACTH）、生长激素（GH）、催乳素（PRL）、卵泡刺激素（FSH）及黄体生成素（LH）的分泌（但不影响其基因表达）。联合使用二甲双胍或SSAs并未增强辛伐他汀的抗肿瘤效果。
<b><i>结论：</i></b> 本研究数据揭示了辛伐他汀对PitNET细胞的直接抗肿瘤作用，为将此类化合物开发为PitNETs潜在治疗手段提供了实验依据与研究方向。