野生型与突变型5-HT2B受体对卡麦角林疗效的影响及其机制研究

Cabergoline (CAB) is the first-line treatment of prolactinomas, however, the mechanism of pharmacotherapeutic resistance observed in approximately 10% of the patients is largely unknown. Our previous studies demonstrated that CAB, in addition to activating the Dopamine receptor D2 (D2DR), also activated the 5-hydroxytryptamine receptor 2B (5-HT2B). Activation of 5-HT2B receptor could promote the cell proliferation; and inhibition of 5-HT2B receptor could induce the cell death in vivo and in vitro. Furthermore, expression of 5-HT2B in resistant prolactinomas was higher than that in sensitive prolactinomas. 5-HT2B knockdown could enhanced sensitivity of prolactinomas to CAB. Therefore, we hypothesize that 5-HT2B is involved in the resistance of prolactinomas to CAB treatment, and inhibition of 5-HT2B receptors may enhance the efficacy of CAB. To investigate the effect and novel mechanism of 5-HT2B in CAB treatment, we will utilize molecular docking to simulate CAB binding with 5-HT2B, and site-directed mutagenesis in 5-HT2B to analyze the effect of CAB to wild type or mutant 5-HT2B, and investigate the mechanism underlying the inhibition of autophagy in response to 5-HT2B receptor activation. This study will provide critical information for clinical applications.

卡麦角林（CAB）为垂体泌乳素瘤的首选治疗，但约10%病例存在耐药性和副作用。我们前期研究发现：CAB除了能激动多巴胺2型受体（D2DR）起治疗作用之外，还激动5-HT2B受体对肿瘤细胞起保护作用，同时5-HT2B受体在耐药泌乳素瘤中往往呈高表达，敲低5-HT2B表达能显著增敏卡麦角林的疗效。由此我们提出初步设想：5-HT2B受体激活导致CAB治疗的耐药性，阻断5-HT2B受体与卡麦角林（配体）结合，有望增敏CAB疗效。为了进一步证实上述推测，本课题拟通过分子对接模拟分析，结合结构生物学、细胞生物学分析，进行不同5-HT2B受体状态（野生型和突变型）泌乳素瘤对CAB敏感性差异研究，并初步探讨5-HT2B抑制自噬参与耐药的机制；最后通过临床敏感和耐药标本分析，明确5-HT2B作为个体化药物治疗的生物标记物，从而为泌乳素瘤的药物治疗开辟新思路。

垂体泌乳素腺瘤首选药物如卡麦角林（CAB）治疗。此外，卡麦角林还用于尝试治疗垂体无功能腺瘤、生长激素腺瘤和ACTH腺瘤。研究发现CAB除了能激动多巴胺2型受体（DRD2）起治疗作用之外，还能激动五羟色胺2B型受体（HTR2B），但其在垂体腺瘤中的功能尚未研究。通过RNA-seq，免疫组织化学染色及PCR方法，发现HTR2B在各肿瘤中表达。通过肿瘤组织与正常垂体比较，发现HTR2B在各亚型垂体腺瘤的表达明显高于正常垂体。大鼠GH3和MMQ细胞株免疫荧光显示HTR2B主要表达在细胞膜，且与DRD2共定位。功能研究上，首先在体内外水平都发现干预HTR2B的功能抑制肿瘤生长，激活HTR2B则促进肿瘤生长；其次在体内外水平及肿瘤原代细胞培养，发现HTR2B抑制剂SB204741能协同CAB抑制肿瘤增殖。我们通过分析CAB与CAB+SB204741两组对比的RNA-seq数据，发现HTR2B通过正向调控ERK信号通路。干预HTR2B表达显著抑制GH3和MMQ细胞的ERK磷酸化，过表达HTR2B则促进ERK磷酸化。另外CAB在HTR2B干预情况下无法激活ERK磷酸化。抑制ERK信号通路减少CAB和SB204741联合应用导致细胞的凋亡和坏死。综上所述，CAB与SB204741联合应用，通过调控ERK信号通路影响垂体瘤自噬水平，导致细胞死亡。上述数据表明，联合HTR2B拮抗剂协同CAB可能为治疗垂体瘤的新策略。