PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor-positive breast cancer

Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors and selective PI3Kα inhibitors are in clinical development. The activity of these agents, however, is not homogenous and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling with different agents results in induction of ER-dependent transcriptional activity as demonstrated by changes in expression in genes containing ER binding sites, enhanced ER transcription and increased occupancy by the ER of promoter regions of upregulated genes. Furthermore, expression of ESR1 mRNA and ER protein levels themselves were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenograft and patient derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition. We propose that increased ER transcriptional activity may be a compensatory mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors. The aim of our study was to explore the mechanism by which combination of PI3K pathway inhibitors and estrogen receptor function blockade results in superior antitumor activity. We aimed to evaluate whether changes in ER function were influencing the clinical response to anti-PI3K therapy in ER-positive breast tumors that harbor PI3K pathway activation. For this purpose, we planned to use various specific PI3K inhibitors, namely: BYL719 (p110α specific catalytic inhibitor), GDC0941 (pan-PI3K inhibitor), GDC0032 and BAY80-6946 (p110sparing PI3K inhibitors) in a panel of ER-positive breast cancer cell lines and xenografts that harbor PIK3CA activating mutations. We also used MK2206 (pan-AKT allosteric inhibitor) to inhibit the PI3K pathway in ER-positive cell lines which activate this pathway through PTEN loss. Finally, in order to evaluate the role of ER up-regulation as a pro-survival signal in our in vitro and in vivo models, we planned to use the selective ER modulator 4-hydroxy-tamoxifen (4-OHT) and degrader fulvestrant. For the in vivo experiments, the number of animals in each group was calculated to measure a 25% difference between the means of placebo and treatment groups with a power of 80% and a p value of 0.01. Host mice carrying xenografts were randomly and equally assigned to either control or treatment groups. Animal experiments were conducted in a controlled and non-blinded manner. Moreover, we evaluated by means of RNAseq gene expression changes breast cancer patients that underwent BYL719 based therapy to validate our in vitro findings in terms of ER expression. In vitro experiments were performed at least two times and at least in triplicate for each replica.

PIK3CA的激活突变是雌激素受体（ER, estrogen receptor）阳性乳腺肿瘤中最为常见的基因组改变，选择性PI3Kα抑制剂目前正处于临床研发阶段。然而此类药物的抗肿瘤活性并不均一，仅少数携带PIK3CA突变的ER阳性乳腺肿瘤患者可从单药治疗中获益。在探寻耐药机制的过程中，我们观察到：通过不同药物抑制PI3K信号通路后，可诱导ER依赖的转录活性，该效应可通过以下现象得以证实：携带ER结合位点的基因表达发生改变、ER转录活性增强，以及ER在上调基因的启动子区域的结合占有率升高。此外，经PI3K抑制处理后，ESR1 mRNA的表达水平与ER蛋白本身的表达量也均有所升高。上述基因表达变化在异种移植模型（xenograft）、患者来源模型（patient derived models）以及接受PI3Kα抑制剂BYL719治疗的患者肿瘤组织中均得到了体内验证。添加雌二醇可增强上述转录调控效应，而抗ER治疗药物氟维司群（fulvestrant）与他莫昔芬（tamoxifen）则可抑制该效应。其中氟维司群可显著增强ER阳性肿瘤对PI3Kα抑制剂的敏感性。我们提出假说：ER转录活性升高或许是一种代偿机制，会限制PI3K抑制剂的抗肿瘤活性；因此联合抑制PI3K与ER通路，是靶向这类肿瘤的合理治疗策略。本研究旨在探究PI3K通路抑制剂与雌激素受体功能阻断剂联合使用时，可产生更强抗肿瘤活性的内在机制。我们还旨在评估：在携带PI3K通路激活的ER阳性乳腺肿瘤中，ER功能的变化是否会影响患者对抗PI3K治疗的临床应答。为此，我们计划在一系列携带PIK3CA激活突变的ER阳性乳腺癌细胞系及异种移植模型中，使用多种特异性PI3K抑制剂，具体包括：BYL719（p110α特异性催化抑制剂（p110α specific catalytic inhibitor））、GDC0941（泛PI3K抑制剂（pan-PI3K inhibitor））、GDC0032与BAY80-6946（对p110β选择性无活性的PI3K抑制剂（p110β-sparing PI3K inhibitors））。此外，我们还使用MK2206（泛AKT变构抑制剂（pan-AKT allosteric inhibitor）），在通过PTEN缺失激活PI3K通路的ER阳性细胞系中抑制该通路。最后，为了评估ER上调作为促存活信号在我们的体外与体内模型中的作用，我们计划使用选择性ER调节剂（selective ER modulator）4-羟基他莫昔芬（4-OHT, 4-hydroxy-tamoxifen）以及ER降解剂氟维司群。针对体内实验，我们通过功效分析计算了每组动物的样本量，以检测安慰剂组与治疗组之间25%的均值差异，检验效能设定为80%，显著性p值为0.01。携带异种移植瘤的宿主小鼠被随机均分至对照组与治疗组。动物实验在受控条件下开展，且未采用盲法设计。此外，我们通过RNA测序（RNAseq）分析了接受BYL719为基础的治疗的乳腺癌患者的基因表达变化，以验证我们体外研究中关于ER表达的发现。体外实验至少重复两次，且每次重复实验均设置至少三次平行样本。