Targeting cell cycle and metabolic pathways of high risk breast cancers using mouse models of hyperinsulinemia

项目概要/摘要 控制肿瘤复发和扩散是乳腺癌的主要挑战。这种情况进一步加剧 对于患有糖尿病的女性来说，大量流行病学研究表明，患有 2 型糖尿病的女性处于 与患有乳腺癌的女性相比，罹患乳腺癌、复发乳腺癌和死于乳腺癌的风险显着增加 不是糖尿病患者。研究表明，与 2 型糖尿病相关的高胰岛素血症是导致 2 型糖尿病的重要因素。 乳腺癌患者死亡率的一个因素，表明迫切需要有效的治疗方法 在高胰岛素条件下抑制肿瘤细胞增殖。我们的早期研究表明 cdk4 敲除 ERBB2 或 RAS 癌基因驱动的小鼠未能患上乳腺癌，这表明 CDK4/6 抑制剂可能是某些乳腺癌的有效治疗剂。 CDK4/6抑制剂获批 乳腺癌治疗验证了这一理论。另一种在胰岛素信号传导中发挥关键作用的激酶 癌症进展是NUAK1/ARK5，属于调节代谢的AMPK基因家族。 研究了 NUAK1/ARK5 在整个生物体中发挥的正常生理作用 肌肉特异性基因敲除小鼠显示 NUAK1 通过以下方式控制葡萄糖代谢： 胰岛素信号传导的调节。因此，当这些基因敲除小鼠被喂食高脂肪饮食时，它们 表现出较低的空腹血糖水平、较高的葡萄糖耐量、较高的胰岛素敏感性，以及 与对照小鼠相比，肌糖原浓度较高，表明抑制 ARK5/NUAK1可以克服高血糖的影响。有趣的是，ARK5最初被识别为 作为转移基因，其过度表达已被证明可促进多种肿瘤的转移 类型。为了达到抑制乳腺肿瘤细胞生长和代谢的目标，我们开发了一种强效的双重 CDK4 和 ARK5 抑制剂 (ON123300)，是一种非常有效的乳腺肿瘤生长抑制剂。在 此外，这种化合物对高糖饮食诱导的肿瘤发展具有深远的影响， 果蝇模型系统中的转移。在此应用中，我们建议将这些研究扩展到小鼠模型 LeRoith 博士开发的高胰岛素血症（MKR 小鼠）和三阴性乳腺癌 PDX 模型 （TNBC）由入江博士开发。目的是：（1）测试ON123300对生长和转移的影响 MKR 小鼠乳腺肿瘤表现出高胰岛素血症、糖尿病前期表型； (2) 检查 高胰岛素血症对 PDX 乳腺肿瘤生长和转移的影响并利用模型 ARK5的最高相对水平来检验ON123300的治疗价值； (3) 使用新的 开发了多重激酶抑制剂珠（MIB）和“癌症工具包功能获得”（CTK）技术 确定乳腺癌细胞在长期接触 ON123300 后是否会对 ON123300 产生耐药性，并 确定可能是这种耐药性根本原因的信号通路的性质。

Project Summary/Abstract Managing tumor recurrence and spread is a major challenge in breast cancer. This scenario is further aggravated for women with diabetes as numerous epidemiological studies show that women with type 2 diabetes are at significantly greater risk of developing, relapsing with, and dying from breast cancer compared to women who are not diabetic. Studies show that hyperinsulinemia associated with type 2 diabetes is a significant contributing factor for the mortality seen in breast cancer patients, suggesting an important need for effective therapies that inhibit tumor cell proliferation under hyperinsulinemic conditions. Our early studies showed that cdk4 knock- out mice fail to develop breast cancers driven by the ERBB2 or RAS oncogenes suggesting that CDK4/6 inhibitors may be effective therapeutic agents for certain breast cancers. The approval of CDK4/6 inhibitors as breast cancer therapeutics validated this theory. Another kinase which plays a critical role in insulin signaling and cancer progression is NUAK1/ARK5 which belongs to the AMPK gene family which regulate metabolism. The normal physiological role played by NUAK1/ARK5 in the whole organism was studied using muscle-specific knock-out mice which showed that NUAK1 controls glucose metabolism through regulation of the insulin signaling. Thus, when these knock-out mice were fed a high fat diet, they exhibited a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentrations of muscle glycogen compared to control mice suggesting that inhibition of ARK5/NUAK1 can overcome the effects of hyperglycemia. Interestingly, ARK5 was originally identified as a metastasis gene and its over-expression has been shown to promote metastasis of several tumor types. To achieve the goal of inhibiting breast tumor cell growth and metabolism, we developed a potent dual inhibitor of CDK4 and ARK5 (ON123300), which was a very effective inhibitor of breast tumor growth. In addition, this compound has shown a profound effect on high sugar diet-induced tumor development and metastasis in a Drosophila model system. In this application, we propose to extend these studies to mouse models of hyperinsulinemia (MKR mice), developed by Dr. LeRoith and PDX models of Triple Negative Breast cancer (TNBCs) developed by Dr. Irie. The aims are: (1) To test the effects of ON123300 on the growth and metastasis of mammary tumors in MKR mice which exhibit hyperinsulinemic, pre-diabetic phenotype; (2) To examine the effects of hyperinsuminemia on the growth and metastasis of PDX breast tumors and utilize the models with the highest relative levels of ARK5 to examine the therapeutic value of ON123300; and (3) To use the newly developed Multiplexed-kinase Inhibitor Beads (MIB) and “Cancer Toolkit gain-of-function” (CTK) technologies to determine whether breast cancer cells develop resistance to ON123300 upon prolonged exposure and to determine the nature of signaling pathways that might be the root cause of such resistance.