Targeting Src Signaling Pathways to Promote Cell Cycle Arrest in Ovarian Cancer

描述(由申请人提供)：完成医学博士学位(1999)，在妇产科住院医师(1999-2003年，Johns Hopkins)，在美国国立卫生研究院(NCI)接受了两年的基础研究培训(2003-05)，然后获得妇科肿瘤学临床和研究奖学金(2005-08，克利夫兰诊所)，Simpkins博士被招募到迈阿密大学(UM)担任妇科肿瘤科和UM Sylvester癌症中心助理教授和内科科学家。在培训期间，她完成了3年的实验室研究，参加了AACR/ASCO临床试验设计研讨会，并在奖学金期间设计并执行了卵巢癌(OVCA)的II期试验。2008年8月，她开始在斯林格兰的实验室指导研究，调查雌激素(E2)以及通过Src、MEK和AKT的信号转导在OVCA中促进生长的机制。辛普金斯博士有75%的受保护研究时间和启动资金，用于支持最初的项目开发，以获得独立资金。申请者未来2-3年的目标是加深对OVCA中雌激素和生长因子信号的了解，获得OVCA临床前药物开发和预测标记物的经验，熟练掌握GRAGSRISHING和I期试验设计。她的职业目标是开发和实施妇科恶性肿瘤靶向治疗的创新策略，并在实验室和临床之间无缝移动。辛普金斯博士的主要导师是J.Slingerland医学博士，他是细胞周期、类固醇激素受体生物学和荷尔蒙抗药性乳腺癌方面的专家，M·佩格拉姆医学博士是分子靶向癌症疗法的临床前和临床开发方面的专家。癌症中心拥有庞大的多种族癌症患者基础，拥有大量具有翻译记录的多学科基础科学家和临床医生，并为临床研究提供行政支持和资源。这款K08将促进辛普金斯博士发展成为一名受过分子机械研究培训的临床科学家，其目标是产生新的疗法并在临床试验中进行测试。提出了详细的培训计划。OVCA是最致命的妇科恶性肿瘤。大多数患者被诊断为晚期III/IV期，并在2年内复发。复发的OVCA是无法治愈的，目标是缓解症状。患者精疲力竭的治疗选择，因此迫切需要新的治疗方法。抗雌激素治疗耐受性很好，因此在这种情况下很有吸引力。这种疗法还没有在早期(IC/II)OVCA中进行测试，在这种情况下可能也是有益的。该项目可能会确定OVCA的新治疗方案。雌二醇在体外和体内促进OVCA生长12-15。雌激素缺乏和雌激素受体(ER)被阻断是因为乳腺癌(BRCA)细胞滞留在细胞周期的G1期16，17。细胞周期的进展是由细胞周期蛋白依赖的激酶(CDK)控制的，CDK被周期蛋白激活并被CDK抑制剂抑制。CDK抑制剂p21和p27是一种周期负性调节因子，在许多癌症中经常降低，包括OVCAs16，17。ER在大多数OVCAs中表达，类似于BRCA18。抗雌激素对OVCAs存活和细胞周期的影响尚不清楚。尽管卵巢癌中ER的表达很高，但抗雌激素疗法的小型试验一直令人失望。抗雌激素的益处还没有在设计良好的大型OVCA试验中进行研究。在卵巢静脉曲张患者中，许多患者表现出对抗雌激素的从头耐药，而那些最初有反应的患者总是发展为耐药。SRC、Ras/Raf/MEK和PI3K通路在卵巢癌中被结构性激活1我的初步数据和其他人的数据支持这样的观点，即雌激素结合的ER和信号通路之间的相互作用导致Src和MEK/MAPK激活，以刺激细胞周期进程，17，34。我们的初步数据显示，在ER+OVCA细胞系中，E_2刺激了Src、MEK和AKT的进一步激活。Src抑制剂联合内质网阻断通过增加p27的水平和作用，使ER+OVCA细胞发生更大程度的G1期阻滞。此外，使用Src和MEK抑制剂药物比单一药物更有效地阻止G1期细胞，抑制Src、MAPK和AKT，并增加p27水平，导致对Cyclin E-CDK2的抑制。虽然大多数OVCA表达ER，但抗雌激素一直令人失望。联合使用抗雌激素和阻断关键的有丝分裂信号通路的药物可以预防或延缓ER+OVCA的抗雌激素耐药性的发展。联合使用阻断不同成分激活的通路的激酶抑制剂也可能更有效地抑制OVCA细胞的生长。我们的假设是，Src和/或Ras/Raf/MEK的结构性激活促进了细胞周期的进展，使ER+OVCA细胞具有抗雌激素抗性。我们进一步推测，Src和MEK抑制剂可能协同作用或与抗雌激素一起逆转耐药，降低OVCA细胞的增殖。目的1在一系列扩增的ER阳性OVCA细胞系中测试不同的Src抑制剂-药物组合的抗增殖活性，并研究细胞周期停滞和凋亡的机制。AIM 2将比较直接从去茎手术中取出的早期和晚期OVCA直接获得的原代培养的药物效果。目的3将利用创新的小鼠OVCA肾被膜模型，测试Src抑制与抗雌激素或激酶抑制剂联合应用于异种移植瘤的抗肿瘤效果。临床前数据将有助于确定具有最大抗肿瘤效果的新药物组合，并可在未来的临床试验中进行测试。虽然不是机械上的新方法，但靶向ER和激活的通路对于迫切需要新疗法的OVCA复发患者来说是一个新的治疗机会。早期OVCA的抗雌激素治疗可能被证明可以预防或延缓复发，这一点从未被探索过。

DESCRIPTION (provided by applicant): On completion of her MD (1999), Residency in OB-GYN (1999-2003, Johns Hopkins), two yrs of basic research training at the NCI (2003-05) followed by a Clinical and Research Fellowship in Gynecologic Oncology (2005-08, Cleveland Clinic), Dr. Simpkins was recruited to the University of Miami (UM) as Assistant Professor and physician scientist in the Division of Gynecologic Oncology and UM Sylvester Cancer Center. During her training, she completed 3 yrs of lab research, attended the AACR/ASCO Clinical Trial Design Workshop, and designed and executed a phase II ovarian cancer (OVCA) trial during fellowship. She began mentored research in Aug, 2008 in Slingerland's lab, investigating mechanisms of growth stimulation in OVCA by estrogens (E2) and signaling via Src, MEK and AKT. Dr. Simpkins has 75% protected research time and start-up funds to support initial project development to garner independent funding. The applicant's goals for the next 2-3 yrs are to deepen her knowledge of estrogen and growth factor signaling in OVCA, gain experience with pre-clinical drug development and predictive markers in OVCA, become proficient in grantsmanship and phase I trial design. Her career goal is to develop and implement innovative strategies in targeted therapy for Gyn malignancies and move seamlessly between lab and clinic. Dr. Simpkins has J. Slingerland MD, PhD as primary mentor, an expert in cell cycle, steroid hormone receptor biology and hormone resistant breast cancer and M. Pegram MD, an expert in pre-clinical and clinical development of molecular targeted cancer therapies, as co-mentor. The Cancer Center has a large multi- ethnic cancer patient base, a critical mass of multi-disciplinary basic scientists and clinicians with a translational track record, and offers admininistrative support and resources for clinical research. This K08 will foster Dr. Simpkins' development as a clinician scientist trained in molecular mechanistic research whose goal is to generate novel therapies and test them in clinical trials. A detailed training plan is proposed. OVCA is the most lethal gynecological malignancy. Most patients are diagnosed with advanced stage III/IV and recur within 2 yrs. Recurrent OVCA is incurable and goals are palliation of symptoms. Patients exhaust treatment options so new therapies are desperately needed. Anti-estrogen therapies are well tolerated and thus appealing in this setting. Such therapies have not yet been tested in early stage (IC/II) OVCAs and maybe of benefit in this setting as well. This project will potentially identify novel treatment options for OVCA. E2 drives OVCA growth in vitro and invivo12-15. E2 deprivation and estrogen receptor(ER) blockade because breast cancer (BRCA) cells to arrest in the G1 phase of the cell cycle16, 17. Cell cycle progression is governed by cyclin-dependent kinases (cdks) that are activated by cyclins and inhibited by cdk inhibitors. The cdk inhibitors, p21 and p27, are cycle negative regulators that are frequently reduced in many cancers, including OVCAs16, 17. The ER is expressed in the majority of OVCAs, similar to BRCA18. Anti-estrogen effects on survival and cell cycle in OVCAs have not been characterized. Despite high ER expression in OVCAs, small trials of anti-estrogen therapies have been disappointing. The benefit of anti-estrogens has not been studied in large well-designed OVCA trials. In OVCAs, many patients manifest de novo resistance to anti-estrogens and those that do initially respond, invariably develop resistance. Src, Ras/Raf/MEK and PI3K pathways are constitutively activated in OVCAs.1 My preliminary data and that of others support the notion that cross talk between estrogen-bound ER and signaling kinases leads to Src and MEK/MAPK activation to stimulate cell cycle progression, 17, 34. Our preliminary data show that E2 stimulates further activation of Src, MEK and AKT in ER+ OVCA lines. Combined Src inhibitor with ER blockade caused a greater G1 arrest of ER+ OVCA cells through increased levels and action of p27. Moreover, use of both Src and MEK inhibitor drugs more effectively arrested cells in G1 than monotherapy, with inhibition of Src, MAPK and AKT and increased p27 levels causing inhibition of cyclin E-Cdk2. While most OVCAs express ER, anti- estrogens have been disappointing. Combined use of antiestrogens with drugs that block critical mitogenic signaling pathways may prevent or delay development of anti-estrogen resistance in ER+ OVCAs.The combined use of kinase inhibitors that block different constitutively activated pathways may also more effectively inhibit OVCA cell growth. Our HYPOTHESIS is that constitutive activation of Src and/or Ras/Raf/MEK promote cell cycle progression, rendering ER+ OVCA cells anti-estrogen resistant. We further postulate that Src and MEK inhibitors may cooperate together or with anti-estrogens to reverse resistance and decrease OVCA cell proliferation. AIM 1 will test different Src inhibitor-drug combinations for anti-proliferative activity in an expanded series of ER positive OVCA cell lines and study the mechanisms of cell cycle arrest and apoptosis. AIM 2 will compare drug effects on primary cultures derived directly from early and advanced stage OVCAs removed at debulking surgery. AIM 3 will test the anti-tumor efficacy of combined Src inhibition with an antiestrogen or kinase inhibitor on xenografts using an innovative mouse OVCA renal capsule model. The pre-clinical data will help to identify novel drug combinations with the greatest anti-tumor efficacy that can be tested in future clinical trial. Although not mechanistically new, targeting ER and activated pathways is a novel therapeutic opportunity for recurrent OVCA patients who desperately need new therapies. Anti-estrogen treatment of early stage OVCA may prove to prevent or delay recurrence, which has never been explored.