PREVENT PRECLINICAL EFFICACY AND INTERMEDIATE BIOMARKER ENDPOINTS TASK ORDER: USE OF CARNOSIC ACID AND VIVOX40 FOR BREAST CANCER PREVENTION

乳腺导管原位癌(DCIS)是最常见的非侵袭性乳腺癌，近几十年来，随着乳腺影像的改善，乳腺导管原位癌的发现率显著提高。如果只进行广泛切除治疗，大约16%-30%的DCIS会在10年内复发。目前治疗DCIS的方法是手术切除联合放射治疗。切除后，乳房放射治疗的复发风险降低了约一半，附加抗激素治疗(如他莫昔芬或芳香酶抑制剂)的复发风险额外减少了三分之一，这是激素受体阳性DCIS患者所独有的好处。抗激素疗法虽然有效，但也伴随着许多副作用，包括潮热、盗汗、血栓形成、骨质丢失、中风和子宫内膜癌。因此，违约率高达50%。与激素受体阳性的DCIS相比，目前没有激素受体阴性乳腺癌的预防选择。与此同时，来自观察性研究的证据表明，很大一部分DCIS(约50%)是惰性的，可能不需要手术切除或放射治疗。因此，对于以导管原位癌(DCIS)为先兆的人类浸润性乳腺癌的预防，开发更安全、更有效的治疗方法的需求尚未得到满足。非侵袭性乳腺肿瘤的非临床模型是有限的，现有的活体模型不能模拟瘤内和瘤内的异质性。Behbod等人普遍认为人类DCIS是在导管内启动的。开发了小鼠导管内(Mind)模型，以显示人类DCIS的亚型是否可能包含不同的肿瘤启动细胞亚群。导管内精神移植模型提供了一个宝贵的工具，它模拟了人类乳房在非侵入性阶段的异质性，并使研究乳腺癌进展的不同分子和细胞机制成为可能。与人类DCIS的演变类似，导管内注射到小鼠体内的DCIS细胞会形成原位病变，然后随着癌细胞渗入肌上皮细胞层和基底膜的自然屏障而侵入周围的间质。思维模型模拟了乳腺肿瘤从非浸润性(导管原位癌)到浸润性疾病的发展过程。这一步被广泛认为是一个关键的转变，因为大多数浸润性乳腺癌被认为是通过DCIS阶段演变的。 以前的研究表明，高核BCL9与高危DCIS的病理特征显著相关。在DCIS思维模型中体内沉默BCL9，可抑制DCIS的侵袭，逆转上皮间充质转化(EMT)，并显著抑制DCIS细胞的增殖。此外，De la Roche及其同事对β-连环蛋白与bcl9结合的小分子抑制剂进行了筛选，发现了鼠尾酸，这是迷迭香提取物(RE)中发现的一种天然化合物。他们的研究表明，鼠尾草酸是无毒的，在HeLa和SW480细胞中诱导蛋白酶体降解活性的β-连环蛋白，并减弱bcl9/β-连环蛋白依赖的转录。在人体中，RE已经被美国食品和药物管理局(USFDA)和欧洲食品安全局批准为安全的食品添加剂。此外，RE已被FDA归类为“公认的安全”或GRAS，迷迭香在美国被广泛用作膳食补充剂。大量的体内研究已经为迷迭香提取物及其主要成分鼠尾草酸在各种癌症类型中的抗癌作用提供了强有力的证据。该项目的总体目标是评估迷迭香提取物VivOX40和鼠尾草酸在DCIS细胞系精神移植、患者来源(PDX)DCIS精神移植模型和概括人类乳腺癌进展的基因工程小鼠模型中的化学预防效果。

Ductal Carcinoma In-Situ (DCIS) of the breast is the most common form of non-invasive breast cancer, with a marked increase in detection rate with improved breast imaging in the last few decades. About 16-30% of DCIS develop a recurrence in 10 years if treated with wide excision alone. DCIS is currently managed by surgical resection combined with radiotherapy. Following resection, the risk of recurrence is reduced by about one-half with breast radiotherapy and by an additional one-third with added anti-hormonal therapy such as tamoxifen or aromatase inhibitors, a benefit that is exclusive to individuals with hormone receptor positive DCIS. Anti-hormonal therapies, while effective, are associated with many side effects including hot flashes, night sweats, thrombogenesis, bone loss, stroke, and endometrial cancers. Consequently, the rate of non-compliance is as high as 50%. In contrast to hormone receptor positive DCIS, there are currently no preventive options for hormone receptor negative breast cancers. At the same time, evidence from observational studies suggests that a large fraction of DCIS, ~50%, are indolent and may not require surgical resection or radiotherapy. Therefore, there is an unmet need for the development of safer and more effective therapies for the prevention of human invasive breast cancer, for which ductal carcinoma in situ (DCIS) is a precursor. Non-clinical models of non-invasive breast tumors are limited, and the existing in vivo models do not mimic inter- and intratumoral heterogeneity. With the prevailing notion that human DCIS initiates inside the ducts, Behbod et al. developed the mouse intraductal (MIND) model to show whether subtypes of human DCIS might contain distinct subpopulations of tumor-initiating cells. The intraductal MIND transplantation model provides an invaluable tool that mimics human breast heterogeneity at the noninvasive stages and allows the study of the distinct molecular and cellular mechanisms of breast cancer progression. Similar to the evolution of human DCIS, DCIS cells injected intraductally into mice form in situ lesions followed by invasion into the surrounding stroma as cancer cells infiltrate the natural barriers of the myoepithelial cell layer and basement membrane. The MIND model mimics the progression of breast neoplasia from non-invasive (ductal carcinoma in situ) to invasive disease. This step is widely recognized as a critical transition, in that most invasive breast cancers are thought to evolve though a DCIS phase. Previous studies showed a significant association between high nuclear BCL9 and pathological characteristics indicative of high-risk DCIS. The in vivo silencing of BCL9 in DCIS MIND models, led to inhibition of DCIS invasion, reversal of epithelial mesenchymal transition (EMT), and a significant reduction in DCIS cellular proliferation. Additionally, de la Roche and colleagues performed a screen for small-molecule inhibitors of β-catenin binding to BCL9 and discovered carnosic acid, a natural compound found in rosemary extract (RE). Their studies showed that carnosic acid was non-toxic, induced proteosomal degradation of active β-catenin and attenuated BCL9/β-catenin-dependent transcription in HeLa and SW480 cells. In humans, RE has already been approved as a safe food additive by the United States Food and Drug Administration (USFDA) and by the European Food Safety Authority. Further, RE has been categorized by the FDA as “generally recognized as safe” or GRAS and Rosemary is widely available as dietary supplement in the United states. Numerous in vivo studies have provided strong evidence for the anti-cancer effects of rosemary extract and its principal component carnosic acid in various cancer types. The overall goal of the project is to evaluate chemopreventive effects of rosemary extract VivOX40 and carnosic acid in DCIS cell lines MIND graft, patient derived (PDX) DCIS MIND graft models and genetically engineered mouse models that recapitulate human breast cancer progression.