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

乳腺原位导管癌（Ductal Carcinoma in situ， DCIS）是最常见的非侵袭性乳腺癌，近几十年来随着乳腺影像学的提高，其检出率显著提高。如果单独进行大面积切除治疗，大约16-30%的DCIS在10年内复发。DCIS目前的治疗方法是手术切除联合放疗。切除后，乳房放射治疗可将复发风险降低约一半，添加抗激素治疗（如他莫昔芬或芳香酶抑制剂）可将复发风险降低三分之一，这种益处仅适用于激素受体阳性DCIS患者。抗激素疗法虽然有效，但也有许多副作用，包括潮热、盗汗、血栓形成、骨质流失、中风和子宫内膜癌。因此，不合规率高达50%。与激素受体阳性DCIS相比，目前尚无激素受体阴性乳腺癌的预防选择。同时，来自观察性研究的证据表明，很大一部分DCIS（约50%）是无痛的，可能不需要手术切除或放疗。因此，对于以导管原位癌（DCIS）为前兆的人类浸润性乳腺癌，开发更安全、更有效的治疗方法是一个尚未满足的需求。非侵袭性乳腺肿瘤的非临床模型是有限的，现有的体内模型不能模拟肿瘤间和肿瘤内的异质性。随着人类DCIS在导管内开始的流行观点，Behbod等人开发了小鼠导管内（MIND）模型，以显示人类DCIS亚型是否可能包含不同的肿瘤启动细胞亚群。导管内MIND移植模型提供了一种宝贵的工具，可以在非侵入性阶段模拟人类乳房的异质性，并允许研究乳腺癌进展的独特分子和细胞机制。与人类DCIS的进化类似，DCIS细胞经导管内注射到小鼠体内，形成原位病变，随后癌细胞浸润到肌上皮细胞层和基底膜的天然屏障中，侵入周围基质。MIND模型模拟乳腺肿瘤从非侵袭性（导管原位癌）到侵袭性疾病的进展。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阶段发展的。

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.