BRIT1-NFYA调控三阴性乳腺癌干细胞特性及其分子机制研究

Triple-negative breast cancers (TNBC) are the tumors that lack the expression of the receptors for oestrogen and progesterone (ER and PR, respectively) and the aberrant expression of HER2. TNBC patients typically have a relative poorer outcome owing to an inherently aggressive clinical behavior and a lack of recognized molecular targets for therapy. Therefore, it is critical to dissect the molecular mechanisms of TNBC in order to facilitate development of potential targeted therapy. Recently, BRIT1 (also known as MCPH1), a chromatin-binding protein, has been identified to be a critical player in homologous recombination repair (HR). We have previously generated the BRIT1-/- mice and demonstrated BRIT1's essential role in tumor suppression in the mouse model. Our preliminary data also indicated that BRIT1 is frequently deleted and aberrantly expressed in TNBC specimens. Moreover, we found that BRIT1 downregulation is able to decrease the expression level of ER/PR proteins. Also, BRIT1 inhibition in T47D by using siRNA technique can result in the accumulation of cancer stem cells (CSCs) determined by mammosphere formation assay. As we previously reported, the accumulation of CSCs in TNBC is attributed to the high-level expression of stem cell-related transcription factors including NFYA, a master transcription factor for stem cell renewal and maintenance. Accordingly, our preliminary data also showed that re-expression of BRIT1 in TNBC cells MDA-MB231 can remarkably inhibit the expression of SC-TFF NFYA. Thus, we hypothesize that BRIT1 acts as a novel suppressor for TNBC via regulating the expression of a master transcription factor of stem cells NFYA. To test our hypothesis, we propose the following specific aims in this proposal: 1) To determine whether BRIT1 deficiency promotes the formation of TNBC in mammary gland-specific BRIT1 conditional knockout mouse model induced by carcinogen DMBA. MMTV-cre;BRIT1flox/flox and BRIT1flox/flox (or BRIT1+/+) will be treated with or without DMBA, and the tumor incidence of TNBC will be monitored and determined by immunohistochemistry of ER/PR and HER2. The association of BRIT1 and NFYA will be simultaneously analyzed. 2) To determine whether BRIT1-NFYA involves in the self-renewal and maintenance of CSCs in TNBC. 3) To dissect the underlying mechanism of BRIT1 regulating NFYA and the resulting enrichment of CSCs. The stem cell accumulation will be determined by using flow cytometry with fluorescent staining of CD24-/lowCD44+/high, and mammosphere formation assay. The ChIP-seq assay will be used to determine whether BRIT1 binds the promoter region of NFYA and whether BRIT1 can regulate other target genes. The protein fraction obtained during ChIP assay will be analyzed to determine whether other transcription factors interact with BRIT1. The microRNA microarray will also be used to determine if BRIT1 can regulate the expression of NFYA via the microRNAs. 4) To discover the small molecular compounds that specifically target BRIT1 deficiency with or without PARP inhibitors and clinical chemodrugs such as cisplatin. Taken together, these proposed studies, if successfully completed, will lead to: (i) reveal BRIT1 acting as an inhibitor for TNBC, (ii) determine the molecular mechanism of BRIT1-NFYA in the self-renewal and maintenance of CSCs, and (iii) develop a novel and effective combination therapy for BRIT1 deficiency-based TNBC. Therefore, this project, if successfully tested, will eventually benefit patients with TNBC who still have few alternative treatment choices.

目前，三阴性乳腺癌（TNBC）仍无有效治疗措施，亟待深入探索以找到有效靶向治疗方法。我们前期工作表明：在TNBC中BRIT1表达降低；BRIT1缺失可致肿瘤干细胞增加，这可能与转录因子NFYA表达上调有关。我们遂提出假说：BRIT1缺失可调控NFYA的表达以促进TNBC进展。为验证此假说，我们首先利用已构建的乳腺组织特异性BRIT1基因敲除小鼠模型阐明BRIT1缺失诱导TNBC的能力及BRIT1-NYFA在TNBC形成中的作用；其次，检测BRIT1-NYFA对TNBC肿瘤干细胞的调控；最后探讨基于BRIT1-NFYA异常的新的有效靶向治疗措施。如能获得资助并顺利完成，本项目将：1)阐明BRIT1是否是一种新的TNBC形成/演进抑制因子；2)确定BRIT1-NFYA在TNBC形成过程中的作用；3)寻找基于BRIT1缺失的靶向性治疗措施，为TNBC临床治疗提供一种新的方法。

目前，三阴性乳腺癌TNBC仍无有效的治疗措施，急需深入探索以找到潜在有效的靶向治疗方法。前期工作显示，在TNBC中BRIT1表达降低；BRIT1缺失可致肿瘤干细胞增加，这可能与转录因子NFYA表达上调有关。在此基础上，我们提出假说：BRIT1缺失可调控NFYA的表达以促进TNBC的进展。为验证此假说，我们从下述3个方面进行验证：首先利用已构建的乳腺组织特异性BRIT1基因敲除小鼠模型阐明BRIT1缺失诱导TNBC的能力及BRIT1-NFYA在TNBC形成中的作用；其次，检测BRIT1-NFYA对TNBC肿瘤干细胞的调控并寻找基于BRIT1-NFYA异常的可能的新的靶向治疗措施。在本项目实施过程中，我们确定BRIT1缺失可以导致小鼠的正常乳腺组织发育迟缓；在致癌物DMBA的作用下，BRIT1缺失性小鼠较野生型小鼠更易发生乳腺肿瘤。在小鼠乳腺发育与肿瘤形成过程中，BRIT1缺失易致重要的调控蛋白表达改变，特别是NFYA，CyclinD与p53等，这都与肿瘤干细胞的形成密切相关。此外，我们利用BRIT1-less缺失性表达标签，获得了数种潜在的抑制BRIT1缺失性TNBC的小分子化合物，正在进行相关细胞与动物的药效试验收尾工作。因此，本项目初步探讨了BRIT1缺失性乳腺癌的形成机制，并有望找到一种潜在的针对BRIT1缺失性TNBC的干预方法。