Transcriptomic effects of curcumin and piperine in breast stem cells

描述(申请人提供)：散发性癌症是一种复杂的疾病，是个体易感性与终身接触营养和有毒化合物相互作用的结果。癌症干细胞理论认为，干细胞自我更新和分化途径中的异常在癌症发生中是不可或缺的。大多数体外癌症研究是在从克隆群体中扩增的癌细胞中进行的，或者是在适应了培养条件的细胞系中进行的。这些研究往往没有考虑到个体间的可变性或先前的暴露，而且它们产生有关化学预防营养治疗的生理学相关数据的能力可能受到限制。在这里，我们提出了一个新的体外模型来理解处理两种已被证明影响干细胞自我更新的化合物的转录效应：饮食中的多酚姜黄素和生物碱胡椒碱。我们以前使用过乳房模型来表明姜黄素和胡椒碱都是针对乳腺干细胞的，并协同抑制自我更新，同时对正常分化的细胞保持无毒。在具体目标1中，我们将在非锚定条件下培养原代乳腺细胞和癌细胞系，并分别和联合使用姜黄素和胡椒碱。我们将使用条形码测序分析方法(Bar-seq)提取RNA进行深度测序。生物信息学分析将确定姜黄素和胡椒碱在癌细胞和正常原代乳腺细胞中作用机制的差异。在特定目标1中确定的差异表达基因将在特定目标2中扩大的原代组织和乳腺癌细胞系样本中得到验证。我们这一新的体外方法学的应用将为理解姜黄素和胡椒碱特异性靶向干细胞的机制以及正常细胞和肿瘤细胞中作用机制的差异提供关键环节。此外，这项研究将为姜黄素和胡椒碱的治疗效果提供干细胞特异性生物标志物，并将扩展到未来的临床研究。这些数据将为姜黄素和胡椒碱等营养化合物是否可用于干预易感人群的乳腺癌预防提供基础证据。

DESCRIPTION (provided by applicant): Sporadic cancer is a complex disease, a consequence of the interaction of individual susceptibility with a lifetime of exposures to both nutritive and toxic compounds. The stem cell theory of cancer postulates that aberrations in stem cell self renewal and differentiation pathways are integral in carcinogenesis. Most in vitro cancer studies are undertaken in cancer cells expanded from a clonal population or in cell lines that have adapted to culture conditions. These studies often do not account for interindividual variability or previous exposures and may be limited in their ability to produce physiologically relevant data about chemopreventive nutrient treatment. Here, we propose a novel in vitro model to understand the transcriptomic effects of treatment of two compounds that have been shown to affect stem cell self renewal: the dietary polyphenol curcumin and the alkaloid piperine. We have previously used the mammosphere model to show that both curcumin and piperine specifically target breast stem cells and synergistically inhibit self-renewal while remaining non-toxic to normal differentiated cells. In Specific Aim 1, we will culture primary breast cells as well as cancer cell lines in anchorage independent conditions, and treat with curcumin and piperine, both individually and in combination. We will extract RNA for deep sequencing using the barcode analysis by sequencing method (Bar-seq). Bioinformatic analyses will indentify differences in mechanism of action of curcumin and piperine in cancer cells and normal primary breast cells. Differentially expressed genes identified in Specific Aim 1 will be validated in an expanded primary tissue and breast cancer cell line sample set in Specific Aim 2. Our application of this novel in vitro methodology will provide a crucial link in understanding the mechanism by which curcumin and piperine specifically target stem cells as well as differences in mechanisms of action in normal vs. tumor cells. Additionally, this study wil provide stem cell specific biomarkers of efficacy of treatment for curcumin and piperine that will be extended to future clinical studies. These data will provide foundational evidence as to whether nutritional compounds, such as curcumin and piperine, can be used as an intervention in breast cancer prevention in susceptible populations.