Identification of Metabolic Liabilities in Breast Cancer

项目摘要/摘要 肿瘤表现出对营养物质的吸收和利用的改变，如葡萄糖和谷氨酰胺，以 以适应肿瘤积累生物量的需要。与正常增殖的组织相比，细胞 由于肿瘤的高增殖率和不可靠的血管系统，肿瘤内的肿瘤经常缺乏营养。 因此，响应营养限制而发生的癌细胞新陈代谢的重新连接可能会出现。 癌症特有的脆弱性，可能成为未来抗癌治疗的目标。在这里，我们建议获得一个 通过应对以下挑战更好地了解癌症新陈代谢：(1)确定肿瘤如何 营养环境影响癌细胞新陈代谢和(2)定义可作为靶点的途径 这种新陈代谢改变的后果。在迎接这些挑战的过程中，我们将实现我们长期的-- 学期目标：描述癌症在体内的新陈代谢，并利用目前存在的因 这改变了新陈代谢，以确定可能成为未来癌症治疗目标的必要基因。 我们建议通过两个相辅相成的目标来应对这些挑战：(1)确定这些酶 以及在乳腺癌的原位模型中对乳腺癌细胞特别重要的通路，以及(2)使用 明确的代谢环境，确定那些对乳房特别重要的酶和途径 癌细胞在营养有限的条件下。前两个目标的实现将使我们有机会(3) 整合两种癌细胞系统的结果，并进行有针对性的随访。 要实现第一个目标，需要在体内实施基于RNAi的功能丧失 屏幕上。这一筛选将使用靶向代谢基因的RNAi载体池进行，从而使 构建一个乳腺癌细胞池，每个细胞都显示出一种酶的抑制作用。在进入时 体内或体外培养，RNAi结构的丰度变化将通过大规模平行测量 DNA测序，并使我们能够确定该构建抑制的基因的本质。 在第二个目标中，我们建议评估小鼠或异种移植瘤的代谢物成分。 模型和患者肿瘤样本，以确定循环提供的关键营养物质 个体肿瘤。然后，实施我们开发的连续介质更换系统 在这些关键营养物质受到限制的特定条件下培养细胞，我们将定义对限制的适应 使用表达谱、代谢物谱和基于RNAi的筛选相结合的关键营养素， 最终发现那些在营养限制下生长所必需的酶或途径。 最后，在第三个目标中，我们将有机会整合来自前两个目标的数据和 确定共同的或独特的代谢物、基因表达或基因依赖情况 所研究的环境，目标是参与有针对性的后续行动，以获得详细的机制 对在这些环境中被确定为必不可少的单个基因或途径的理解。

Project Summary / Abstract Tumors exhibit altered uptake and utilization of nutrients, such as glucose and glutamine, to accommodate the tumor’s need to accumulate biomass. In contrast to normally proliferative tissues, cells within a tumor are frequently starved for nutrients due to their high proliferation rate and unreliable vasculature. Therefore, the rewiring of cancer cell metabolism that occurs in response to nutrient limitation may present cancer specific vulnerabilities that can be the target of future anti-cancer therapies. Here, we propose to gain a better understanding of cancer metabolism by meeting the challenges of (1) determining how the tumor nutrient environment impacts cancer cell metabolism and (2) defining pathways which can be targeted as a consequence of this altered metabolism. In meeting these challenges, we will enable the fulfillment of our long- term goal: to characterize the metabolism of cancer in vivo and take advantage of the liabilities present due to this altered metabolism to identify essential genes which can be the target of future cancer therapies. We propose to address these challenges by two complementary Aims: (1) Determine those enzymes and pathways specifically essential to breast cancer cells in an orthotopic model of breast cancer and (2) using defined metabolic environments, determine those enzymes and pathways specifically essential to breast cancer cells in nutrient limited conditions. Completion of these first two Aims will give us the opportunity to (3) integrate the results from the two cancer cell systems and conduct targeted follow-up. Accomplishing the First Aim will require the implementation of an in vivo RNAi-based loss-of-function screen. This screen will be conducted using a pool of RNAi vectors targeting metabolic genes, enabling the construction of a pool of breast cancer cells, each of which exhibits suppression of a single enzyme. Upon in vivo or in vitro culture, the change in abundance of the RNAi construct will be measured by massively parallel DNA sequencing, and allow us to determine the essentiality of the gene which that construct suppresses. In the Second Aim we propose assessing the metabolite composition of murine or xenograft tumor models and patient tumor samples to identify key nutrients provided by the circulation that are depleted from individual tumors. Then, implementing a continuous medium replacement system that we have developed to grow cells in defined conditions where such key nutrients are limiting, we will define the adaptation to limiting key nutrients using a combination of expression profiling, metabolite profiling and RNAi-based screening, ultimately uncovering those enzymes or pathways essential for growth upon nutrient limitation. Finally, in the Third Aim we will have the opportunity to integrate the data from the first two Aims and identify metabolite, gene expression, or gene dependency profiles which are common or unique to the environments studied, with the goal of engaging in a targeted follow-up to gain a detailed mechanistic understanding of individual genes or pathways identified as essential in these environments.