Identification of Metabolic Liabilities in Breast Cancer

项目总结/摘要 肿瘤表现出改变的营养物质的摄取和利用，如葡萄糖和谷氨酰胺， 以满足肿瘤积累生物量的需要。与正常增殖的组织相反，细胞 由于它们的高增殖率和不可靠的脉管系统，肿瘤内的细胞经常缺乏营养。 因此，癌细胞代谢的重新布线发生在响应营养限制可能存在 癌症特异性的脆弱性，可以成为未来抗癌疗法的目标。在这里，我们建议获得一个 更好地了解癌症代谢的挑战（1）确定肿瘤如何 营养环境影响癌细胞代谢和（2）定义可以作为靶向的途径， 这是新陈代谢改变的结果。在迎接这些挑战的过程中，我们将能够实现我们长期以来的目标- 术语目标：描述体内癌症代谢的特征，并利用由于 这改变了新陈代谢，以确定可能成为未来癌症治疗靶点的必需基因。 我们建议通过两个互补的目标来解决这些挑战：（1）确定这些酶 和乳腺癌原位模型中乳腺癌细胞特异性必需的途径，和（2）使用 确定的代谢环境，确定这些酶和途径，特别是必不可少的乳腺癌 癌细胞在营养有限的条件下。完成前两个目标将使我们有机会（3） 整合两个癌细胞系统的结果，并进行针对性的随访。 实现第一个目标将需要实施体内基于RNAi的功能丧失， 屏幕该筛选将使用靶向代谢基因的RNAi载体库进行，使得能够进行基因组测序。 构建一个乳腺癌细胞库，每个细胞都表现出对单一酶的抑制。在 在体内或体外培养中，RNAi构建体丰度的变化将通过大规模平行测定来测量。 DNA测序，并允许我们确定该结构抑制的基因的重要性。 在第二个目标中，我们提出评估小鼠或异种移植肿瘤的代谢物组成 模型和患者肿瘤样本，以确定由循环提供的关键营养素， 个体肿瘤然后，实施我们开发的连续培养基更换系统， 在限定的条件下生长细胞，在这些关键营养素是有限的，我们将定义适应限制 使用表达谱、代谢物谱和基于RNAi的筛选的组合， 最终发现那些酶或途径对营养限制下的生长至关重要。 最后，在第三个目标中，我们将有机会整合前两个目标的数据， 确定代谢物、基因表达或基因依赖性谱，这些谱是共同的或独特的， 环境研究，目的是从事有针对性的后续行动，以获得详细的机制， 了解在这些环境中被确定为必不可少的单个基因或途径。

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.