Identification of Metabolic Liabilities in Breast Cancer

描述（由申请人提供）：肿瘤表现出对营养物（如葡萄糖和谷氨酰胺）的摄取和利用的改变，以适应肿瘤积累生物质的需要。与正常增殖的组织相反，肿瘤内的细胞经常处于饥饿状态 因为它们的高增殖率和不可靠的脉管系统。 因此，响应于营养限制而发生的癌细胞代谢的重新布线可能呈现癌症特异性脆弱性，这可能是未来抗癌疗法的目标。在这里，我们建议通过应对以下挑战来更好地了解癌症代谢：（1）确定肿瘤营养环境如何影响癌细胞代谢，以及（2）定义由于这种代谢改变而可以靶向的途径。在应对这些挑战的过程中，我们将能够实现我们的长期目标：描述癌症在体内的代谢，并利用由于这种改变的代谢而存在的责任来识别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）确定乳腺癌原位模型中乳腺癌细胞特别必需的酶和途径，以及（2）使用定义的代谢环境，确定营养有限条件下乳腺癌细胞特别必需的酶和途径。前两个目标的完成将使我们有机会（3）整合两个细胞系统的结果并进行有针对性的随访。 实现第一个目标将需要实施基于体内RNAi的功能丧失筛选。该筛选将使用靶向代谢基因的RNAi载体库进行，从而能够构建乳腺癌细胞库，每个细胞都表现出对单一酶的抑制。在体内或体外培养时，RNAi构建体的丰度变化将通过大规模平行DNA测序来测量，并允许我们确定该构建体抑制的基因的必要性。 在第二个目标中，我们提出评估小鼠或异种移植肿瘤模型和患者肿瘤样本的代谢物组成，以确定由循环提供的从个体肿瘤中耗尽的关键营养素。然后，实施我们已经开发的连续培养基更换系统，以在这些关键营养素受限的特定条件下生长细胞，我们将使用表达谱分析，代谢物谱分析和基于RNAi的筛选的组合来定义对限制关键营养素的适应，最终揭示营养限制后生长所必需的酶或途径。 最后，在第三个目标中，我们将有机会整合前两个目标的数据，并确定所研究环境中常见或独特的代谢产物，基因表达或基因依赖性特征，目的是进行有针对性的随访，以获得对这些环境中确定为必不可少的单个基因或途径的详细机制理解。 公共卫生相关性： 了解癌细胞如何适应营养限制条件将有助于识别 代谢基因和途径在转化状态下特别需要。这些必需的代谢基因和途径的鉴定预计将有助于发现新的抗癌靶点，并与生物信息学方法相结合，确定最有可能从抑制这些靶点中获益的患者。最后，对人类肿瘤中限制的那些关键营养素的鉴定以及这种营养素限制对总体癌细胞代谢的影响将增加对体外使用患者来源的细胞系进行癌症代谢研究的任何限制的理解。

DESCRIPTION (provided by applicant): 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 cancr 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. PUBLIC HEALTH RELEVANCE: Understanding how cancer cells adapt to nutrient limiting conditions will enable the identification of metabolic genes and pathways specifically required in the transformed state. The identification of these essential metabolic genes and pathways is anticipated to facilitate the discovery of novel anti-cancer targets, and in combination with bioinformatic methods, identify patients who would be most likely to benefit from inhibition of such targets. Finally, identificatin of those key nutrients limiting in human tumors and the impact of this nutrient limitation on overall cancer cell metabolism will increase understanding of any limitations in the in vitro use o patient derived cell lines for cancer metabolism research.