Phosphocholine modulation by oncognenic signaling - MRS studies of mechanism

描述(由申请人提供)：新的抗癌疗法越来越多地涉及到针对导致肿瘤发生的分子遗传异常。然而，对这种治疗的反应通常与肿瘤停滞有关，而不是缩小，限制了传统成像方法监测早期反应的效用。我们的长期目标是开发非侵入性的、局部的、基于磁共振(MR)的方法来检测对靶向治疗的分子反应。我们已经证明，通过丝裂原活化蛋白激酶(MAPK)和磷脂酰肌醇3-激酶(PI3K)通路抑制信号会导致磁共振波谱(MRS)可检测到的含有胆碱的代谢物的调制，特别是磷胆碱(PC)。然而，我们的数据显示，根据治疗的不同，抑制可以导致PC的增加或减少，限制了其作为反应的强大生物标志物的使用。因此，这项应用的目标是确定信号通路影响细胞代谢的机制，从而调节PC和其他含有胆碱的代谢物。第二个目标是识别和验证生物标记物对以前未探索的靶向治疗的反应。这项临床前研究将有助于更好地理解导致胆碱代谢产物变化的机制。因此，有可能以更可靠、更稳健和更可预测的方式使用这些代谢物中的调制来评估针对信号转导的治疗的反应。MAPK和PI3K信号可以通过直接影响参与胆碱代谢的酶或间接影响控制脂肪酸合成的脂肪酸合成酶(FASN)来影响含有胆碱的代谢物。因此，我们建议同时研究胆碱代谢和脂肪酸合成。我们将结合1H、31P和13C MRS，并监测信号的调节如何影响这两条代谢途径，从而影响含有胆碱的代谢物。具体目的1.确定脂肪酸合成对胆碱代谢的影响。我们将首先确定抑制FASN如何影响FA和胆碱代谢。我们将研究活细胞和提取物，并使用1H，31P，13C MRS来监测代谢。具体目的2.确定信号通路和新陈代谢之间的机制联系。使用与上述相同的方法，我们将监测1)MAPK抑制，2)PI3K抑制，3)通过HSP90抑制多个信号通路对FA和胆碱代谢的影响。具体目的3.证实细胞中的机制发现在体内转化为肿瘤。皮下移植肿瘤将通过1H、31P、13C MRS进行研究，以确认在特定目标2中的发现在体内是正确的。公共卫生相关性磁共振波谱可用于非侵入性监测针对特定致癌突变的新型癌症疗法的反应。然而，之前报道的治疗后磁共振可检测到的代谢变化背后的确切机制尚不清楚。在这项应用中，我们将研究这一联系，我们的研究将产生更强大、可靠和可预测的、基于磁共振的肿瘤治疗反应指标，最终改善患者护理。

DESCRIPTION (provided by applicant): Novel anti-cancer therapies increasingly involve targeting of the molecular-genetic abnormalities that result in oncogenesis. However, response to such therapies is often associated with tumor stasis, rather than shrinkage, limiting the utility of conventional imaging methods to monitor early response. Our long-term goal is to develop noninvasive, localized, magnetic resonance (MR)-based methods that detect molecular response to targeted treatments. We have shown that inhibition of signaling via the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways results in modulation of MR spectroscopy (MRS)-detectable choline-containing metabolites and in particular phosphocholine (PC). However, our data show that, depending on treatment, inhibition can result either in an increase or in a decrease in PC, limiting its use as a robust biomarker of response. The goal of this application is therefore to determine the mechanisms by which signaling pathways affect cellular metabolism resulting in modulation of PC and other choline-containing metabolites. A secondary goal is to identify and validate biomarkers of response to previously unexplored targeted therapies. This pre-clinical research will result in a better understanding of the mechanisms that lead to changes in choline-containing metabolites. As a result, it will be possible to use the modulation in these metabolites in a more reliable, robust and predictable way to assess response to therapies that target signaling. MAPK and PI3K signaling can affect choline-containing metabolites either directly, by affecting the enzymes involved in choline metabolism, or indirectly, by affecting fatty acid synthase (FASN), which controls fatty acid (FA) synthesis. Thus, we propose to investigate both choline metabolism and fatty acid synthesis. We will combine 1H, 31P and 13C MRS and monitor how modulation of signaling affects the two metabolic pathways, and, consequently, choline-containing metabolites. Specific Aim 1. To determine the effect of fatty acid synthesis on choline metabolism. We will first determine how inhibition of FASN affects FA and choline metabolism. We will study live cells and extracts, and use 1H, 31P, 13C MRS to monitor metabolism. Specific Aim 2. To determine the mechanistic link between signaling pathways and metabolism. Using the same methods as above us will monitor the effect on FA and choline metabolism of 1) MAPK inhibition 2) PI3K inhibition and 3) inhibition of multiple signaling pathways via HSP90. Specific Aim 3. To confirm that the mechanistic findings in cells translate to tumors in vivo. Subcutaneous tumor xenografts will be investigated by 1H, 31P, 13C MRS to confirm that the findings made in Specific Aim 2 hold true in vivo. PUBLIC HEALTH RELEVANCE Magnetic resonance spectroscopy can be used to noninvasively monitor response to novel cancer therapies that target specific oncogenic mutations. However, the exact mechanism behind the magnetic resonance-detectable metabolic changes previously reported following treatment is not clear. In this application we will investigate this link, and our research will result in more robust, reliable, and predictable, noninvasive, magnetic resonance-based indicators of tumor response to treatment, ultimately improving patient care.