Genetic and pharmacological manipulation of system xc in pancreatic cancer

描述（由申请人提供）：RAS是人类基因组中最有效的致癌基因之一，在所有人类癌症中超过四分之一发生突变[1]。特别是在胰腺癌中，KRAS以惊人的高速率突变，在超过95%的病例中被激活[2]。虽然在胰腺癌的转基因小鼠模型中消除RAS信号传导和突变型RAS蛋白表达完全消除了肿瘤[3]，但开发针对胰腺癌患者的特异性RAS抑制剂或其他有用的疗法已被证明几乎是不可能的。胰腺导管腺癌（PDA）仍然是最致命的癌症之一，5年生存率仅为5%。很明显，在胰腺癌领域开发新的治疗方法以确定更有效和毒性更小的治疗方法是一个关键的问题。 目前的努力已经确定的途径，其操作可以证明致命的，特别是在含有RAS突变的细胞的情况下。这种“合成致死”组合可以在临床环境中提供巨大的效用，限制通常与放射和化学治疗方法相关的毒性，并以特定有效的方式靶向RAS驱动的肿瘤。在2003年，一个合成的致命化学筛选进行了博士。 Stockwell鉴定了Ras突变细胞的特定杀手[4]。这种化合物，称为“erastin”，通过抑制一种名为系统xc-的胱氨酸-谷氨酸反向转运蛋白，引起一种铁依赖性形式的氧化性细胞死亡，称为“铁凋亡”[5]。然而，这种方法在体内的实用性和系统xc-在Ras驱动的肿瘤中的确切作用仍然未知。 鉴于突变型RAS在PDA的发展和维持中的作用，所提出的研究的总体目标是检验系统xc-功能是KRAS突变型胰腺癌的发展和维持所需的假设。具体来说，我们的目标是询问的后果，遗传和药理学操纵系统xc-对胰腺癌细胞的增殖和生存。 利用分子遗传学、小鼠模型和临床前治疗学方面的专业知识，我们将探讨系统xc-在PDA生长和维持中的作用，并评估靶向该途径作为胰腺癌患者治疗干预手段的临床可行性。具体来说，我们将使用shRNA和病毒技术来调节系统xc-在体外。为了研究体内系统xc-基因消除的效果，我们将系统xc-功能缺陷的小鼠与我们的PDA临床前模型KPC小鼠杂交。最后，我们将使用一种化合物，来自erastin，以评估系统xc-在体外和KPC模型中对胰腺肿瘤细胞的药理学抑制作用。 总之，本文提出的实验将帮助我们理解氧化还原状态维持在癌症生长和存活中的精确作用，以及验证系统xc-抑制剂在胰腺癌的有效治疗中的用途。

DESCRIPTION (provided by applicant): RAS is among the most potent oncogenes in the human genome and is mutated in over one in four of all human cancers [1]. In pancreatic cancer specifically, KRAS is mutated at an alarmingly high rate, activated in over 95% of all cases [2]. While extinguishing both RAS signaling and mutant RAS protein expression in a transgenic mouse model of pancreatic cancer completely ablates tumors [3], developing specific RAS-inhibitors or other useful therapies for pancreatic cancer patients has proven near impossible. In keeping with this lack of progress, pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal cancers, with a 5-year survival rate of just 5%. It is clear that there is a critical ned to develop novel therapeutic approaches in the field of pancreatic cancer in order to identify more effective and less toxic treatments. Current efforts have been made to identify pathways whose manipulation could prove lethal specifically in the context of cells containing RAS mutations. Such "synthetic lethal" combinations could provide great utility in a clinical setting, limiting the toxicities commonly associated with both radiation and chemotherapeutic approaches, and targeting RAS-driven tumors in a specific, effective way. In 2003, a synthetic lethal chemical screen carried out by Dr. Stockwell identified a specific killer of Ras-mutant cells [4]. This compound, called "erastin," causes an iron-dependent form of oxidative cell death, termed "ferroptosis," through the inhibition of a cystine-glutamate antiporter named system xc- [5]. However, the utility of this approach in vivo and the precise role of system xc- in Ras-driven tumors remains unknown. Given the role of mutant RAS in the development and maintenance of PDA, the overall goal of the proposed research is to test the hypothesis that system xc- function is required for the development and maintenance of KRAS mutant pancreatic cancers. Specifically, we aim to interrogate the consequences of genetic and pharmacological manipulation of system xc- on the proliferation and survival of pancreatic cancer cells. Leveraging expertise in molecular genetics, mouse modeling, and preclinical therapeutics, we will interrogate the role of system xc- in the growth and maintenance of PDA and evaluate the clinical viability of targeting this pathway as a means of therapeutic intervention for pancreatic cancer patients. Specifically, we will use shRNA and viral technologies to modulate system xc- in vitro. To study the effects of genetic ablation of system xc- in vivo, we will cross mice deficient for system xc- function to our preclinical model of PDA, the KPC mouse. Finally, we will use a compound, derived from erastin, to evaluate the effect of pharmacological inhibition of system xc- on pancreatic tumor cells both in vitro and in the KPC model. In summary, the experiments proposed herein will aid our understanding of the precise role of redox state maintenance in cancer growth and survival as well as validate the use of system xc- inhibitors in the effective treatment of pancreatic cancer.