MK-STYX: A Requisite Gatekeeper to Mitochondrial Function and Death

描述（由申请人提供）：尽管临床医生可使用的靶向治疗选择越来越多，但耐药转移性疾病对癌症患者构成了最严重的威胁。虽然对晚期癌症进展的深入研究揭示了许多导致化疗耐药性的机制，但对使细胞对细胞毒性化疗脱敏的细胞内信号传导机制知之甚少。为了解决这一知识缺口，我们最近进行了一项大规模的RNA干扰（RNAi）筛选，旨在全面鉴定人类基因组中改变或改变肿瘤细胞对化疗药物敏感性的关键激酶和磷酸酶。在这个RNAi筛选中，我们鉴定了一种新的磷酸酶MK-STYX，它有效地抑制了肿瘤细胞对各种化疗药物的反应。我们的中心假设是MK-STYX通过调节ATP合成所需机制的磷酸化来特异性控制线粒体功能，从而在诱导化疗诱导的细胞死亡中发挥重要作用。本项目的目的是确定MK-STYX如何调节细胞ATP水平，从而调节内源性凋亡。我们提出以下具体目标来解决这一假设，并了解其在转移性结直肠癌中的意义：（1）确定MK-STYX在线粒体中的催化机制;（2）确定MK-STYX调节化疗耐药性的机制;（3）确定MK-STYX在结直肠癌进展和化疗耐药性中的作用。与我们的中心假设一致，我们已经表明，MK-STYX的损失增加ATP的生产。因此，我们预测，由于MK-STYX的损失而导致的细胞ATP的升高足以抑制线粒体的形成和进入凋亡。我们已经表明，MK-STYX与两个额外的线粒体蛋白相互作用，我们将机械地确定线粒体功能和这些相互作用的分子后果。我们还发现MK-STYX表达的缺失与结直肠癌进展相关。为了确定MK-STYX的丧失是否介导体内化学抗性，我们将使用表现出可变的MK-STYX表达的细胞系或已被操作以降低内源性MK-STYX水平的细胞系来测试标准化疗对结肠直肠异种移植模型的功效。我们还将确定MK-STYX蛋白水平在结直肠癌患者队列中的预后意义。公共卫生相关性：我们最近鉴定了13种抑制化疗耐药性或其表达驱动化疗敏感性的磷酸酶，为更好地理解化疗耐药性的分子基础提供了途径。如果我们能够确定这些磷酸酶的表达缺失如何导致化疗耐药性的发展，我们将能够更好地筛选化疗耐药性并设计合理的药物策略来治疗癌症患者的化疗耐药肿瘤。

DESCRIPTION (provided by applicant): Chemoresistant metastatic disease presents the most serious threat to cancer patients despite the increased arsenal of targeted therapeutic options available to clinicians. While intense study into late-stage cancer progression has revealed a number of mechanisms that contribute to chemoresistance, little is known about the intracellular signaling mechanisms that desensitize cells to cytotoxic chemotherapy. In an effort to address this knowledge gap, we recently performed a large-scale RNA-interference (RNAi) screen intended to comprehensively identify critical kinases and phosphatases in the human genome that alter or modify tumor cell sensitivity to chemotherapeutic agents. In this RNAi screen, we identified a novel phosphatase, MK-STYX, which potently suppressed the response of tumor cells to a wide variety of chemotherapeutic drugs. Our central hypothesis is that MK-STYX specifically controls mitochondrial function by regulating phosphorylation of the machinery required for ATP synthesis, and thereby serves an essential role in the induction of chemotherapeutic-induced cell death. The objective of this project is to determine how MK-STYX regulates cellular ATP levels, and thus modulates intrinsic apoptosis. We propose the following specific aims to address this hypothesis and to understand its significance in the context of metastatic colorectal carcinoma: (1) Identify the catalytic mechanism of MK-STYX in the mitochondria; (2) Identify the mechanism whereby MK-STYX regulates chemoresistance; (3) Establish the role of MK-STYX in colorectal cancer progression and chemoresistance. Consistent with our central hypothesis, we have shown that loss of MK-STYX increases ATP production. Therefore, we predict that the elevation in cellular ATP due to loss of MK-STYX is sufficient to inhibit apoptosome formation and entry into apoptosis. We have shown that MK-STYX interacts with two additional mitochondrial proteins and we will mechanistically determine the mitochondrial function and the molecular consequences of each of these interactions. We have also shown that loss of MK-STYX expression correlates with colorectal cancer progression. To determine whether loss of MK-STYX mediates chemoresistance in vivo, we will test the efficacy of standard chemotherapies on a colorectal xenograft model using cell lines that demonstrate variable expression of MK-STYX, or have been manipulated to decrease endogenous MK-STYX levels. We will also determine the prognostic significance of MK-STYX protein levels in a cohort of patients with colorectal cancer. PUBLIC HEALTH RELEVANCE: Our recent identification of thirteen phosphatases that suppress chemoresistance, or whose expression drives chemosensitivity, has provided an avenue for a better understanding of the molecular basis of chemoresistance. If we can determine how loss of expression of these phosphatases leads to the development of chemoresistance, we will be better able to screen for chemoresistance and design rational drug strategies to treat chemoresistant tumors in cancer patients.