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

描述(由申请人提供)：耐化疗的转移性疾病是对癌症患者最严重的威胁，尽管临床医生可用的靶向治疗选择增加了。虽然对晚期癌症进展的密集研究揭示了许多导致化疗耐药的机制，但对使细胞对细胞毒性化疗不敏感的细胞内信号机制知之甚少。为了解决这一知识鸿沟，我们最近进行了一项大规模的RNA干扰(RNAi)筛查，旨在全面识别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，我们发现了一种新的磷酸酶，MK-Styx，它能有效地抑制肿瘤细胞对多种化疗药物的反应。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地控制线粒体的功能，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如何调节细胞内的ATP水平，从而调节内在的细胞凋亡。我们提出以下具体目标来解决这一假说，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调节化疗耐药的机制；(3)确定MK-Styx在结直肠癌进展和化疗耐药中的作用。与我们的中心假设一致，我们已经表明MK-Styx的丢失增加了ATP的产生。因此，我们预测由于MK-Styx的缺失而导致的细胞内ATP的升高足以抑制凋亡体的形成和进入细胞凋亡。我们已经证明MK-Styx与另外两种线粒体蛋白相互作用，我们将从机械上确定线粒体的功能和每一种相互作用的分子后果。我们还发现MK-Styx表达缺失与结直肠癌的进展相关。为了确定MK-Styx的缺失是否在体内介导了化疗耐药，我们将在结直肠癌异种移植模型上测试标准化疗的有效性，使用的细胞株表现出MK-Styx的不同表达，或者已经被操纵以降低内源性MK-Styx的水平。我们还将确定MK-Styx蛋白水平在一组结直肠癌患者中的预后意义。公共卫生相关性：我们最近发现了13种抑制化疗耐药或其表达驱动化疗敏感性的磷酸酶，这为更好地理解化疗耐药的分子基础提供了一条途径。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的发生，我们将能够更好地筛选化疗耐药，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.