The role of the E3 igase TRIM-25 in the control of caspase-2 translation and its implication for chemotherapy resistance of colon carcinoma cells

结直肠癌是西方世界最常见的癌症之一。尽管目前有改进的治疗方案，但耐药性的发生仍然限制了抗肿瘤治疗的疗效。逃避凋亡性细胞死亡被认为是癌细胞治疗抗性的主要原因。一些实验证据表明，caspase-2（同义词：Nedd 2或Ich 1 L）表达的缺陷与各种实体瘤的耐药性相关，支持这种进化高度保守的caspase在人类癌症中的肿瘤抑制功能。为了与caspase-2作为“损伤传感器”的已知功能保持一致，我们先前报道了药物诱导的对caspase-2的内部核糖体进入片段（IRES）依赖性翻译的抑制通过增加caspase-2-5 'UTR与普遍存在的RNA结合蛋白人抗原R（HuR）的结合而发生，并赋予结肠癌细胞化疗药物抗性。与半胱天冬酶-2活化的机制相比，对半胱天冬酶-2翻译的控制及其对治疗抗性的可能影响知之甚少。使用RNA亲和层析，我们已经鉴定了包含三部分基序的蛋白TRIM 2 - 5（同义词：雌激素反应指蛋白，Efp），一种E3连接酶，作为另一种caspase-2-5 ′ UTR结合蛋白，其抑制caspase-2翻译，进而保护结肠癌细胞免受药物诱导的凋亡。重点将放在识别IRES反式作用因子（ITAFs）作为TRIM 25依赖性泛素化的潜在靶点。该项目部分的数据可以提供有关E3连接酶在控制包括IRES依赖性翻译在内的多种RNA功能中的功能作用的新信息。由于半胱天冬酶-2已知由DNA损伤诱导的激酶如共济失调-毛细血管扩张突变（ATM）或ATM-/Rad 3-相关（ATR）激酶控制，我们想要阐明TRIM 25本身是否是ATM和/或ATR依赖性磷酸化的直接靶点，因此对这两种激酶的药理学抑制剂敏感。该项目的第二部分将强调TRIM 25抑制caspase-2对人结肠癌细胞化疗耐药性的功能后果，特别关注caspase-2触发和化疗诱导的DNA损伤反应，包括细胞周期阻滞和凋亡。该项目的数据有助于评估靶向TRIM 25用于新型肿瘤治疗的潜在益处及其在当前癌症治疗方案中的实施。

Colorectal cancer is one of the most common cancers in the western world. Despite current improved treatment options, the occurrence of drug resistance does still limit the efficacy of antitumour treatments. An evasion of apoptotic cell death is thought as a major reason for therapy resistance of cancer cells. Several lines of experimental evidence implicate that a deficiency in the expression of caspase-2 (synonymic: Nedd2 or Ich 1L) expression correlates with drug resistance of various solid tumours supporting the tumoursuppressive function of this evolutionary highly conserved caspase in human carcinomas. In keeping with the known function of caspase-2 to act as a “damage sensor”, we previously reported that drug-induced inhibition of internal ribosome entry segment (IRES)-dependent translation of caspase-2 occurs through an increased caspase-2-5´UTR binding of the ubiquitous RNA binding protein human antigen R (HuR) and confers chemotherapeutic drug resistance in colon carcinoma cells. In comparison to the mechanisms of caspase-2 activation, the control of caspase-2 translation and its possible impact for therapy resistance is only poorly understood. Using RNA affinity chromatography, we have identified the tri-partite motif-containing protein TRIM25 (synonym: estrogen response finger protein, Efp) an E3 ligase as a further caspase-2-5´UTR binding protein which inhibits caspase-2 translation and in turn protects colon carcinoma cells from drug-induced apoptosis. The major emphasis will be put on the identification of IRES -trans -acting factors (ITAFs) as potential targets of TRIM25-dependent ubiquitination. Data from this project part could provide novel information about the functional role of E3 ligases in the control of diverse RNA functions including IRES-dependent translation. Since caspase-2 is known to be controlled by the DNA damage-induced kinases such as ataxia-telangiectasia mutated (ATM) or ATM-/Rad3-related (ATR) kinase, we want to elucidate whether TRIM25 itself is a direct target of ATM and/or ATR-dependent phosphorylation and consequently, is sensitive to pharmacological inhibitors of both mentioned kinases. The second part of the project will emphasize on the functional consequences of caspase-2 suppression by TRIM25 for chemotherapy resistance of human colon carcinoma cells with a particular focus on caspase-2-triggered and chemotherapeutics-induced DNA damage responses including cell cycle arrest and apoptosis. Data from this project help to evaluate potential benefits of targeting TRIM25 for novel tumour therapies and their implementation in current cancer therapy regimen.