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。尽管目前的治疗方案有所改进，但耐药的发生仍然限制了抗肿瘤治疗的疗效。逃避凋亡细胞死亡被认为是肿瘤细胞耐药的主要原因。一些实验证据表明，caspase-2（同义词：Nedd2或Ich 1L）表达的缺乏与各种实体肿瘤的耐药相关，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。根据caspase-2作为“损伤传感器”的已知功能，我们之前报道了药物诱导的caspase-2内部核糖体进入段（IRES）依赖性翻译的抑制发生在caspase-2-5´UTR与普遍存在的RNA结合蛋白人抗原R （HuR）的增加，并赋予结肠癌细胞化疗耐药。与caspase-2激活的机制相比，caspase-2翻译的控制及其对治疗耐药的可能影响尚不清楚。利用RNA亲和层析，我们已经鉴定了含有三方基元的蛋白TRIM25（同义：雌激素反应指蛋白，Efp）和E3连接酶作为caspase-2-5´UTR结合蛋白，抑制caspase-2的翻译，从而保护结肠癌细胞免受药物诱导的凋亡。重点将放在鉴定IRES -反式作用因子（ITAFs）作为trim25依赖性泛素化的潜在靶点。该项目部分的数据可以为E3连接酶在控制包括ires依赖性翻译在内的多种RNA功能中的功能作用提供新的信息。由于已知caspase-2由DNA损伤诱导的激酶（如ataxia-毛细血管扩张突变（ATM）或ATM-/ rad3相关（ATR）激酶）控制，我们想要阐明TRIM25本身是否是ATM和/或ATR依赖性磷酸化的直接靶标，因此，对上述两种激酶的药理学抑制剂敏感。该项目的第二部分将强调TRIM25抑制caspase-2对人类结肠癌细胞化疗耐药的功能后果，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TRIM25用于新型肿瘤治疗及其在当前癌症治疗方案中的实施的潜在益处。

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.