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效果。对凋亡细胞死亡的逃避被认为是癌细胞产生治疗抵抗的主要原因。多条实验证据表明，caspase-2(同义词：Nedd2或ICH 1L)表达不足与各种实体肿瘤的耐药性有关，支持这种进化上高度保守的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，药物诱导抑制依赖于内部核糖体进入片段(IRES)的caspase-2翻译是通过增加普遍存在的RNA结合蛋白人类抗原R(Hur)与caspase-2-5‘UTR的结合而实现的，并导致结肠癌细胞对化疗药物的耐药性。与caspase-2激活的机制相比，caspase-2翻译的调控及其对治疗耐药的可能影响还知之甚少。利用RNA亲和层析，我们已经鉴定了含有三个基序的蛋白TRIM25(同义词：雌激素反应手指蛋白，EFP)和E3连接酶是另一个caspase-2-5‘UTR结合蛋白，它抑制caspase-2的翻译，进而保护结肠癌细胞免受药物诱导的凋亡。主要的重点将放在确定IRES反式作用因子(ITAFs)作为TRIM25依赖的泛素化的潜在靶标上。来自该项目部分的数据可能提供有关E3连接酶在控制包括IRES依赖的翻译在内的多种RNA功能中的功能作用的新信息。由于已知caspase-2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-/RAD3相关(ATR)激酶控制，我们想要阐明TRIM25本身是否是ATM和/或ATR依赖的磷酸化的直接靶点，从而对这两种激酶的药物抑制剂敏感。该项目的第二部分将侧重于TRIM25抑制caspase-2对人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，包括细胞周期停滞和细胞凋亡。来自该项目的数据有助于评估将TRIM25作为新的肿瘤疗法的靶点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.