UBE2S通过内质网应激蛋白GRP78影响GBM对TMZ耐药性分子机制研究

Glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor of the central nervous system, is characterized by a relentless disease recurrence despite continued advancement in surgery, radiotherapy, and chemotherapy. Resistance to temozolomide (TMZ), a standard chemotherapeutic agent for GBM, remains a major challenge. In our previous study, we found that Akt1 enhanced the stability of UBE2S by phospharylation in PTEN mutant GBM cells. A retrospective/follow-up study showed that UBE2S was significantly upregulated in GBM of chemo- and radiotherapy insensitivity patients. However, the molecular mechanism of UBE2S resistance in GBM is still unclear. Based on the result of protein mass spectrometry, we found that UBE2S interacted with an ER stress response protein (GRP78) which plays an important role in the TMZ chemotherapy resistance in several reports. We hypothesized that the phosphorylated UBE2S interacts with GRP78, which is involved in TMZ resistance to chemotherapy in GBM. To confirm this hypothesis, we plan to study the impacts of UBE2S in temozolomide resistance in GBM and the role of GRP78 in this process by using genomics and proteomics techniques. In this research we will elucidate the molecular mechanism of TMZ resistance in GBM by endoplasmic reticulum stress, and provide a theoretical basis for the molecular pathological classification and Precision Medicine in GBM.

多形性胶质母细胞瘤（GBM）是中枢神经系统最常见的恶性肿瘤。尽管手术切除并辅以放化疗，GBM仍容易复发。替莫唑胺（TMZ）是GBM临床常用化疗药物，但耐药性局限了TMZ的疗效，是GBM化疗失败的主要原因之一。我们前期研究发现PTEN突变的GBM中，Akt1磷酸化UBE2S增强其稳定性。随访发现术后放化疗抵抗的GBM中UBE2S高表达。然而UBE2S参与GBM化疗耐药的分子机制不清。我们通过质谱分析发现UBE2S与内质网应激蛋白GRP78形成复合物，而文献报道GRP78在TMZ化疗耐药中起重要作用。我们推测在GBM中，UBE2S与GRP78相互作用参与TMZ的化疗耐药。为证实此假说，本课题拟用基因组和蛋白组学技术研究UBE2S对GRP78的调控并观察二者在GBM应用TMZ化疗时的反应。本课题将从内质网应激角度阐明GBM对TMZ耐药的分子机制，为GBM分子病理分型及个体化精准治疗提供理论基础。

背景：恶性胶质瘤尤其是胶质母细胞瘤（GBM）是中枢神经内系统中致死率最高的肿瘤类型。现行的胶质瘤治疗标准尚未取得满意的结果，且放化疗耐药已经成为目前制约胶质瘤疗效的主要症结。泛素结合酶E2S（UBE2S）是调控蛋白泛素化修饰的一种关键蛋白，本研究的目的就是进一步探索UBE2S调控胶质瘤对替莫唑胺（TMZ）耐药的机制。.方法：本实验利用蛋白组学实验筛选UBE2S的互作蛋白，并通过免疫共沉淀实验和免疫荧光实验进行机制验证。其次，研究还利用短发夹RNA（shRNA）敲低实验对目的基因进行敲低，验证其对胶质瘤恶性特征的作用及对异种移植模型皮下成瘤的影响。最后使用药物刺激实验，验证UBE2S介导胶质瘤对TMZ耐药的作用机制。.结果：敲低UBE2S可以明显抑制胶质瘤细胞的增殖和侵袭，并且损害了裸鼠皮下注射的胶质瘤细胞的成瘤能力。蛋白组学结果鉴定出UBE2S可以和葡萄糖调节蛋白78（GRP78）相互作用且共定位于细胞膜，并验证了UBE2S的E2酶活性和GRP78的C端是两者结合所必须的。其次，UBE2S可以通过招募泛素特异性蛋白酶15（USP15）抑制GRP78的K48位泛素化修饰，从而维持其稳定性。敲低UBE2S可以导致GRP78蛋白水平降低，并且在面对TMZ刺激时可以激活内质网（ER）应激促凋亡通路，促进CHOP和caspase-3/7的表达和活化，使胶质瘤细胞对化疗更敏感。.结论：本研究首次证实了UBE2S可以招募USP15调控GRP78的K48位泛素化水平降低，抑制其蛋白水解并维持在胶质瘤中的高表达水平，从而持续激活内质网应激反应，使得胶质瘤在面对TMZ刺激时产生抵抗作用。本研究揭示了胶质瘤对放化疗耐药的潜在调控机制，丰富了UBE2S在胶质瘤耐药机制中生物学作用，为克服肿瘤耐药提供了全新的研究方向。UBE2S可能作为有效的药物靶点进而改善胶质瘤的治疗现状。