Experimental Pathology and Therapeutics Group, Research Center (CI-IPOP), Portuguese Institute of Oncology, 4200-072, Porto, Portugal.
Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313, Porto, Portugal.
J Exp Clin Cancer Res. 2021 Jun 9;40(1):191. doi: 10.1186/s13046-021-01988-6.
Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential.
A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals.
Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues.
HOMER3-glycoforms allow the identification of patients' subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established.
由于治疗失败和广泛的分子异质性，肌肉浸润性膀胱癌（MIBC）仍然是最致命的泌尿生殖系统恶性肿瘤之一，延迟了有效的靶向治疗。缺氧和营养缺乏、过度唾液酸化和 O-聚糖缩短是侵袭性肿瘤的显著特征，形成具有治疗潜力的细胞表面糖蛋白组指纹图谱。
使用侵袭性膀胱癌细胞模型，采用糖组学指导的糖蛋白质组学工作流程来鉴定潜在的可靶向生物标志物。通过质谱（MS）对 5637 和 T24 细胞的 O-聚糖组进行了表征，并将获得的信息用于指导糖蛋白质组学实验，结合唾液酸酶、凝集素亲和和自上而下的纳升液相色谱-电喷雾-串联质谱（nanoLC-ESI-MS/MS）蛋白鉴定。通过内部开发的生物信息学方法对数据进行了整理，根据人类蛋白质图谱的见解对基于临床相关的分子特征进行分类。通过 MS 和免疫测定在细胞模型、膀胱癌肿瘤和转移中验证了排名靠前的靶标和糖型。在缺氧和葡萄糖剥夺下生长的细胞揭示了肿瘤微环境对相关生物标志物表达的贡献。通过 20 种不同个体的组织/细胞的免疫组织化学和 MS 在健康组织中验证了癌症特异性。
发现唾液酸化 T（ST）抗原是细胞系中最丰富的聚糖，鉴定出超过 900 种可能携带这些聚糖的糖蛋白。通常定位于细胞质的 HOMER3 作为一种排名靠前的可靶向糖蛋白出现在细胞膜上，携带短链 O-聚糖。在更具侵袭性的原发性肿瘤和远处转移中观察到质膜 HOMER3，是最差预后的独立预测因子。这种表型是由营养缺乏和细胞侵袭增加触发的。T24 HOMER3 敲低显著降低了正常氧和缺氧条件下的增殖，在一定程度上降低了侵袭性；而 HOMER3 敲入增加了其膜表达，在葡萄糖剥夺下更为明显。HOMER3 的过表达与正常氧条件下的细胞增殖增加有关，并在缺氧条件下增强了侵袭。最后，通过 EThcD-MS/MS 在膀胱癌中对 HOMER3 糖基位点进行映射，揭示了在健康组织中未检测到的潜在可靶向结构域。
HOMER3-糖型可用于鉴定预后最差的患者亚群，有可能针对缺氧细胞，且具有有限的脱靶效应。已经确定了鉴定新的膀胱癌分子靶标的分子原理。
