Zhou Shu-Min, Cheng Li, Guo Shu-Juan, Wang Yang, Czajkowsky Daniel M, Gao Huafang, Hu Xiao-Fang, Tao Sheng-Ce
Breast Cancer Res. 2015 Mar 11;17(1):36. doi: 10.1186/s13058-015-0544-9.
Triple-negative breast cancer (TNBC) patients often face a high risk of early relapse characterized by extensive metastasis. Previous works have shown that aberrant cell surface glycosylation is associated with cancer metastasis, suggesting that altered glycosylations might serve as diagnostic signatures of metastatic potential. To address this question, we took TNBC as an example and analyzed six TNBC cell lines, derived from a common progenitor, that differ in metastatic potential.
We used a microarray with 91 lectins to screen for altered lectin bindings to the six TNBC cell lines. Candidate lectins were then verified by lectin-based flow cytometry and immunofluorescent staining assays using both TNBC/non-TNBC cancer cells. Patient-derived tissue microarrays were then employed to analyze whether the staining of Ricinus communis agglutinin I (RCA-I), correlated with TNBC severity. We also carried out real-time cell motility assays in the presence of RCA-I. Finally, liquid chromatography-mass spectrometry/tandem spectrometry (LC-MS/MS) was employed to identify the membrane glycoproteins recognized by RCA-I.
Using the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity. Tissue microarray experiments showed that the intensity of RCA-I staining is positively correlated with the TNM grades. The real-time cell motility assays clearly demonstrated RCA-I inhibition of adhesion, migration, and invasion of TNBC cells of high metastatic capacity. Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.
We discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects. These results thus indicate that RCA-I-specific cell surface glycoproteins may play a critical role in TNBC metastasis and that the extent of RCA-I cell binding could be used in diagnosis to predict the likelihood of developing metastases in TNBC patients.
三阴性乳腺癌（TNBC）患者常面临早期复发的高风险，其特征为广泛转移。先前的研究表明，异常的细胞表面糖基化与癌症转移相关，这表明糖基化改变可能作为转移潜能的诊断标志物。为解决这一问题，我们以TNBC为例，分析了源自共同祖细胞、转移潜能不同的六种TNBC细胞系。
我们使用含有91种凝集素的微阵列筛选六种TNBC细胞系中凝集素结合的改变。然后通过基于凝集素的流式细胞术和免疫荧光染色试验，使用TNBC/非TNBC癌细胞对候选凝集素进行验证。接着使用患者来源的组织微阵列分析蓖麻凝集素I（RCA-I）的染色是否与TNBC严重程度相关。我们还在RCA-I存在的情况下进行实时细胞运动分析。最后，采用液相色谱-质谱/串联质谱（LC-MS/MS）鉴定RCA-I识别的膜糖蛋白。
使用凝集素微阵列，我们发现RCA-I与TNBC细胞的结合与其转移能力成正比。组织微阵列实验表明，RCA-I染色强度与TNM分级呈正相关。实时细胞运动分析清楚地证明RCA-I抑制高转移能力的TNBC细胞的黏附、迁移和侵袭。此外，通过LC-MS/MS鉴定出一种膜糖蛋白，即POTE锚蛋白结构域家族成员F（POTEF），在高/低转移TNBC细胞中具有不同程度的半乳糖基化，是RCA-I的结合蛋白。
我们发现RCA-I与TNBC细胞的结合程度与其转移能力成正比，且这种结合抑制细胞侵袭、迁移和黏附，并鉴定出一种膜蛋白POTEF，其可能在介导这些效应中起关键作用。因此，这些结果表明，RCA-I特异性细胞表面糖蛋白可能在TNBC转移中起关键作用，且RCA-I与细胞的结合程度可用于诊断，以预测TNBC患者发生转移的可能性。
