Laboratory of Biochemistry, Chulabhorn Research Institue, Bangkok 10210, Thailand.
Int J Oncol. 2020 Jun;56(6):1387-1404. doi: 10.3892/ijo.2020.5022. Epub 2020 Mar 20.
Breast cancer is the most common type of cancer and leading cause of cancer‑associated mortality in women worldwide. O‑linked N‑acetyl glucosaminylation (O‑GlcNAcylation) is a dynamic post‑translational modification of nuclear, cytoplasmic and mitochondrial proteins. Mounting evidence suggests that abnormal O‑GlcNAcylation status is associated with cancer malignancy. In our previous study, it was reported that O‑GlcNAc and O‑GlcNAc transferase (OGT; an enzyme responsible for the addition of O‑GlcNAc) were upregulated in breast cancer tissues and cells. Moreover, O‑GlcNAcylation was required for resistance to anoikis and the anchorage‑independent growth of breast cancer cells. However, the precise roles of this modification on the development of malignancy are yet to be elucidated. Therefore, in the present study, the effects of inhibiting O‑GlcNAc on the malignant transformation of MCF‑7 breast cancer cells under different culture conditions were determined, using monolayer (primary growth), anoikis resistance (spheroid growth) and reseeding (secondary growth) to mimic the metastatic process. Decreasing O‑GlcNAc levels using small interfering (si)RNA targeting OGT resulted in a reduction in cell viability and invasiveness in anoikis resistant and reseeding conditions. Furthermore, gel‑free quantitative proteomics was performed to identify the proteins affected by a reduction of O‑GlcNAc. A total of 317 proteins were identified and compared, and the expression of 162 proteins was altered >1.5 fold in the siOGT treated cells compared with the siScamble (siSC) treated cells. Notably, 100 proteins involved in cellular metabolism, cellular localization, stress responses and gene expression were significantly altered in the reseeding condition. Among these differentially expressed proteins, the levels of small nuclear ribonucleoprotein Sm D1 exhibited the largest decrease in expression following knockdown of OGT, and this reduction in expression was associated with a significant decrease in the levels of mTOR expression, a protein which promotes tumor growth and progression. Taken together, the results of the present study demonstrate that decreasing O‑GlcNAcylation altered protein expression, and ultimately influenced the metastatic processes, particulary regarding the invasion and reattached growth of MCF‑7 breast cancer cells.
乳腺癌是全球女性最常见的癌症类型和癌症相关死亡的主要原因。O-连接的 N-乙酰氨基葡萄糖基化 (O-GlcNAcylation) 是核、细胞质和线粒体蛋白的一种动态翻译后修饰。越来越多的证据表明，异常的 O-GlcNAcylation 状态与癌症恶性程度有关。在我们之前的研究中，据报道 O-GlcNAc 和 O-GlcNAc 转移酶 (OGT；负责添加 O-GlcNAc 的酶) 在乳腺癌组织和细胞中上调。此外，O-GlcNAcylation 是乳腺癌细胞抵抗凋亡和锚定非依赖性生长所必需的。然而，这种修饰对恶性发展的确切作用仍有待阐明。因此，在本研究中，通过使用单层（原代生长）、抗凋亡（球体生长）和再接种（继发生长）来模拟转移过程，确定抑制 O-GlcNAc 对 MCF-7 乳腺癌细胞恶性转化的影响。使用靶向 OGT 的小干扰 (si)RNA 降低 O-GlcNAc 水平可降低抗凋亡和再接种条件下细胞活力和侵袭性。此外，还进行了无胶定量蛋白质组学分析，以鉴定受 O-GlcNAc 减少影响的蛋白质。共鉴定和比较了 317 种蛋白质，与 siScamble (siSC) 处理细胞相比，siOGT 处理细胞中有 162 种蛋白质的表达变化 >1.5 倍。值得注意的是，在再接种条件下，有 100 种参与细胞代谢、细胞定位、应激反应和基因表达的蛋白质发生了显著变化。在这些差异表达的蛋白质中，小核核糖核蛋白 Sm D1 的水平在 OGT 敲低后表达下降最大，这种表达下降与 mTOR 表达水平的显著下降有关，mTOR 是一种促进肿瘤生长和进展的蛋白质。总之，本研究结果表明，降低 O-GlcNAcylation 改变了蛋白质表达，最终影响了 MCF-7 乳腺癌细胞的转移过程，特别是细胞侵袭和再附着生长。
