Quantitative proteomic analysis of the association between decreasing O‑GlcNAcylation and metastasis in MCF‑7 breast cancer cells

Breast cancer is the most common type of cancer and leading cause of cancerassociated mortality in women worldwide. Olinked Nacetyl glucosaminylation (OGlcNAcylation) is a dynamic posttranslational modification of nuclear, cytoplasmic and mitochondrial proteins. Mounting evidence suggests that abnormal OGlcNAcylation status is associated with cancer malignancy. In our previous study, it was reported that OGlcNAc and OGlcNAc transferase (OGT; an enzyme responsible for the addition of OGlcNAc) were upregulated in breast cancer tissues and cells. Moreover, OGlcNAcylation was required for resistance to anoikis and the anchorageindependent 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 OGlcNAc on the malignant transformation of MCF7 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 OGlcNAc levels using small interfering (si)RNA targeting OGT resulted in a reduction in cell viability and invasiveness in anoikis resistant and reseeding conditions. Furthermore, gelfree quantitative proteomics was performed to identify the proteins affected by a reduction of OGlcNAc. 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 OGlcNAcylation altered protein expression, and ultimately influenced the metastatic processes, particulary regarding the invasion and reattached growth of MCF7 breast cancer cells.