Decreasing O-GlcNAcylation affects the malignant transformation of MCF-7 cells via Hsp27 expression and its O-GlcNAc modification

Abstract

O-GlcNAcylation is a dynamic posttranslational modification of nucleoplasmic proteins. Previously, we reported that the O-GlcNAcylation level was increased in primary breast and colorectal cancer tissues. However, its precise roles in cancer development and progression are still largely unexplored. The aim of the present study was to investigate the roles of O-GlcNAcylation in the malignant transformation of cancer cell lines. O-GlcNAcylation level was examined in six cancer cell lines including breast (MCF-7 and MDA-MB-231), colorectal (SW480 and SW620), and liver (SK-Hep1 and HepG2). We found that the levels of O-GlcNAcylation and O-GlcNAc transferase (OGT), an O-GlcNAc catalyzing enzyme, were obviously increased in all cancerous cells, except SK-Hep1, when compared to normal cells. Reducing O-GlcNAcylation using RNA interference against OGT showed a marked reduction in OGT and O-GlcNAcylation levels. Surprisingly, siOGT had no effect on cell growth under conventional monolayer cultures. However, it inhibited anchorage-independent growth in soft agar cultures of all cancer cells, except SK-Hep1. Under anoikis resistance conditions performed by spheroid cultures, siOGT treatment decreased viability only in MCF-7, SW480, and SW620 cells. Among them, OGT knockdown in MCF-7 cells revealed a high inhibitory effect on colony and spheroid cultures. Using two-dimensional gel electrophoresis and mass spectrometric analysis, heat shock protein 27 (Hsp27) was found to be the highest upregulated protein upon OGT knockdown. Immunoblots revealed that the Hsp27 protein level was increased but its O-GlcNAc modification level was decreased in siOGT-treated cells. These changes were associated with the inhibition of MCF-7 cell transformation. Notably, double knockdown of OGT and Hsp27 showed a reversal in the inhibitory effect on colony and spheroid cultures. Collectively, these results indicate that O-GlcNAcylation is required for anoikis resistance and anchorage-independent growth of MCF-7 cells. Blocking this glycosylation by OGT knockdown may regulate both Hsp27 protein expression and its O-GlcNAc modification levels. This alteration may play vital roles in malignant transformation.

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

“…Reducing NPM1 expression using siRNA resulted in a significant decrease in cell proliferation and induced cell cycle arrest in leukemia, colon and breast cancer (30)(31)(32). Moreover, the present study supports the results of previous studies which have demonstrated that O-GlcNAc regulates the cellular stress responses via the expression of numerous HSPs, including HSP27 (14,33). HSP27 expression levels affect cell proliferation, migration and invasion abilities in several types of cancer, such as liver, prostate and breast cancer (34)(35)(36).…”

“…OGT is essential for cell viability and OGT depletion results in embryonic lethality (20). Moreover, our previous study revealed that OGT knockdown caused a marked decrease in cell viability under the anoikis resistant conditions (14). Furthermore, OGT silencing reduces the migratory and invasive capacities in a number of different types of cancer including breast (21), prostate (22), ovarian (23) and colon cancer (24).…”

“…In our previous studies, it was demonstrated that O-GlcNAcylation was increased in primary breast and colorectal cancer tissues (12,13). Moreover, O-GlcNAcylation is required for anoikis resistance and anchorage-independent growth, which are vital steps in the progression of breast cancer (14). However, the precise roles of this modification regarding malignant transformation are yet to be elucidated.…”

“…no. mAb3700; Cell Signaling Technology, Inc.), with overnight incubation at 4˚C in 1% PBS-casein blocking buffer (Bio-Rad Laboratories, Inc.) as previously described (14). The membranes were incubated with the corresponding secondary antibodies, including swine anti-rabbit (1:5,000; cat.…”

“…no. 7074; Cell Signaling Technology, Inc.) secondary antibody and the detection was performed, as described previously (14).…”