Proteomic analysis and abrogated expression ofO-GlcNAcylated proteins associated with primary breast cancer

Abstract

O-GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, which catalyze the addition and removal of O-GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor-associated proteins are O-GlcNAc modified, the total O-GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O-GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O-GlcNAc immnoblotting and LC-MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O-GlcNAcylated or associated with O-GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O-GlcNAcylated or associated with O-GlcNAcylation. Moreover, OGT knockdown showed that decreasing O-GlcNAcylation was related to inhibition of the anchorage-independent growth in vitro. These data indicate that aberrant protein O-GlcNAcylation is associated with breast cancer. Abnormal modification of these O-GlcNAc-modified proteins might be one of the vital malignant characteristics of cancer.

Aberrant O-GlcNAcylated Proteins: New Perspectives in Breast and Colorectal Cancer

“…We have shown that OGT silencing led to a reduction of anchorage-independent growth of a breast cancer cell line, MDA-MB-231 (25). Caldwell et al also reported that reduction of OGT in breast cancer cells caused inhibition of tumor growth, both in vitro and in vivo (43).…”

“…Blocking of O -GlcNAcylated PFK1 led to a reduction of cancer cell proliferation in vitro and impaired tumor formation in vivo . Other glycolytic enzymes also reported to be modified by O -GlcNAc include triose phosphate isomerase (TPI) (21), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (22–25), enolase 2 (Eno2) (22, 25–27), and pyruvate kinase M2 (PKM2) (25). Although the glycosylation sites of these glycolytic enzymes have not been identified, their modifications could potentially modulate tumor cell metabolism promoting proliferation.…”

“…Accumulating evidence reveals that the levels of O -GlcNAcylation and its cycling enzymes in malignant tissues are altered in various cancers. Recently, we showed that O -GlcNAcylation and OGT expression levels are increased in both breast and CRC (25, 40). As mentioned above, the O -GlcNAc cycling enzymes OGT and OGA tightly regulate the level of O -GlcNAcylation.…”

O-GlcNAc signaling in cancer metabolism and epigenetics

“…Cancer cells exhibit increased HBP flux and O-GlcNAcylation of multiple metabolic enzymes [18, 70]. The O-GlcNAc moiety has been detected on a majority of glycolytic enzymes.…”

“…Pyruvate kinase catalyzes the last committed step in glycolysis. O-GlcNAcylated pyruvate kinase muscle isozyme 2 (PKM2) is present in breast cancer but not the normal tissues; however, whether O-GlcNAcylation of PKM2 plays a regulatory role remains unknown [18]. Additionally, proteomic analysis reveals the presence of O-GlcNAc moieties on many enzymes involved in amino acid and nucleotide metabolism, such as, phosphoglycerate dehydrogenase (PHGDH), argininosuccinate synthetase (ASS), and thymidylate synthase (TYMS) [80].…”