This study explores the relationship between anti-proliferative signaling by transforming growth factor-beta (TGF-beta) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-beta1. To investigate the mechanism, we used T47D cells that lack type II TGF-beta receptor (TGF-betaRII) and are insensitive to TGF-beta1. After introducing the TGF-betaRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-beta1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-beta1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-betaRII-expressing cells when exogenous TGF-beta1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-beta1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-betaRII and exogenous TGF-beta1. To investigate this synergism, the phosphorylation of TGF-beta signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-beta1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-beta signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.

Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-LC-MS/MS-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6±13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by Western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an over-representation of high mannose, hybrid and paucimannosidic type N-glycans and an under-representation of complex N-glycans (P<0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated (P<0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N-glycome profiles. EGFR-specific N-glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P<0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N-glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.