Wnt/B-catenin signaling plays an essential role in colon carcinogenesis. Galectin-3, a B-galactoside-binding protein, has been implicated in Wnt signaling, but the precise mechanisms by which galectin-3 modulates the Wnt pathway are unknown. In the present study, we determined the effects of galectin-3 on the Wnt/B-catenin pathway in colon cancer cells, as well as the mechanisms involved. Galectin-3 levels were manipulated in human colon cancer cells by stable transfection of galectin-3 antisense, short hairpin RNA, or fulllength galectin-3 cDNA, and effects on B-catenin levels, subcellular distribution, and Wnt signaling were determined. Galectin-3 levels correlated with B-catenin levels in a variety of colon cancer cell lines. Down-regulation of galectin-3 resulted in decreased B-catenin protein levels but no change in B-catenin mRNA levels, suggesting that galectin-3 modulates B-catenin by another mechanism. Reduction of galectin-3 led to reduced nuclear B-catenin with a concomitant decrease in TCF4 transcriptional activity and expression of its target genes. Conversely, transfection of galectin-3 cDNA into colon cancer cells increased B-catenin expression and TCF4 transcriptional activity. Down-regulation of galectin-3 resulted in AKT and glycogen synthase kinase-3B (GSK-3B) dephosphorylation and increased GSK activity, increasing B-catenin phosphorylation and degradation. Ly294002, an inhibitor of phosphatidylinositol 3-kinase, and dominant-negative AKT, suppressed TCF4 transcriptional activity induced by galectin-3 whereas LiCl, a GSK-3B inhibitor, increased TCF4 activity, mimicking the effects of galectin-3. These results suggest that galectin-3 mediates Wnt signaling, at least in part, by regulating GSK-3B phosphorylation and activity via the phosphatidylinositol 3-kinase/AKT pathway, and, thus, the degradation of B-catenin in colon cancer cells. [Cancer Res 2009;69(4):1343-9]

TRAIL induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors resulting in the internalization of ligand-receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL-death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL-resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL-sensitivity and promotes TRAIL-mediated endocytosis of TRAIL-death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand-receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.

Notch pathway plays an important role in the growth of high-grade serous ovarian (HGS-OvCa) and other cancers, but its clinical and biological mechanisms are not well understood. Here, we found that the Notch pathway alterations are prevalent and significantly related to poor clinical outcome in patients with ovarian cancer. Particularly, Notch3 alterations, including amplification and upregulation, were highly associated with poor patient survival. Targeting Notch3 inhibited OvCa growth and induced apoptosis. Importantly, we found that DNM-mediated endocytosis was required for selectively activating Jagged-1-mediated Notch3 signaling. Cleaved Notch3 expression was the critical determinant of response to Notch-targeted therapy. Collectively, these data identify previously unknown mechanisms underlying Notch3 signaling and identify new, biomarker-driven approaches for therapy.

Dll4, one of the Notch ligands, is overexpressed in ovarian cancer, especially in tumors resistant to anti–VEGF therapy. Here, we examined the biological effects of dual anti-Dll4 and anti-VEGF therapy in ovarian cancer models. Using Dll4-Fc blockade and anti-Dll4 antibodies (murine REGN1035 and human REGN421), we evaluated the biological effects of Dll4 inhibition combined with aflibercept or chemotherapy in orthotopic mouse models of ovarian cancer. We also examined potential mechanisms by which dual Dll4 and VEGF targeting inhibits tumor growth using immunohistochemical staining for apoptosis and proliferation markers. Reverse phase protein arrays were used to identify potential downstream targets of Dll4 blockade. Dual targeting of VEGF and Dll4 with murine REGN1035 showed superior anti-tumor effects in ovarian cancer models compared to either monotherapy. In the A2780 model, REGN1035 (targets murine Dll4) or REGN421 (targets human Dll4) reduced tumor weights by 62% and 82%, respectively; aflibercept alone reduced tumor weights by 90%. Greater therapeutic effects were observed for Dll4 blockade (REGN1035) combined with either aflibercept or docetaxel P < 0.05 for the combination vs. aflibercept). The superior anti-tumor effects of REGN1035 and aflibercept were related to increased apoptosis in tumor cells compared to the monotherapy. We also found that GATA3 expression was significantly increased in tumor stroma from the mice treated with REGN1035 combined with docetaxel or aflibercept, suggesting an indirect effect of these combination treatments on the tumor stroma. These findings identify that dual targeting of Dll4 and VEGF is an attractive therapeutic approach.

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