Activation of Vitamin D/VDR Signaling Reverses Gemcitabine Resistance of Pancreatic Cancer Cells Through Inhibition of MUC1 Expression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to its therapeutic resistance. Inactivation of vitamin D/vitamin D receptor (VDR) signaling may contribute to the malignant phenotype of PDA and altered expression of oncoprotein mucin 1 (MUC1) may be involved in drug resistance of cancer cells. AIM: To determine whether vitamin D/VDR signaling regulates the expression and function of MUC1 and its effect on acquired gemcitabine resistance of pancreatic cancer cells. METHODS: Molecular analyses and animal models were used to determine the impact of vitamin D/VDR signaling on MUC1 expression and response to gemcitabine treatment. RESULTS: RPPA analysis indicated that MUC1 protein expression was significantly reduced in human PDA cells after treatment with vitamin D3 or its analog calcipotriol. VDR regulated MUC1 expression in both gain- and loss-of-function assays. Vitamin D3 or calcipotriol significantly induced VDR and inhibited MUC1 expression in acquired gemcitabine-resistant PDA cells and sensitized the resistant cells to gemcitabine treatment, while siRNA inhibition of MUC1 was associated with paricalcitol-associated sensitization of PDA cells to gemcitabine treatment in vitro. Administration of paricalcitol significantly enhanced the therapeutic efficacy of gemcitabine in xenograft and orthotopic mouse models and increased the intratumoral concentration of dFdCTP, the active metabolite of gemcitabine. CONCLUSION: These findings demonstrate a previously unidentified vitamin D/VDR-MUC1 signaling axis involved in the regulation of gemcitabine resistance in PDA and suggests that combinational therapies that include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA.

Galectin-3 Mediates Tumor Cell-Stroma Interactions by Activating Pancreatic Stellate Cells to Produce Cytokines via Integrin Signaling.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by activated pancreatic stellate cells (PSCs), abundance of extracellular matrix (ECM), and production of cytokines and chemokines. Galectin 3 (GAL3), a ß-galactoside-specific lectin, contributes to PDAC development but its effects on the stroma and cytokine production are unclear. METHODS: The effect of recombinant human GAL3 (rGAL3) on activation of PSCs, production of cytokines, and ECM proteins was determined by proliferation, invasion, cytokine array, and quantitative polymerase chain reaction. We assessed co-cultures of PDAC cells with GAL3 genetic alterations with PSCs. Production of interleukin 8 (IL8) and activities of nuclear factor (NF)-κB were determined by enzyme-linked immunosorbent assay and luciferase reporter analyses. We studied the effects of inhibitors of NF-κB and integrin-linked kinase (ILK) on pathways activated by rGAL3. RESULTS: In analyses of the Gene Expression Omnibus database and our dataset, we observed higher levels of GAL3, IL8, and other cytokines in PDAC than in nontumor tissues. Production of IL8, granulocyte-macrophage colony-stimulating factor, chemokine ligand 1, and C-C motif chemokine ligand 2 increased in PSCs exposed to rGAL3 compared with controls. Culture of PSCs with PDAC cells that express different levels of GAL3 resulted in proliferation and invasion of PSCs that increased with level of GAL3. GAL3 stimulated transcription of IL8 through integrin subunit beta 1 (ITGB1) on PSCs, which activates NF-κB through ILK. Inhibitors of ILK or NF-κB or a neutralizing antibody against ITGB1 blocked transcription and production of IL8 from PSCs induced by rGAL3. The GAL3 inhibitor significantly reduced growth and metastases of orthotopic tumors that formed from PDAC and PSC cells co-implanted in mice. CONCLUSION: GAL3 activates PSC cells to produce inflammatory cytokines via ITGB1signaling to ILK and activation of NF-κB. Inhibition of this pathway reduced growth and metastases of pancreatic orthotopic tumors in mice.

Low expression of galectin-3 is associated with poor survival in node-positive breast cancers and mesenchymal phenotype in breast cancer stem cells.

BACKGROUND: Galectin-3 (Gal3) plays diverse roles in cancer initiation, progression, and drug resistance depending on tumor type characteristics that are also associated with cancer stem cells (CSCs). Recurrence of breast carcinomas may be attributed to the presence of breast CSCs (BCSCs). BCSCs exist in mesenchymal-like or epithelial-like states and the transition between these states endows BCSCs with the capacity for tumor progression. The discovery of a feedback loop with galectins during epithelial-to-mesenchymal transition (EMT) prompted us to investigate its role in breast cancer stemness. METHOD: To elucidate the role of Gal3 in BCSCs, we performed various in vitro and in vivo studies such as sphere-formation assays, Western blotting, flow cytometric apoptosis assays, and limited dilution xenotransplant models. Histological staining for Gal3 in tissue microarrays of breast cancer patients was performed to analyze the relationship of clinical outcome and Gal3 expression. RESULTS: Here, we show in a cohort of 87 node-positive breast cancer patients treated with doxorubicin-based chemotherapy that low Gal3 was associated with increased lymphovascular invasion and reduced overall survival. Analysis of in vitro BCSC models demonstrated that Gal3 knockdown by small hairpin RNA (shRNA) interference in epithelial-like mammary spheres leads to EMT, increased sphere-formation ability, drug-resistance, and heightened aldefluor activity. Furthermore, Gal3negative BCSCs were associated with enhanced tumorigenicity in orthotopic mouse models. CONCLUSIONS: Thus, in at least some breast cancers, loss of Gal3 might be associated with EMT and cancer stemness-associated traits, predicts poor response to chemotherapy, and poor prognosis.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner.

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics.

Recurrence of gastrointestinal adenocarcinomas after surgery and chemotherapy may be attributed, in part, to the presence of a small population of tumor-initiating cancer stem cells (CSC). The expression of galectin-3 (Gal3), a multifunctional oncolectin, has been associated with biological behaviors associated with CSC. We examined the ability of Gal3 to characterize the CSC phenotype, and to identify a clinically important gastrointestinal cancer CSC population. Human colorectal and pancreatic cancer cell lines were sorted to identify subpopulations expressing commonly used CSC markers, and Gal3-positive CSC subpopulations. The association of Gal3 with the stem cell properties and alterations of these phenotypes by manipulation of Gal3 expression was examined. Gastrointestinal cancer cell lines contain both Gal3-positive and Gal3-negative subpopulations. Gal3-positive CSCs are characterized by high ALDH activity, enhanced self-renewal ability in vitro (sphere formation) and tumor forming ability in vivo, and resistance to chemotherapeutic agents and death-receptor-mediated apoptosis compared to Gal3-negative CSCs. Silencing Gal3 modifies this behavior. Cell surface Gal3 expression identifies a subset of CSCs in gastrointestinal cancers with high levels of stem cell characteristics, including chemoresistance. This may provide a platform for developing treatment strategies that target CSC.

Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding Ras and activating Ras signaling.

Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3-5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional ß-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.