MUC1 carrying core 2 O-glycans functions as a molecular shield against NK cell attack, promoting bladder tumor metastasis.

Core 2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) forms an N-acetylglucosamine branch in O-glycans (core 2 O-glycans) of cell surface glycoproteins. C2GnT-expressing bladder tumors acquire highly metastatic phenotypes by surviving longer in host blood circulation. However, the detailed mechanisms underlying this increased survival remain unclear. In this study, we report that the expression of C2GnT in bladder tumors positively correlates with tumor progression and that bladder tumor cell-surface mucin 1 (MUC1) carrying core 2 O-glycans plays an important role in the evasion from natural killer (NK) cell attack. In C2GnT-expressing bladder tumor cells, heavily core 2 O-glycosylated MUC1 carries poly-N-acetyllactosamine in its O-glycans and galectin-3 binds to MUC1 through this poly-N-acetyllactosamine. The binding of galectin-3 to poly-N-acetyllactosamine in MUC1 core 2 O-glycans attenuates the interaction of the tumor cells with NK cells and interferes with the access of tumor necrosis factor-related apoptosis-inducing ligand to the tumor cell surface. These effects of MUC1 carrying core 2 O-glycans on NK cell attack facilitate C2GnT-expressing tumor cells to evade NK cell immunity and survive longer in host blood circulation. We reveal that MUC1 carrying core 2 O-glycans thus functions as a molecular shield against NK cell attack, thereby promoting bladder tumor metastasis.

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O-glycans.

The O-glycan branching enzyme, core2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT), forms O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (core2 O-glycans) on cell-surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT-expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT-expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK-activating receptor, NKG2D, by its tumour-associated ligand, Major histocompatibility complex class I-related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT-expressing bladder tumour cells, poly-N-acetyllactosamine was present on core2 O-glycans on MICA, and galectin-3 bound the NKG2D-binding site of MICA through this poly-N-acetyllactosamine. Galectin-3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT-expressing bladder tumour cells, resulting in tumour metastasis.

Requirement for FBP17 in invadopodia formation by invasive bladder tumor cells.

PURPOSE: Invadopodia (protrusions of the plasma membrane formed by invasive tumor cells) have an essential role in bladder tumor invasion. To understand the process of bladder tumor invasion it is crucial to investigate the molecular mechanisms of invadopodia formation. We found that invasive bladder tumor cells express FBP17. In this study we examined the role of FBP17 in bladder tumor cell invadopodia formation and invasion. MATERIALS AND METHODS: We used the 3 bladder tumor cell lines YTS-1, T24 and RT4 (ATCC®), and primary culture of bladder tumors from patients. Cells were stained with phalloidin for invadopodia formation. FBP17 knockdown cells were tested for invadopodia formation and subjected to invasion assay using a Transwell® cell culture chamber. We also examined the role of the extended FER-CIP4 homology and Src homology 3 domains of FBP17 in invadopodia formation in FBP17 mutant constructs. RESULTS: Invadopodia formation was observed in invasive bladder tumor cells and FBP17 was localized to invadopodia in invasive cells. FBP17 knockdown decreased invadopodia formation in invasive cells to 13% to 14% (p <0.0005) and decreased their invasive capacity to 14% to 16% (p <0.001). The extended FER-CIP4 homology and Src homology 3 domains of FBP17 were necessary for invadopodia formation and invasion. CONCLUSIONS: Invadopodia formation requires membrane deformation activity and recruitment of dynamin-2 mediated by FBP17. FBP17 has a critical role in the process of bladder tumor cell invasion by mediating invadopodia formation.

Invadopodia formation by bladder tumor cells.

A major cause of death in patients with bladder tumors is recurrence with metastasis. Bladder tumor metastasis is largely dependent upon the invasive capacity of tumor cells. Tumor cell invasion is mainly mediated by actin-rich protrusive membrane structures called invadopodia. The formation of invadopodia was observed in various types of invasive tumors such as breast cancer and melanomas. However, invadopodia formation so far has not been described in bladder tumor cells. We here report that human bladder tumor cells form functionally active invadopodia. By using a confocal laser scanning microscope, we demonstrated that invasive bladder tumor cell lines, YTS-1 and T24, with high Matrigel degradation activity form invadopodia but that noninvasive bladder tumor cell lines, RT4 and KK-47, form no detectable invadopodia. Invadopodia formed by YTS-1 cells had the ability to secrete matrix metalloproteases and degrade extracellular matrix to invade surrounding areas. Moreover, we observed that primary tumor cells obtained from patients with invasive bladder tumors also form invadopodia, validating the results from bladder tumor cell lines. Our results provide evidence that invasive human bladder tumor cells form invadopodia for tumor invasion.

Hepatoid carcinoma of the skin: spontaneous rat skin hepatoid carcinoma with eosinophilic globules and crystals immunoreactive to alpha-1-antitrypsin.

We present a case of hepatoid carcinoma of the abdominal skin in a male Wistar rat. Histopathologically, this carcinoma resembled human hepatocellular carcinoma with respect to trabecular-sinusoidal structures. Carcinoma tissues contain numerous eosinophilic globules and crystals, and in this case, we found the characteristic eosinophilic globules in the hepatoid carcinoma cells and the crystals in the extracellular portions. Vivid carcinoma cells full of eosinophilic globules were present near the necrotic areas in tumor tissue, wherein quadrate crystals unstained with eosin were observed. PAS staining after diastase digestion revealed that the globules were PAS positive and diastase resistant. In addition, we found that the hepatoid carcinoma cells were immunoreactive for alpha-1-antitrypsin (anti-A1AT) antibody with the globules and crystals staining peripherally, and a central unstained region. Ultrastructural study of intracytoplasmic globules and extracellular crystals revealed that the fringe of each globule and crystal had no limiting membrane and showed the same level of electron density. These findings suggest that the characteristic crystals in this tumor may have originated from the globules that were emitted from the carcinoma cells after their death as a result of saturation with intracytoplasmic globules.