Preferential inhibition of bone metastases by 5'-deoxy-5-fluorouridine and capecitabine in the 4T1/luc mouse breast cancer model.

5'-deoxy-5-fluorouridine (5'-DFUR) and capecitabine are oral anti-cancer agents, which are enzymatically converted to 5-fluorouracil (5-FU) by thymidine phosphorylase in humans and uridine phosphorylase in mice. Since the activity of these phosphorylases is higher in cancerous tissue than in normal tissue, systemic administration of 5'-DFUR and capecitabine achieves high intratumoral 5-FU levels and low adverse effects on non-tumoral tissue. Accordingly, 5'-DFUR and capecitabine are widely used for the treatment of cancer patients. In the present study, we examined the effects of 5'-DFUR and capecitabine on bone metastases, one of the most common complications of breast cancer, using an animal model in which inoculation of 4T1/luc mouse breast cancer cells into the mammary fat pads of female BALB/c mice developed spontaneous metastases in distant organs including bone, lung and liver. Mice received 4T1/luc cell inoculation in the mammary fat pad at day 0 and oral 5'-DFUR (31, 62, 123 or 246 mg/kg) or capecitabine (90, 180 or 359 mg/kg) daily from day 7 to day 21. Both 5'-DFUR and capecitabine significantly inhibited orthotopic tumor formation and distant metastases to bone, lung and liver in a dose-dependent manner. Of note, the lowest dose of 5'-DFUR (31 mg/kg) and capecitabine (90 mg/kg), which failed to inhibit orthotopic tumor development and the lung and liver metastases, significantly reduced the bone metastases. In conclusion, our results suggest that oral 5'-DFUR and capecitabine are effective for the treatment of primary and secondary breast tumors. Most notably, they also suggest that these agents are preferentially beneficial for bone metastases.

Developmental regulation of Wnt/beta-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification.

Studies have suggested that continuous Wnt/beta-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/beta-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of beta-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated beta-catenin mutant (delta-beta-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/beta-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Delta-beta-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.