Exosomal tumor necrosis factor-α from hepatocellular cancer cells (Huh-7) promote osteoclast differentiation

Bone is the common extra-hepatic site for cancer metastasis. Hepatic cancer is associated with a higher incidence of pathological fracture. However, this important regulatory mechanism remains unexplored. Thus, exosome-mediated cell-cell communication between hepatocellular cancer and bone might be key to osteolytic bone destruction. Huh-7 exosomes were characterized for size and exosome marker expressions (CD63, Alix). Exosome mediated osteoclast differentiation in the RAW 264.7 cells was monitored from day 1 to 6 and multinucleated osteoclast formation and bone resorption activity were analyzed. The osteoclastogenic factor expressions in the exosomes and osteoclast differentiation markers such as tumor necrosis factor receptor 6 (TRAF6), nuclear factor κB (NF-κB), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and cathepsin K (CTSK) were analyzed using western blot. Exosomes released by liver cancer cells (Huh-7) promoted osteoclast differentiation in RAW 264.7 cells. Analysis of osteoclastogenic factors in the exosomes showed that exosomes were specifically enriched with tumor necrosis factor α (TNF-α). Huh-7 exosomes promoted osteoclast differentiation by significantly increasing the number of TRAP-positive multi nucleated osteoclasts and resorption pits. Importantly, exosomes upregulated osteoclast markers TRAF6, NF-κB, and CTSK expressions. Further, neutralizing exosomal TNF-α reverted exosome-mediated osteoclast differentiation in RAW 264.7 cells. Collectively, our findings show that cellular communication of exosomal TNF-α from hepatocellular cancer cells (Huh-7) regulates osteoclast differentiation through NF-κB/CTSK/TRAP expressions. Thus, exosomal TNF-α might act as an important therapeutic target to prevent hepatocellular cancer mediated pathological bone disease.