Background
Non-alcoholic steatohepatitis (NASH) is an increasingly important trigger for the development of hepatocellular carcinoma (HCC). Due to its limited therapeutic options, NASH-induced HCC (NASH-HCC) is among the most lethal cancers. Ample data points towards the involvement of cathepsins, lysosomal proteases, in many metabolic diseases, including many types of cancers. We previously determined that cathepsin D (CTSD), is increased in plasma of NASH patients and can serve as an early and sensitive marker for this condition. Furthermore, we have demonstrated that increased plasma (extracellular) CTSD contributes to metabolic dysregulation and low-grade chronic inflammation. However, information regarding the role of cathepsins in NASH-induced HCC (NASH-HCC) is lacking. In the current study, we investigate the role of these extracellular cathepsins in NASH-HCC.

Methods
Plasma CTSD levels were determined in healthy controls (N = 87), NASH (N = 63) and HCC (N = 15) patients. In addition, plasma cathepsin S (CTSS) levels were measured in healthy controls (N = 20), NASH (N = 20) and HCC (N = 16) patients. Furthermore, the effect of cathepsin B (CTSB) inhibition on cell viability and migration was studied in a set of cancer cells in vitro. Lastly, the effect of dietary and hormonal factors was determined on the CTSD activity in vitro and in (HCC) ASV-B mice.

Results
Plasma levels of CTSD and CTSS were significantly increased in HCC patients compared to healthy controls and to NASH patients. As expected, extracellular CTSB inhibition in cancer cells, led to a significantly lower viability and migration compared to intracellular CTSB inhibition and the control. In line with their function in metabolism, cortisol and glucose increased the activity of CTSD, whereas melatonin decreased the CTSD activity.

Conclusion
Our data demonstrate that dietary and hormonal factors affect NASH-HCC progression through extracellular cathepsins.