The term “cathepsin” (derived from the Greek word “Καθεψίνη”, which means to “boil down” or “digest”) was first coined in 1929 by Nobel Prize winner Richard Willstätter to describe all proteases that degrade proteins [1]. Cathepsins are now recognized as proteases that are predominantly active in the endo-lysosomal compartment [2]. These proteases are subdivided into three distinguished families based on which amino acid is present at their active site, i.e., the serine proteases (cathepsin A, G), the aspartic proteases (cathepsin D, E), and the cysteine proteases (cathepsin B, C, F, H, K, L, O, S, V, X, W) [3]. The most abundant cathepsins are cathepsin B, D, and L (CTSB, CTSD, CTSL respectively) [4]. Although most cathepsins are ubiquitously expressed throughout the body, some show tissue-specific expression [5]. This tissue-specific expression is important since the function of cathepsin can differ per tissue type.

Cathepsins execute diverse functions during homeostasis in which they cleave proteins within essential processes such as the immune response and energy metabolism [6]. They are expressed as inactive precursors and are activated through proteolytic cleavage of the N-terminal pro-peptide ^[7][8][7,8]. For optimal activity, cathepsins require an acidic pH, which can be found inside the endo-lysosomal compartments ^[3][5][3,5]. However, several cathepsins are also stable in more neutral pH conditions outside of their endo-lysosomal compartment and even outside of the cell [9]. While extracellular cathepsins have physiological functions in processes such as wound healing, they are mainly associated with pathological conditions ^[2][9][2,9], including several metabolic diseases and various types of cancer ^[3][10][3,10], where increased cathepsin concentrations have been associated with a poor prognosis ^[11][12][11,12]. Among others, the pathogenic role of cathepsins was demonstrated in hepatocellular carcinoma (HCC) ^[11][13][11,13]. In addition, cathepsins were found to participate in the disease progression of non-alcoholic steatohepatitis (NASH), an increasingly important risk factor for the development of HCC ^[14][15][14,15]. The role of cathepsins in NASH-induced HCC (NASH-HCC), however, has not been elucidated yet. Since NASH-HCC is a condition with limited therapeutic options and taking into account the involvement of cathepsins in both processes, targeting specific cathepsins might be a viable therapeutic option.

2. NASH-HCC Is a Global Health Problem

Worldwide, the prevalence of non-alcoholic fatty liver disease (NAFLD) in the adult population is 25% ^[16][19] and is expected to rise to more than 30% in the next decade ^[17][20]. NALFD is defined by excessive hepatic steatosis (≥5% in hepatocytes), not caused by excessive alcohol ingestion or by other chronic hepatic diseases such as viral hepatitis ^[18][19][21,22]. Furthermore, the disease is closely associated to conditions such as metabolic syndrome and type 2 diabetes ^[19][22]. NAFLD patients have an increased risk of developing liver-related morbidity and mortality ^[20][23]. In addition, NAFLD patients are at high risk of developing cardiovascular disease, chronic kidney disease, and extrahepatic malignancies (e.g., colorectal, uterine, and breast cancer), among others ^[21][22][24,25]. For about 30% of the patients affected by NALFD, the disease can progress into the more severe non-alcoholic steatohepatitis (NASH) ^[16][23][19,26]. Besides hepatic fat accumulation, NASH entails lobular inflammation and injury to the hepatocytes [15]. In addition, the liver of these patients can become fibrotic, making them at risk of the development of liver cirrhosis (5–18% for NASH patients without fibrosis, up to 38% for NASH patients with fibrosis ^[23][26]) and hepatocellular carcinoma (HCC) (2.4–12.8% for cirrhotic NASH patients ^[24][27]) ^[25][26][28,29]. HCC is the most common type of primary liver cancer ^[27][30]. Furthermore, it is the third leading cause of cancer-related deaths worldwide ^[28][31]. Currently, NASH is the most emergently growing cause for the development of HCC in patients that receive liver transplantations or that are on the waiting list ^[29][30][32,33]. A subset of patients develops cirrhosis as a result of NASH. These cirrhotic patients are at risk for developing HCC and they enter a surveillance program in order to detect the tumor on time ^[31][32][34,35]. However, the tumor is missed in one-fifth of these cirrhotic patients during these screening sessions, resulting in late-stage diagnosis of HCC ^[33][36]. In addition, it is now acknowledged that NASH patients without the presence of cirrhosis can also develop HCC ^[34][35][36][37,38,39]. In the absence of cirrhosis, these patients do not enter the surveillance program, which often results in a late-stage diagnosis as well. As curative treatment options require the cancer to be diagnosed in an early stage, many patients therefore miss this opportunity and are subjected to systemic treatment or palliative care ^[37][40]. As such, HCC poses a great burden on health and the economy ^[38][41]. CTSD has been found to be elevated in the plasma of patients with NASH compared to healthy individuals [14]. In addition, CTSB, CTSD, CTSL, and CTSS were found to be increased in HCC patients and associated with poor prognosis ^[11][39][40][11,42,43]. Ample studies have been performed regarding the role of cathepsins in several types of cancers. In the next section, the mechanism of these cathepsins in NASH will be addressed.

3. Mechanisms of Cathepsins in NAFLD/NASH

3.1. Cathepsins Induce Apoptosis in NASH

Apoptosis is a critical process in the disease progression of NASH and other liver diseases ^[41][44]. Apoptosis is mediated through extrinsic or intrinsic factors (classical pathway) that lead to the activation of effector caspases, resulting in apoptosis ^[42][45]. Next to the classical pathway, apoptosis can also be triggered alternatively through lysosomal disruption, which leads to the release of lysosomal proteases into the cytosol. It is generally accepted that cathepsins, subsequent to lysosomal membrane permeabilization (LMP), enter the cytosol to induce the apoptotic machinery by cleaving several proteins involved in the cascade ^[43][46]. In NALFD patients, disease severity has been associated with lysosomal permeability, and therefore the release of cathepsins into the cytosol ^[44][47]. An imbalance in the rate of cell proliferation and apoptosis is also thought to contribute to the NASH pathology, since an excess of apoptosis results in increased rates of regeneration and damage ^[44][47].

3.2. Cathepsins Play a Role in the Disease Progression of Conditions with Lipid Accumulation

Cathepsins play a role in the pathology of several diseases related to lipid accumulation, such as metabolic syndrome and NAFLD/NASH. Lipids that accumulate in tissues other than adipose tissue enter the cells and build up in the lysosomes, which results in the release of cathepsins and other lysosomal enzymes. This process culminates in cell death, also known as lipotoxicity ^[39][42]. Therefore, cathepsins are thought to be involved in conditions where excess lipid accumulation plays a role. Many patients that suffer from metabolic syndrome and NAFLD present with insulin resistance, which increases the risk of the development of type 2 diabetes (T2D) ^[45][48]. Cathepsins play a role in insulin sensitivity as well. Insulin sensitivity, CTSD levels, and activity were measured in overweight and obese individuals ^[46][49]. The activity of plasma CTSD was inversely associated with the whole-body sensitivity of insulin, i.e., the more likely it is that these overweight/obese individuals will develop type 2 diabetes (T2D), the higher the activity of their plasma CTSD. Additionally, in T2D patients, plasma CTSD activity appeared to be significantly higher compared to healthy individuals ^[47][50]. In addition, T2D markers (plasma glucose, HbA1c) were significantly correlated to plasma CTSD activity. Moreover, CTSS levels were higher in a population of T2D patients that had cardiovascular disease compared to T2D patients that did not have cardiovascular disease and the plasma CTSS levels were positively associated to components of the metabolic syndrome in obese adults ^[48][49][51,52]. In addition to insulin resistance, cathepsins have been linked to the deregulation of the liver metabolism in NAFLD and NASH. Apart from the observation that lipid accumulation in the liver and muscle in NALFD patients is positively associated with the levels of CTSD ^[50][53], multiple studies also point towards the involvement of other cathepsins in the more progressed form of NALFD, NASH. Increased levels of CTSD were observed in NASH patients compared to healthy individuals [14]. Furthermore, the involvement of cathepsins in NASH was also demonstrated in mice. Wild-type (WT) mice that were fed a fructose-palmitate-cholesterol (FPC) diet developed NASH within 18 weeks. Fang et al. demonstrated that these NASH mice showed elevated levels of CTSB compared to mice on a low-fat diet ^[51][54]. In addition, they showed that CTSB knockout mice on an FPC diet had lower levels of plasma total cholesterol, lower levels of liver triglyceride, and higher levels of plasma HDL compared to WT mice on an FPC diet. In CTSB knockout mice, decreasing inflammatory cytokine and chemokine levels were also observed, resulting in lower inflammation ^[51][54]. Improved liver function upon CTSB inhibition was also observed in NASH mice that were fed a methionine choline diet (MCD) compared to MCD-fed mice without the CTSB inhibitor ^[52][55]. In line, inhibition of CTSD in a model of murine NASH (LDLR^−/− mice on a high-fat, high-cholesterol (HFC) diet) resulted in decreased levels of plasma cholesterol and triglycerides, in addition to a decrease in hepatic cholesterol and total triglycerides compared to NASH mice in which CTSD was not inhibited. Additionally, genes related to hepatic lipid metabolism, such as PPAR-γ, were decreased in expression upon CTSD inhibition ^[53][56]. More specifically, when the extracellular fraction of CTSD was inhibited in HFC LDLR^−/− mice, the hepatic total cholesterol and triglycerides were significantly decreased. Furthermore, when the intracellular CTSD inhibitor was compared to the extracellular CTSD inhibitor, the hepatic total triglyceride levels were lower and the levels of fecal bile acids (indicating increased cholesterol breakdown) were higher in the extracellular inhibitor group, suggesting that extracellular CTSD inhibition leads to a better metabolic profile in NASH mice ^[54][57]. Not only mice demonstrated a beneficial effect on NASH progression after CTSB or CTSD inhibition; additionally, in Sprague-Dawley rats on a high-fat diet (simulating NASH), inhibition of CTSB (through exercise) and CTSD (through compound) improved liver function ^[55][56][58,59]. In summary, CTSB and CTSD seem to play an important role in the progression of pathologies related to the metabolic syndrome, including NASH. While CTSB and CTSD clearly influence NALFD/NASH progression, CTSS did not seem to have an influence. Even though obese patients and patients who had T2D combined with cardiovascular disease showed increased levels of plasma CTSS, NALFD patients did not have different levels of plasma CTSS compared to healthy controls ^[57][60]. Furthermore, genetic variants of CTSS need to be taken into consideration since certain genetic variants seemed to be associated with traits related to obesity, such as BMI ^[58][61]. However, there is not much information regarding the role of CTSS and CTSL in NAFLD/NASH and in metabolic syndrome development or progression and thus further research should be conducted in this area. While most of the studies mentioned above regarding cathepsins and metabolism are descriptive, mechanistic studies have demonstrated that the amelioration of NASH-related symptoms upon CTSB inhibition might be linked to the restoration of the levels of the master metabolic regulator sirtuin-1 (SIRT1) ^[59][60][61][62,63,64], which plays an essential (beneficial) role in the metabolic regulation of lipids, glucose, and insulin ^[62][65].

3.3. Cathepsins Exacerbate NASH by Stimulating the Immune System

In NASH, the immune system is deregulated and proinflammatory ^[63][66]. Cathepsins play a central role in this deregulation and the involvement of various cathepsins has been demonstrated in several studies. In an in vivo model of NASH, mice had upregulated expression of inflammatory genes, such as TNF-α and IFN-γ, and increased serum levels of proinflammatory cytokines such as IL-1β and IL-18. This gene expression and the serum levels of these proinflammatory cytokines decreased upon administration of a CTSB inhibitor ^[52][61][55,64]. Not only CTSB inhibition decreased proinflammatory markers; additionally, the inhibition of CTSD resulted in decreased expression of the proinflammatory markers TNF-α, CCL2, and Caspase 1 ^[53][56]. A possible mechanism of action for CTSB to induce a proinflammatory phenotype in NASH is through the abovementioned SIRT1 (see Section 3.2.Section 3.2). SIRT1 exerts an inhibitory effect on the inflammatory regulator NFκΒ ^[64][67]. Upon inhibition of SIRT1 by CTSB, NFκΒ activates the NLRP3 inflammasome ^[61][65][64,68], leading to increased secretion of the proinflammatory cytokines IL-1β and IL-18. Notably, several cathepsins (e.g., CTSB, CTSS, and CTSL) can also directly activate the NLRP3 inflammasome ^[66][67][68][69,70,71]. Macrophages play a central role in the pathogenesis of NASH through the creation of a proinflammatory environment by producing various cytokines and chemokines and thereby activating other surrounding (immune) cells ^[63][66]. Cathepsins have been shown to play a role in macrophage polarization in NASH. CTSB knockout NASH mice (FPC-fed mice) demonstrated higher expression of the anti-inflammatory phenotype macrophage “M2” marker Arg1 and decreased expression of the proinflammatory phenotype macrophage “M1” markers iNOS and F4/80 compared to NASH mice with normal CTSB expression, suggesting that in NASH mice, cathepsins induce proinflammatory M1 macrophages ^[51][54].