Cathepsins: emerging targets in the tumor ecosystem to overcome cancers

Abstract

Cathepsins, a group of lysosomal peptidases, have traditionally been recognized as tumor facilitators. Recent research, however, highlights their critical role in orchestrating cancer and the tumor microenvironment (TME). Primality, cathepsins degrade extracellular matrix, enabling cancer cells to invade and metastasize, while also promoting vascular endothelial infiltration and subsequent angiogenesis. Additionally, cathepsins boost fibroblast growth, thereby supporting tumor progression. More importantly, cathepsins are pivotal in modulating immune cells within the TME by regulating their recruitment, antigen processing and presentation, differentiation, and cell death, primarily contributing to immune suppression. Given their overexpression in tumors and elevated levels in the circulation of cancer patients, it is crucial to consider the systemic effects of cathepsins. Although the comprehensive role of cathepsins in cancer patients’ bodies remains underexplored, they likely influence systemic immunity and inflammation, cellular metabolism, muscle wasting, and distant metastasis through their unique proteolytic functions. Notably, cathepsins also confer resistance to chemoradiotherapy by rewriting the cellular profile within the TME. In this context, promising results are emerging from studies combining cathepsin inhibitors with conventional therapies to suppress tumor development effectively. This review aims to decipher the cathepsin-driven networks within cancer cells and the TME, detailing their contribution to chemoradioresistance by reshaping both micro- and macroenvironments. Furthermore, we explore current and future perspectives on therapies targeting cathepsins’ interactions, offering insights into innovative treatment strategies.

1. INTRODUCTION

Cathepsins, categorized as lysosomal peptidases, span three distinct groups depending on their catalytic profiles: serine cathepsins (A and G), aspartic cathepsins (D and E), and cysteine cathepsins (B, C, F, H, K, L, O, S, V, W, and X). Cathepsins are localized in the cytosol, nucleus, and extracellular space and show proteolytic activity in district subcellular compartments. Cathepsin B, C, F, H, L, and O are broadly expressed throughout the entire human body, while the others display a more specific distribution within particular organs or cells, such as immune cells or osteoclasts. Cathepsins recognize a variety of substrates, akin to other protease species, and mediate the development of numerous diseases, including Alzheimer’s disease, auto-immune diseases, atherosclerosis, and cancers. Indeed, the profound roles of cathepsins in cancer cells have been investigated in hundreds of basic and clinical literatures.

Nowadays, cathepsins have emerged as critical regulators in the cancer landscape, serving dual roles: ensuring cell survival and orchestrating an environment conducive to the enhancement of malignancy. Although cathepsins operate effectively within an acidic environment of lysosomes, the neutral cytoplasm, as well as an acidic tumor microenvironment (TME) may be advantageous for their function [1–3]. In addition, the hypoxic milieu in the TME also endows a higher expression and a stronger proteolytic capability of cathepsins via HIF-1α regulation [2, 4]. Cathepsins’ abilities extend to the degradation of the extracellular matrix (ECM) to facilitate cancer invasion and metastasis, promote neovascularization, and modulate tumor-associated immune cells. Furthermore, excess circulating cathepsins have been known to impact systemic immunity, leading to enhanced inflammation, altered cellular metabolism, and extensive vascular and tissue damages, thereby creating an optimal environment for cancer colonization. Recent studies suggest that these cathepsin functions are widely associated with chemo-radioresistance in cancers, lending credence to the potential of targeting cathepsins as a promising therapeutic approach (Figure. 1) [reviewed in [5]]. This strategy could be employed to curtail cancer proliferation, either as a monotherapy or synergistically with conventional chemotherapeutic agents, immunotherapy, or radiotherapy. However, considering cathepsins not only fuel cancer proliferation by activating numerous signaling pathways, but also play a pivotal role in maintaining cellular homeostasis both within the TME and the body at large, a thorough understanding of cathepsin-regulated homeostasis is crucial to the design of innovative therapeutic interventions. This review aims to decipher how cathepsins refine the TME and contribute to chemoradioresistance by modifying this microenvironment (Table. 1).

Figure 1.

The role of cathepsins in the tumor microenvironment (TME). Cathepsins are highly expressed in cancer cells and upregulated by hypoxic, acidic conditions present in the TME. They stimulate the tumor growth (not discussed in this review article), forming a positive feedback loop that further increases cathepsin expression. Cathepsins affect various components of the extracellular matrix (ECM) as well as different cells within the TME, including fibroblasts, vascular epithelial cells, and immune cells. The primary function of cathepsins in degrading substances in the ECM grants cancer cells the capacity for invasion and metastasis. As tumors grow, fibroblasts in the TME undergo differentiation, facilitating further tumor progression. Cathepsins amplify this reciprocal feedback between cancer-associated fibroblasts and tumor cells, imparting specific functions to the fibroblasts, including antigen presentation. Additionally, cathepsins induce angiogenesis and recruit leukocytes, causing local inflammation that fosters a more conducive environment for tumor progression. Conversely, cathepsins regulate leukocytes and myeloid cells to help cancers evade anti-tumor immunity. Lastly, cathepsins contribute to shield cancers from chemoradiotherapy by creating optimal conditions for tumor survival.

Table 1.

Cathepsins and their role in the tumor microenvironment.

Subtype	Catalytic type	Localizations	Substrates	Roles	Descriptions, References
A	Serine	Ubiquitously	Superoxide dismutase	Inflammation, tissue damage	Mitigates the accumulation of superoxide radicals and prevent inflammation and cardiomyopathy [82].
B	Cysteine	Ubiquitously	Laminin, fibronectin, collagen type 2	ECM degradation	Promotes the ECM remodeling and subsequent metastasis [6, 7, 11].
				Angiogenesis	Enhances MM9 and VEGF secretion [13].
			CXCR3	Impaired chemotaxis	Cleaves and inactivates CXCR3, reducing number of tumor-infiltrating lymphocytes [28].
			CCL20	Induced chemotaxis	Generates a fully-functional CCL20 isoform. [27]
			MHC-I	Impaired antigen presentation	Degrades MHC-I and decreases tumor-antigen presentation [44].
				Autophagy	Regulates autophagy in myeloid cells [57–60, 62].
				Cellular differentiation	M2 macrophage polarization [68, 69].
			TLR-9	Cellular differentiation	Regulates caspase-induced cell death of pro-B cells through TLR-9 signaling [115].
				Fibrosis	Promotes α-smooth muscle actin expression and fibroblast differentiation [157]
				Osteoclastogenesis and bone metastasis	[152]
C	Cysteine	Ubiquitously	PR3, TSP-1	NET formation	Promotes NET formation, and subsequent tumor metastasis [74].
				Cellular differentiation	M1 macrophage polarization [72, 73].
D	Aspartic	Ubiquitously	SPARC	ECM degradation	SPARC fragments promote invasion and migration of tumor cells [3].
			Mucin		Proteolyzes fully glycosylated mucin [10]
				Angiogenesis	Activates VEGF-C and -D [14].
			LDL receptor-related protein 1	Fibrosis	Enhances fibroblast overgrowth, and subsequent tumor progression [23, 24].
				Autophagy	Regulates autophagy in myeloid cells [57, 60].
			C5	Complement activation	Cleaves C5 into C5a, promoting tumorigenesis by stabilizing KCTD5/cullin3/Roc-1 complex [80].
E	Aspartic	Immune cells	TRAIL	Immune cell proliferation	Proteolytically releases soluble TRAIL and activates macrophage-mediated anti-tumor immune response [65].
F	Cysteine	Ubiquitously
G	Serine	Myeloid cells	Fibronectin	ECM degradation	[6, 7]
			MHC-I	Antigen presentation	Upregulates cell surface MHC-I presentation [45, 46].
H	Cysteine	Ubiquitously		NET formation	Composes of NET [75].
K	Cysteine	Osteoclasts Fibroblasts	Elastin and collagen type 1	ECM degradation	[6, 7]
			Notch1 and VEGF	Angiogenesis	Promotes neovascularization to recover from ischemia [15, 16].
			CD18		Facilitates leucocyte recruitment and subsequent IL-1ß-induced angiogenesis [17].
			CCL3, 4, and 21	Impaired chemotaxis Cellular differentiation	Cleaves and inactivates chemokines [26].
					M2 macrophage polarization [70].
			Heparanase	Hematopoiesis and myelopoiesis	Sustains the ECM homeostasis in bone marrow [108, 109].
				Bone metastasis	Increase bone resorption process [150, 151].
			IRS1	Cachexia	Decreases skeletal muscle mass [148].
L	Cysteine	NK cells CD8+ T cells	Fibronectin	ECM degradation	[6, 7]
			ELR chemokines	Chemotaxis	Cleaves and activates the ELR chemokines, contributing to leukocyte recruitment [33].
			C3	Complement activation	Cleave C3 into C3a and C3b, mediating effector T-cell differentiation [78].
			CD1d	Cellular differentiation	Processes CD1d in the thymus, influencing NKT cell selection and maturation [118, 119].
O	Cysteine	Ubiquitously
S	Cysteine	Macrophages, APCs, B cells	Laminin, fibronectin, collagen type 2	ECM degradation	[6, 7]
			Canstatin, arrestatin, and laminin-5	Angiogenesis	Eliminates anti-angiogenic canstatin and arrestatin, and produce pro-angiogenic peptides by cleaving laminin-5 [19].
			Fractalkine	Chemotaxis	Enhances the secretion of fractalkine [30–32].
			(CX3CL1)
			Tumor antigens	Antigen presentation	Enhances tumor-antigen processing and presentation, activating CD4+ T cells and reducing CD8+ T cell infiltration [36, 37, 41].
				Cellular differentiation	M2 macrophage polarization [68].
			JAM-B	Brain metastasis	Degrade a component of blood-brain barrier [154].
V	Cysteine	Thymus, Testis
W	Cysteine	Cytotoxic cells
X	Cysteine	Myeloid cells	Integrin, LFA-1		Activates Mac-1, supporting LFA-1-mediated T cell proliferation and migration as well as phagocytosis in dendritic cells and macrophages [49–51].

2. CATHEPSINS IN THE TUMOR MICROENVIRONMENT

2.1. Extracellular matrix (ECM)

The extracellular matrix (ECM) disintegration, a hallmark of cathepsin activity in cancer, empowers tumor cells with heightened invasive and metastatic capacities. The ECM consists of elastin, collagen, fibronectins, etc., most of which are primarily targeted by cathepsins [6]. For instance, mature cathepsin K released from osteoclasts degrades type 1 collagen and elastin, whereas cathepsin B, L, G, and S specialize in cleaving fibronectin [6, 7]. Observations from clinical studies indicate a marked inverse correlation between cathepsin B expression in cancers and tumor-associated laminin, a component of the ECM [8–10]. In addition, therapeutic inhibition of cathepsin B by 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) suppressed the ECM remodeling, epithelial-mesenchymal transition, and subsequent metastasis in colorectal cancer [11].

Apart from cysteine cathepsins, cathepsin D plays a pivotal role in ECM degradation. In the acidic TME, tumor-derived cathepsin D cleaves fibroblast- or cancer-derived secreted protein acidic and rich in cysteine (SPARC) protein into multiple fragments. Among these fragments, 9-kDa SPARC fragment exhibit oncogenic potential, and promotes invasion and migration of tumor cells [3]. Recently, a study identified the role of cathepsin D in mucin degradation [12]. Since mucins are densely O-glycosylated proteins located mostly on cell surfaces to form a complex connection from cell-to-cell or cell-to-ECM, this finding will enlighten a new research perspective to understand the role of cathepsins within the ECM.

2.2. Angiogenesis

Angiogenesis, or neovascularization, enables cancerous tumors to secure nutrient supply and a conduit for distant metastases. Angiogenesis is another important contribution of cathepsins for tumor development (Figure. 2). Numerous studies corroborate the ability of cathepsins to trigger the production of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) to promote angiogenesis [13, 14]. It seems a very simple, clear answer of the cathepsins’ responsibility for angiogenesis. But now, it has become apparent that cathepsins also work more broadly on vascular endothelial cells (ECs), smooth muscle cells, or immune cells to modify an advantageous environment for vascular growth. The role of cathepsin K in neovascularization by hypoxia-induced ischemia was firstly indicated by Jiang et al., who demonstrated that cathepsin K deficiency reduced the levels of cleaved Notch1, VEGF, and phosphorylated Akt (p-Akt) in ischemic muscle cells, all of which were required for angiogenesis [15]. In addition, cathepsin K knockdown in ECs decreased their proliferation, invasion, and tubal formation. The group also found that cathepsin deficiency is associated with reduced number of endothelial progenitor-like CD31⁺/c-Kit⁺ cells in peripheral blood [16]. An interesting interaction of cathepsin B and leukocyte recruitment to facilitate angiogenesis was reported by Nakao et al [17]. The study shows that cathepsin B cleaves CD18 on immune cells and regulates leukocyte extravasation into tissues, which subsequently promotes IL-1β-induced angiogenesis. There is strong evidence indicating that cathepsin S also contributes significantly to angiogenesis within TME. Microvascular ECs exposed to inflammatory cytokines or angiogenic factors increase cathepsin S expressions, and therefore secreted cathepsin S degrades the ECM, resulting in more profound invasion of ECs [18]. Cathepsin S has also been shown to eliminate anti-angiogenic peptides, including canstatin and arrestatin, while at the same time triggers the production of pro-angiogenic γ2 fragments by cleaving laminin-5 [19]. Another study investigating the angiogenetic effect of cathepsin S by using a lower limb ischemia mode revealed increased levels of phosphorylated endothelial nitric oxide synthase, p-mTOR, p-Akt, and p-ERK1/2 in the ischemic muscles [20]. Pharmacological inhibition of cathepsin S by cystatin C or Fsn0503 has been proven effective in blocking angiogenesis and curbing tumor invasion [19, 21]. Contrastingly, caneCPI-5, a variant of sugarcane cystatin that inhibits cathepsins B, K, and L, has been shown to enhance angiogenesis in ischemic muscles through increased production of VEGF and fibroblast growth factor (FGF) [22]. Taken together, cathepsin S has distinct roles among cell types or tissues balancing pro- or anti-inflammatory function to stimulate neovascularization in the TME, and targeting cathepsin S in the TME may contribute to suppress angiogenesis, leading to tumor growth inhibition.

Figure 2.

The schematic summary of cathepsins’ role in inducing angiogenesis. A: Generally, cathepsins expressed in cancer cells significantly stimulate the expression of matrix metalloprotease-9 (MMP-9) and vascular endothelial growth factor (VEGF). B: They enhance cathepsins’ ability to degrade the extracellular matrix (ECM) and promote the infiltration of vascular endothelial cells. C: Additionally, certain cathepsins specifically induce angiogenesis by creating an optimal environment for vascular endothelial cells. Cathepsin B in immune cells cleaves surface CD18, enabling leukocytes to detach from endothelial ICAM-1-mediated adhesion to vessels. The shedding of CD18 facilitates leukocyte extravasation and subsequent IL-1β-induced angiogenesis. D: In endothelial cells under hypoxia, cathepsin K activates cleaved Notch1 and its downstream signaling pathways, including phosphorylated Akt. Cathepsin K also enhances the proliferation, invasion, and tubal formation of endothelial cells as well as the presence of CD31⁺/c-Kit⁺ progenitor-like cells. E: Cathepsin S is linked with the activation of endothelial progenitor-like cells and the phosphorylation of Akt and mTOR in ischemic tissues. Uniquely, cathepsin S cleaves anti-angiogenic peptides and facilitates the production of pro-angiogenic peptide by cleaving laminin-5.

2.3. Fibroblasts

The crosstalk between tumor cells and fibroblasts is a well-recognized mechanism to promote cancer development. They mutually secrete various cytokines or growth factors to boost their proliferation, invasion, and migration in the TME. For example, overexpression and hypersecretion of cathepsin D from cancer cells formed a positive feedback loop for tumor progression by enhancing fibroblast overgrowth [23]. One proposed mechanism for this effect involves the underlying inhibition of the proteolysis of the LDL receptor-related protein 1 (LRP1) by cathepsin D [24]. In breast cancer-associated fibroblasts, secreted pro-cathepsin D binds to the β-chain extracellular domain of LRP1, but does not stimulate its tyrosine phosphorylation. Instead, both catalytically active and inactive forms of cathepsin D inhibit LRP1-regulated intramembrane proteolysis by preventing LRP1β-carboxyl-terminal fragment production, thereby modulating fibroblast growth independently of its catalytic activity. Despite these findings, our understanding of the role of cathepsins in cancer-associated fibroblasts remains limited compared to their established role in immune cells. There is a clear need for further research, especially with regard to the function of cathepsins F, K, L, and O, which are known to be highly expressed in fibroblasts.

2.4. Immune cells -recruitment and antigen presentation

Cathepsins are believed to be associated with the recruitment of lymphoid cells and myeloid cells with or without excessively secretion of cytokines or chemokines [25]. Many chemokines, including CCL3[26], CCL4[26], CCL20[27], CCL21[26], CXCR3[28], CXCL9[28], CXCL10[28], CXCL12[29], and CX3CL1[30–32], are known to be regulated by cathepsins. In this regard, cathepsin K, L, and S have been found to cleave and activate the chemokines with glutamate-leucine-arginine motif (ELR chemokines) while inactivating non-ELR chemokines, contributing to leukocyte recruitment [33].

Antigen presentation and processing within the TME are important mechanisms for cancer cells to evade immune response. Several studies have shown that major histocompatibility complex class I (MHC-I) is degraded primarily in lysosomes, facilitating tumor escape from immune cells (Figure. 3A) [34, 35]. Recent studies demonstrated that cathepsin S emerges as a key protease to mediate antigen presentation in cancer immunity. In a non-Hodgkin lymphoma model, the activity of cathepsin S, either increased by amplification or Y132 mutation, enhances antigen processing while reducing antigen diversity in tumor cells. This process activates CD4⁺ T follicular helper cells, promoting lymphoma progression. Contrastingly, loss of cathepsin S activity encourages CD8⁺ T cell infiltration by inhibiting cathepsin S-dependent CD74 cleavage, a critical regulator of antigen presentation via the MHC-II pathway [36, 37]. The involvement of cathepsins L and S in MHC-II-mediated antigen presentation, a critical process for initiating adaptive immunity, has also been demonstrated in many studies [38–40]. Another study revealed that Bacillus Calmette–Guérin (BCG) vaccine treatment on melanoma activates cathepsin S in macrophages and dendric cells, promoting tumor-antigen processing and presentation [41]. However, in colorectal cancer, the cross-presentation of synthetic long peptides dependent on the lysosomal cathepsin S inhibits cytotoxic T-cell function by reducing activation and inducing exhaustion [42]. Furthermore, YTHDF1(m6A reader protein)-mediated cathepsin expressions govern the cross-presentation of tumor antigens in dendritic cells, and inhibiting cathepsins remarkably promotes anti-cancer immunity by optimizing the cross-presentation of dendritic cells [43]. Interestingly, cathepsin S also endows non-immune cells, such as fibroblasts, with antigen-presenting capacity, thus suppressing normal T-cell function [42]. These studies imply the complex nature of cathepsin S-mediated antigen processing and presentation in the TME, where cathepsins appear to undermine intrinsic anti-tumor immunity. Consequently, cathepsins are the promising targets to enhance anti-tumor immunity, necessitating further exploration of potential cathepsin inhibitors. Supporting this therapeutic potential, Deng et al. demonstrated a potential therapeutic application of cathepsin inhibition for anti-tumor treatment by controlling cathepsin-mediated antigen presentation. They revealed that the inhibition of cathepsin B in cancer cells attributes to the MHC-I degradation in lysosomes and promotes MHC-I presentation on the cell membrane [44]. Moreover, the authors revealed that cycloastragenol, which inhibited cathepsin B-mediated MHC-I degradation, remarkably curtailed tumor growth in an animal model, and when combined with anti-PD-1 antibody therapy, synergistically intensified CD8⁺ T cells cytotoxicity. This resulted in a significant accumulation of CD8⁺, CD45⁺, and H2-Kd⁺ cells within the tumor. Lastly, cathepsin G, commonly secreted by neutrophils on inflammation, also accelerates MHC-I presentation on both immune and glioblastoma cells, underscoring the critical and generalized function of the cathepsin family in facilitating antigen presentation in cancer.[45, 46]

Figure 3.

Multiple efforts of cathepsins on tumor-surrounding immune cells. A. Cathepsins play a critical role in regulating antigen processing and presentation in cancer cells. Lysosomal cathepsin B and S primarily degrade MHC-I, impeding CD8⁺ T cells infiltration into the TME. Additionally, cathepsin S breaks down tumor antigens to enhance antigen presentation by MHC-II, facilitating the recruitment of CD4⁺ follicular T cells and further secretion of pro-inflammatory cytokines in the TME. B: Cathepsin X plays a key role in leukocyte recruitment. It activates lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) to boost T cell proliferation and migration. Moreover, cathepsin X triggers NOD-like receptor family, pyrin domain containing-3 protein (NLRP) inflammasome formation, leading to systemic inflammation through increased IL-1β production. C: Numerous cathepsins determine the fate of immune cells. For instance, tumor-associated macrophages (TAMs), significantly influenced by cathepsins, exhibit altered survival and differentiation. M2 polarization of TAMs is extensively regulated by cathepsins through metabolic or signaling modifications. M2 TAMs enhance cathepsin B maturation and IL-17 secretion, promoting cancer metastasis and chemoresistance. Cathepsins also regulate cell survival and death by controlling autophagy in myeloid cells, typically maintaining normal autophagy flux by adjusting the number and formation of autolysosomes. Furthermore, cathepsin B cleaves lysosomal calcium channel TRPML1, blocking calcium efflux from lysosomes and suppressing transcription of autophagy-related gene via transcription factor EB (TFEB) in bacterial infection models.

Cathepsin X stands out from other cathepsins due to its unique substrates (Figure. 3B) [47]. It has been identified as a pivotal regulator of integrin signaling, primarily through targeting lymphocyte function-associated antigen 1 (LFA-1), a type of integrin necessary for T cell migration and activation [reviewed in [48]]. Its main target is integrin β2 subunit, which subsequently activates macrophage antigen-1 (Mac-1) and supports LFA-1-mediated T cell proliferation and migration [49–51]. This cascade also supports Mac-1 receptor-dependent adhesion and phagocytosis in dendritic cells or macrophages [51]. On the contrary, cathepsin X does not affect NK cell activity although LFA-1 has a central role in their activation [52, 53]. In addition to the essential function to suppress anti-tumor immunity, cathepsin X is also involved in systemic inflammation by activating NOD-like receptor family, pyrin domain containing-3 protein (NLRP3) inflammasome via integrin α₅-regulated IL-1β production [54]. Given these insights, cathepsin X appears to be responsible for both the recruitment of lymphocytes and myeloid cells and the initiation of local and systemic immune responses.

2.5. Immune cells -survival and differentiation

Cathepsins have a significant role in the regulation of immune suppression within tumor-associated immune cells (Figure. 3C). In addition to modulating chemotaxis, antigen processing, and cytokine secretion, they critically facilitate immune cell proliferation by leveraging autophagy. Given their lysosomal localization, they are intricately involved in autophagy regulation to prevent apoptosis in various cells [reviewed in [55]. Normally, the initiation of autophagy requires cathepsin release from lysosomes with highly permeabilized membrane [56]. For instance, an increased number of autophagosomes and autolysosomes with a significant accumulation of autophagy proteins, including LC3 and p62, were observed in cathepsin B and D double knock-out mice, leading to chronic inflammation in pancreas [57]. Similarly, pharmacological inhibition of cathepsin B and L impaired the autophagy and promoted caspase-dependent apoptosis of pancreatic β-cells, showing lysosomal localization of accumulated pro-cathepsins and the subsequent enlargement of lysosomes [58]. On the other hand, the genetic ablation of cathepsin B in bone marrow-derived macrophages resulted in an increased number of lysosomes, enlargement of autophagosomes, and enhanced transcription of lysosomal and autophagy genes in response to Francisella novicida infection [59]. This suggests that cathepsin B negatively regulates host defense against organisms by interrupting the normal flux of autophagy. In myeloid-derived suppressor cells (MDSCs), cathepsins B and D potentially inhibit autophagy and trigger endoplasmic reticulum stress-mediated cell death upon exposure to the acid ceramidase inhibitor LCL-521 [60]. Specifically, cathepsin B cleaves the lysosomal calcium channel TRPML1 and blocks calcium efflux from lysosome, suppressing the nuclear translocation of transcription factor EB (TFEB). This process is further modulated by mTORC1-mediated TFEB phosphorylation, which subsequently impacts the transcription of autophagy-related genes [59, 62].

Since autophagy essentially regulates macrophage differentiation, cathepsin activity also appears to play a role in myeloid cell differentiation within the TME [48, 63, 64]. Myeloid cells are strongly impacted by the cathepsin family to display their anti-tumor immunity in the TME. In older days, for example, cathepsin E was shown to bolster macrophage-mediated tumor immunity of melanoma cells by releasing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [65]. In another in-vivo study, cathepsin B or S supplied by tumor-associated macrophages (TAMs) was discovered to promote pancreatic cancer growth, neovascularization and cancer invasion [66]. Moreover, recent research highlighted the importance of cathepsins in macrophage polarization in the TME [63, 67–71]. Increased glucose uptake in M2-like TAMs has been shown to regulate O-GlcNAcylation for cathepsin B maturation, which promotes cancer metastasis and chemoresistance by degrading the ECM [69]. M2 polarization is regulated by cathepsin K in prostate cancers, where an increase in the release of cathepsin K and the subsequent secretion of IL-17 from cancers accelerates M2 TAM polarization [70]. Another study revealed that blocking cathepsin B, L, and S attributes the shift from M2 to M1 TAM by altering lysosomal signaling and lipid metabolism [68]. Interestingly, the opposing activity of cathepsin C in promoting M1 macrophage polarization has been demonstrated in several studies. High expression of cathepsin C in atherosclerotic plaques was shown to accelerate the phosphorylation of focal adhesion kinase (FAK), subsequently activating p38-MAPK/nuclear factor kappa B (NF-κB) signaling pathways, which drive M1 macrophage polarization and inflammation [72, 73].

Neutrophils are also influenced by cathepsins. While they inhibit tumor development by releasing anti-tumor factors, including hydrogen peroxide and thrombospondin-1 (TSP-1), they can promote cancer metastasis with a unique web-like structure called neutrophil extracellular traps (NETs). Cathepsin C has been demonstrated to promote breast cancer metastasis by regulating the NETs formation and by degrading TSP-1 via activated proteinase 3 (PR3)-IL-1β axis in neutrophils [74]. An intriguing positive feedback loop was also observed in this study, where neutrophil recruitment by cathepsin C released from tumor cells reciprocally reinforces tumor colonization through the excess secretion of IL-6 and CCL3 from neutrophils, both of which are regulated by membrane-bound PR3. In this context, cathepsin G is also known to promote cancer progression and metastasis via pro-tumor effect of NET [75].

The differentiation of MDSCs is also impacted by cathepsins. In an in-vitro study, functional CD14⁺ MDSCs significantly increased by cathepsin X and L in the presence of breast cancer cells [76]. Furthermore, a cathepsin L inhibitor, CLIK-148, successfully reduced breast cancer cell invasion and tumor growth in this study. Compared to myeloid cells, less is known about the interplay between cathepsin and immunosuppression in leukocytes. However, when regulatory T cells (Tregs) were treated with a cathepsin S inhibitor, they displayed reduced proliferation in vitro and caused more apoptosis in cancer cells co-cultured with these Tregs [77].

Finally, cathepsins can also target complement in the TME. Intracellular cathepsin L in human CD4⁺ T cells cleaves C3 into activated forms of C3a and C3b, regulating cellular survival and mediating effector differentiation in T cells [78]. Although the role of complement modification by cathepsins within the TME has not been fully investigated, a cathepsin L inhibitor potentially reduces C3b surface level in cytotoxic T lymphocyte, which could jeopardize their maximum cytotoxic activity [79]. Complement degradation associated with oncogenesis in colonic epithelial cells was reported by Ding et al. They mention that intra-lysosomal C5 cleavage by cathepsin D, and stabilizing KCTD5/cullin3/Roc-1 by C5aR1 activation promotes the tumorigenesis of colorectal cancers.[80]

An abundance of studies has underscored the potential centrality of cathepsins in anti-tumor immunity. The equilibrium between pro-tumor and anti-tumor immunity is meticulously managed by cathepsins, and the integration of therapeutic interventions remains a contentious subject. Unquestionably, the moment has been getting ripe for clinical trials to probe anti-cathepsin therapies, particularly in combination with checkpoint inhibitors.

3. SYSTEMIC EFFECT OF CATHEPSINS IN CANCER

3.1. Circulating cathepsins in cancer patients

Beyond their established roles in tumor progression at the primary site, cathepsins released into circulation become key mediators of systemic pathology. This systemic distribution of tumor-derived cathepsins favors cancer progression through effects on inflammation, metabolism, and tissue remodeling.

The systemic impact of elevated cathepsins extends beyond localized effects, potentially affecting multiple organ systems throughout cancer patients’ bodies. These circulating proteases orchestrate widespread tissue remodeling and inflammatory responses, creating microenvironments conducive to tumor progression and metastatic spread. Importantly, elevated cathepsin expression and concentration across multiple tumor types and their association with poor outcomes have been demonstrated in several clinical studies [90–93]. We analyzed the mRNA expression and protein levels of major cathepsins, including cathepsins B, D, K, L, and S, using data from The Cancer Genome Atlas and the Clinical Proteomic Tumor Analysis Consortium, respectively. The analysis revealed distinct transcriptomic patterns across tissues, showing a generalized pattern with relatively higher expression of cathepsins B and D compared to others (Figure. 4A). On the other hand, protein levels of these cathepsins did not differ significantly between normal and tumor tissues (Figure 4B), suggesting that the complexity of their expression may be balanced in the TME.

Figure 4.

mRNA and protein expression levels of major cathepsins in various tumors and their comparison to normal tissues. A. Boxplots representing normalized log2(TPM + 1) values for the expression of cathepsin genes (CTSB, CTSD, CTSK, CTSS and CTSL) across different cancer types in The Cancer Genome Atlas (TCGA). Cancer types include Breast invasive carcinoma (BRCA), Colon adenocarcinoma (COAD), Ovarian serous cystadenocarcinoma (OV), Clear cell renal cell carcinoma (ccRCC), Uterine corpus endometrial carcinoma (UCEC), Lung adenocarcinoma (LUAD), Pancreatic adenocarcinoma (PAAD), Head and neck squamous cell carcinoma (HNSC), Glioblastoma multiforme (GBM), Liver hepatocellular carcinoma (LIHC), and Prostate adenocarcinoma (PRAD). B. Boxplots showing normalized protein expression (Z-values) of cathepsin genes (CTSB, CTSD, CTSK, CTSS, and CTSL) across normal and tumor samples for the same cancer types from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset. Boxplots depict protein expression of each gene in both normal (blue) and tumor (red) tissues. Tumor samples show significantly higher expression of cathepsin genes in several cancer types compared to normal samples (p < 0.0001). CTSB, cathepsin B; CTSD, cathepsin D; CTSK, cathepsin K; CTSL, cathepsin L; CTSS, cathepsin S. Asterisk marks indicate a significant difference (p value < 0.05) between normal and tumor sample.

Investigations looking into circulating cathepsin levels and their correlation with early diagnosis or patient prognosis remain limited. A clinical study indicated the serum cathepsin B concentration was significantly higher in patients with gastrointestinal cancers and even with gastric epithelial dysplasia or colorectal adenoma, suggesting a strong predictable value of serum cathepsin B for cancer patients [94, 95]. Additionally, serum cathepsin S level was significantly higher in patients with gastric cancer, and the elevated level of circulating cathepsin S was associated with unfavorable tumor behavior [96]. Likewise, substantial elevations in serum concentrations of cathepsin B, L, and S were identified in colorectal cancer patients, and the elevated cathepsins were correlated with higher stage of cancers [97, 98]. Conversely, serum cathepsin X in colorectal cancer did not differ from the healthy control, but was associated with overall survival [99]. These findings have underscored the significance of systemic cathepsins emanating from increased cathepsin expression in the TME. In the following sections, we delve into how circulating cathepsins exert influence over the entire body in cancer patients (Figure. 5).

Figure 5.

Systemic impact of cathepsins in cancer patients. While the systemic functions of cathepsins amplified by cancers remain underexplored, they are believed to influence systemic inflammation, metabolism, and cancer-associated cachexia. First, cathepsins play a critical role in maintaining the hematopoietic stem cell (HSC) niche in the bone marrow, spleen, and lymph nodes, where they regulate the selection and differentiation of lymphocytes and myeloid cells. Cathepsins also contribute to systemic inflammation by enhancing cytokine secretions from circulating immune cells, including IL-1β, TNFα, and IL-8, and by activating NLRP3 inflammasome. Additionally, they can alter cellular metabolisms among various cells in cancer patients, potentially leading to increased blood sugar levels and insulin resistance. Systemic lipid profiles may also be disrupted. The changes in systemic metabolism subsequently contribute to the initiation of cancer-associated cachexia. Cathepsins primarily cause proteolysis in skeletal muscle cells, leading to muscle atrophy and muscular degeneration. These effects collectively contribute to further cancer progression and distant metastasis. Furthermore, cathepsin-specific functions, including ECM degradation, extensive tissue damage followed by fibrosis, neutrophil extracellular trap (NET) formation, and proteolysis, facilitate cancer cell colonization in distant organs, such as the lungs, brain, and bone.

3.2. Systemic immunity and inflammation

Cathepsins’ extracellular proteolytic activities have been shown to impact the development and differentiation of immune cells in distant places, including hematopoietic and lymphoid organs. Of note, cathepsin K modifies the hematopoietic stem cell (HSC) niche by altering the ECM in bone marrow. Cathepsin K degrades several ECM compositions [100], and some are critical for the optimal survival of HSCs in bone marrow, including collagen type I [101, 102], osteopontin [103], and proteoglycans, such as heparan sulfate [104–107]. Heparanase, processed and activated by cathepsin L, degrades heparan sulfate in the ECM and plays an important role to sustain the ECM homeostasis [108, 109]. Additionally, CXCL12, which is primarily digested by cathepsin B, L, and X, maintains HSCs and lymphoid progenitors by acting as a chemoattractant, enabling their migration and residence within specialized bone marrow niches while regulating their proliferation and differentiation through PI3K-AKT or JAK-STAT pathways [29, 110, 111]. Cathepsin K influences the differentiation of myeloid cells, including macrophages and dendritic cells, from osteoclast precursor cells, the same progenitor as other myeloid immune cells, in bone marrow [38, 112]. Genetic ablation of cathepsin K in mice led to increased HSCs in the spleen and decreased HSCs in bone marrow [113]. Studies have demonstrated that cathepsin K deletion causes splenomegaly in mice, suggesting its critical role in regulating hematopoietic site switching between spleen and bone marrow [100, 113]. While cathepsin K deletion increased CD11b⁺ osteoclast precursors in bone marrow, it significantly impaired their ability to migrate to fractured bone during injury repair [113]. Another study demonstrated that increased cathepsin K expression in mice increased osteoclast formation, indicating its regulatory role in osteoclast precursor differentiation [114]. Cathepsins also regulate B cell maturation and production. Toll-like receptor (TLR)-9 expressed on immature and mature B cells control the production of pro-B cells in bone marrow. TLR9 signaling directly induced caspase-independent cell death and inhibited the expansion of pro-B cells through a cathepsin B-dependent mechanism [115]. In this context, cathepsin L has been shown to negatively affect B-cell production by acting both on bone marrow stem cells and B-cell progenitors, and it further suppressed migration of B-cells from bone marrow, decreasing the number of peripheral B-cells [116]. These data indicate cathepsins serve a negative impact on B-cell production and output in bone marrow, maintaining systemic B-cell population homeostasis.

Cathepsins have also been involved in immune cell dynamics across various organs. For example, cathepsin L deficiency in mice exhibited significant enlargement of lymph nodes and an increased number of mature B cells, whilst cathepsin K deletion decreased bone marrow cellularity and caused splenomegaly, suggesting cathepsins control the production and homing of lymphocytes in lymphoid organs as cathepsins do in bone marrow [38, 100, 116]. The ECM component in lymph nodes, including laminin, fibronectin, and collagens, was also degraded by cathepsin L [38]. On the other hand, in the human thymus, cathepsin S, which is highly expressed in thymic dendritic cells, participates in the negative selection of autoreactive T cells through two mechanisms: processing autoantigens and destroying specific T cell epitopes. This dual function balances the education of CD4⁺ T cells, with outcomes depending on the specific antigens involved and the level of cathepsin S expression [117]. Lastly, cathepsin L plays a critical role in processing CD1d molecules in the thymus, which is essential for the presentation of lipid antigens to developing NKT cells, thereby influencing their selection and maturation [118, 119].

It is worth noting that extracellular cathepsins also serve as an important orchestrator of systemic inflammation by modulating cytokines and chemokines. As aforementioned, cathepsins regulate leukocyte recruitment by activating several chemokines. For example, cathepsin G secreted from macrophages, neutrophils, and epithelial cells exacerbates systemic inflammation by stimulating IL-8 secretion, activating IL-1β and TNFα-related signaling, and inactivating IL-6 [reviewed in [120]]. Mirroring cathepsin G, other cathepsins, including B, C, S, L, and X propel the secretion of IL-1β to evoke systemic inflammation and the NLRP3 inflammasome activation is involved in this mechanism [121–123]. Pharmacological or genetic inhibitions of cathepsins are known to suppress the NLRP inactivation and inhibited the ensuing pro-IL-1β and IL-1β synthesis at an ER level [123–125]. Cathepsin K plays a crucial role in regulating bone marrow hematopoiesis and lack of cathepsin K dramatically impairs lymphocyte homeostasis [126]. However, crosstalk between systemic immune cells and cathepsins, especially in cancer patients, has yet to be clarified. Unraveling the general function of cathepsins on systemic immunity presents the next challenge to surmount.

3.3. Metabolism

Cathepsin production is subject to metabolic regulation. Recently, Vidergar et al, showed the glucose-dependent metabolic regulation of active cathepsin secretion by M2-like TAMs in a preclinical melanoma model [69]. Tumor-derived inflammation (IL-4) can induce O-GlcNAc transferase (OGT) expression in macrophage, which helps in serine 210 O-GlcNAcylation of cathepsin B. Absence of glucose or OGT abrogated matured cathepsin B levels in IL-14-induced M2-like TAMs. Conversely, cathepsins has a strong impact on cellular proliferation by modulating the glycolytic state. Cathepsin L has been shown to regulate cell proliferation and growth by enhancing the transcription and activity of lactate hydrogenase A (LDHA), thereby stimulating glycolysis [127]. In addition to cathepsin’s role in cellular metabolism, they have been shown to affect systemic metabolic profiles, contributing to the development of metabolic disorders, such as obesity and diabetes mellitus [128, 129]. A study by Yang et al, showed that cathepsin knock-out mice display reduced serum insulin and glucose levels. Also, these mice showed reduced turnover of insulin receptors in the skeletal muscle and pharmacological inhibition of Cathepsin reduced weight gain in mice. Similarly, a recent study demonstrated that circulating cathepsin S increased plasma insulin and benefited to maintain blood sugar level by decreasing glucose output from hepatocytes via mRNA expression suppression of glucose-6 phosphate catalytic subunit 1 and phosphoenolpyruvate carboxykinase 1 [130]. In an in-vitro model, cathepsin S did not affect insulin secretion from pancreatic β-cells or glucose metabolism in myotubes and adipocytes, but inhibited glucose release from hepatocytes. Interestingly, however, this glycemic regulation by cathepsin S was canceled in insulin resistant states, such as a high-fat-fed mouse or a db/db mouse model. Rather, a cathepsin inhibitor improved the insulin resistance, suppressed adipocyte formation, and reduced macrophage infiltration in the white adipose tissue in the obese mice [131]. In this context, cathepsins have also been associated with dysregulation of lipid metabolism in several organs. Mizunoe et al. reported that overexpression of cathepsin B in white adipocytes led to increased release of glycerol and futile basal lipolysis due to the perilipin 1 reduction in an obese mouse model [135]. Additionally, chronic inflammation and fat accumulation in the liver are associated with an enhanced activity and expression of cathepsin D; its inhibition resulted in diminished hepatic inflammation, alleviated lipid dysregulation, and elevated excretion of cholesterol via bile acids in non-alcoholic steatohepatitis mouse models.

In the context of cancer, systemic metabolic regulation by cathepsins remains largely unexplored. Cancer patients generally demonstrate insulin resistance characterized by increased hepatic glucose production and gluconeogenesis (reviewed in [132]). It indicates that high level of systemic cathepsins may worsen insulin resistance in cancer patients. Similarly, dysregulation lipid metabolism is one of the major hallmarks of cancer patients. Chronic inflammation triggered by pro-inflammatory cytokines in response to high metabolic demand of cancer cells contributes to increased energy expenditure and insulin resistance [132–134]. The abovementioned role of cathepsin in obesity and diabetes model indicates the possible role of cathepsins in lipid metabolism in cancer patients which remains unexplored warrants further investigations [135, 136].

In conclusion, cathepsins play integral roles in regulating metabolism across multiple organ systems, in particular in a liver and adipose tissues. Cathepsins largely contribute to altered glucose metabolism, insulin tolerance, and lipotoxicity, further leading to exacerbation of systemic inflammation and even distant metastasis [137]. Metabolic disorder-induced carcinogenesis, such as hepatocellular carcinoma, may be tightly affected by the level of circulating cathepsins [138]. In addition, their involvement in protein turnover, lipid metabolism, and glucose homeostasis positions them as crucial mediators of cancer-associated cachexia. The dysregulation of cathepsins in various metabolic tissues contributes to the complex interplay between cancer and systemic metabolism, highlighting their potential as therapeutic targets in cancer-related metabolic disorders.

3.4. Cancer-associated cachexia

Cancer-associated cachexia is a complex metabolic disorder that typically occurs alongside cancer progression and is characterized by severe muscle wasting and body weight loss [139, 140]. This devastating condition does not only undermine the patients’ quality of life but also impedes the continuation of chemotherapy. Clinical trials to treat cachexia with multimodal intervention, amino acid supplementation, or anamorelin have been conducted, but the outcomes have not reached a satisfactory level [141–143]. Recently, the role of cathepsins in the initiation and progression of cachexia has been discovered.

Cathepsin B and L have been identified as key players in protein degradation during muscle atrophy since earlier days [144, 145]. The cathepsin L up-regulation has been confirmed in various muscle atrophy models, including cancer-associated cachexia, insulin deficiency, and fasted mouse models [146, 147]. While an increased proteolytic intensity in muscles is suggested, the exact targeting substrates or altered downstream pathways remain unclear.

The latest findings concerning the link between cachexia and cathepsins are the involvement of cathepsin K in muscle atrophy and regeneration process. Cathepsin K in skeletal muscles degrades insulin receptor substrate 1, which phosphorylates protein kinase B, and induces skeletal muscle mass reduction, muscle dysfunction, and loss of muscular fibers in both in-vivo and in-vitro cachexia models [148]. Cathepsin K also functions to prevent injured skeletal muscles from regeneration. In a cardiotoxin-induced muscle injury model, cathepsin K deletion does not only reduce muscle cell apoptosis but also alleviate interstitial fibrosis, thereby improving muscle regeneration [149]. In conclusion, while cathepsins appear to be a promising target for treating cancer-associated cachexia, a more detailed understanding of the underlying mechanisms is essential.

3.5. Distant metastasis

In general, systemic inflammation and an insulin resistant state favor the migration of cancers in the bloodstream and their colonization in distant locations. Given that cathepsins amplify the inflammatory response, they could potentially facilitate distant metastasis in cancer patients. Unfortunately, there is limited evidence to suggest that cathepsins act as potent facilitators of distant metastasis, but some cathepsins have been found to aid tumor colonization in specific manners. Cathepsin K, for instance, is a major contributor for cancers to establish nests in bones due to its robust resorption and destructive function [150, 151]. Not only cathepsin K but also other cathepsins play a role in osteoclasteogenesis and subsequent bone metastasis [152, 153]. Cathepsin B suppression by CST6 up-regulates sphingosine kinase 1, a hydrolytic substrate of cathepsin B, inhibiting receptor activator of nuclear factor kappa-B ligand (RANKL)-induced p38 activation in osteoclasts, and the diminished bone differentiation prevents bone metastasis of breast cancers [153]. On the contrary, an inhibition of cysteine cathepsin secretion from MDSCs promotes osteoclast differentiation and fusion, possibly enhancing bone resorption and the following bone metastasis [112]. Evidently, cathepsins play important roles in cancer-associated bone resorption and bone metastasis. However, the complexities of involved cathepsin regulation in bone microenvironment have not been fully investigated and an optimized treatment strategy to inhibit cathepsin is mandatory. Considering the current investigations, including clinical studies, a selective cathepsin K inhibition may be a good option to control bone metastasis.

There is also evidence for the tissue-specific function of cathepsin in tumor metastasis, particularly in the brain. High expression of cathepsin S in the primary site is associated with diminished brain metastasis-free survival in patients with breast cancer [154]. In the same study, junctional adhesion molecule-B (JAM-B), a blood-brain barrier component, was identified as a substrate of cathepsin S. This highlights that tissue-specific substrates of cathepsins could serve as strategic targets to hinder the establishment of cancers in localized regions.

Though there is no evidence that cathepsins facilitate cancer metastasis in distant organs, except bone and brain, in a specific manner, their generalized function can be a cause of them. For instance, cathepsin C was recently found to induce neutrophils to form NETs so that cancer cells are able to colonize in lungs as previously described [74]. However, this mechanism is not restricted to the lungs but could occur in any tissue in the body. As highlighted in prior basic research, cathepsins play crucial roles in degrading the healthy structure of the ECM, recruiting lymphocytes, and inducing local inflammation and tissue damage (fibrosis). Consequently, the increased expression of local cathepsins and elevated levels of circulating cathepsins can create an optimal metastatic environment in any organ. Several studies revealed that cathepsin induced damage to blood vessels, causing atherosclerosis and cardiomyopathy, possibly creating ideal niches for cancer colonization. Moreover, tissue scarring in the lungs or livers, which can progress to liver cirrhosis, is a well-established function of cathepsin B and L, ultimately contributing to subsequent carcinogenesis [155, 156]. Kasabova et al. reported an interesting mechanism of cathepsins to develop lung fibrosis. This study demonstrated that cathepsin B promoted α-smooth muscle actin expression and fibroblast differentiation via driven-TGF-β1/Smad canonical signaling pathway, while increased cathepsin B-induced TGF-β1 expression conversely inhibit cathepsins by enhancing cystatin C secretions to avoid excess fibrosis [157]. These findings suggest that dysregulated cathepsin secretion originating from the TME can disrupt the physiological balance of proteolytic activity in various organs, resulting in extensive tissue injuries and scarring, which can create a favorable environment for cancer metastasis.

3.6. Cathepsins and chemo-radio resistance acquired through the tumor microenvironment

Chemo-radioresistance via cathepsins is considered to be acquired through the epithelial-mesenchymal transition (EMT) by the ECM degradation or regulating cellular adhesion molecules [69, 158–160]. Cathepsin B, L and S secreted into the TME by TAMs instigate the degradation of ECM and cleavage of E-cadherin, thereby disrupting cell-to-cell junctions among cancer cells. This ultimately confers high metastatic capability and subsequent chemoradioresistance upon cancer cells. Cathepsins also directly influence intracellular signaling pathways involved in chemotherapy resistance. A previous study demonstrated that increased cathepsin S expression in triple-negative breast cancer led to upregulation of MMP-9 that promoted ECM degradation, thus contributing to enhanced chemotherapy resistance [161]. Similarly, cathepsin L was shown to play a pivotal role in EMT by activating NF-κB signaling, upregulating EMT-related transcription factors, such as Snail and ZEB1. This resulted in the loss of epithelial markers, including E-cadherin, and boosted chemoresistance, as elevated cathepsin L levels were observed in paclitaxel- and cisplatin-resistant lung cancer cells [162].

Recent studies propose that cathepsins can impede the effects of chemoradiation on cancers via other pathways as well. Several studies revealed a robust defense mechanism of cathepsin E, S, and K against conventional chemotherapeutic reagents by regulating TRAIL-mediated apoptosis triggered by mitochondrial ROS [163–166]. More specifically, these cathepsins down-regulates a key deubiquitinase, ubiquitin-specific protease 27x (Usp27x), expression which culminates in cellular FLICE-like inhibitory protein (cFLIP) expression. These events block death receptor signaling-mediated apoptosis in cancer cell. Additionally, cathepsin S was associated with cytoplasmic BRCA1 degradation in breast cancer cells. The increase in BRCA1 by cathepsin S inhibition led to the downregulation of anti-apoptotic BCL2, which was responsible for reinforced radiation sensitivity [167, 168]. Furthermore, ionizing radiation-induced DNA damages can cause increased expression of cathepsin S, which ultimately work protectively to a further irradiation [5, 169]. On the other hand, Wang et al. suggested a pivotal role of cathepsin L for radiation-induced lysosome-mediated necrosis in cervical cancer cells [170]. In their study, cathepsin L was required to enhance radiation-induced DNA damage and cell death activation through the inhibition of the endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) [170].

Beyond their direct effects on cancer cells, cathepsin activity is also instrumental in bolstering the TME to deter cell death induced by chemoradiotherapy. Conventional chemotherapies, such as gemcitabine and 5-FU, activate the NLRP3-dependent caspase-1 activation complex (inflammasome) in MDSCs and promote lysosomal permeabilization that enables the cytosolic release of cathepsin B. This, in turn, leads to an increased release of IL-1β from the MDSCs, which results in an overproduction of IL-17 by CD4⁺ T cells within the TME, thereby blunting the effectiveness of chemotherapy-induced cell death [121]. TAMs are also reported to shield breast cancer cells from the cytotoxic effects of paclitaxel in an in-vitro co-culture model, which in turn can be reversed by cathepsin B and S inhibition [171]. Of note, cathepsins may be involved in innate and acquired resistance to immune checkpoint inhibitors (ICIs) since cathepsins tightly regulate autophagy in tumor-associated immune cells which is significantly linked to the effectiveness of ICIs. Antigen-presenting molecules, including MHC-I, are representative targets of autophagic degradation, impairing CD8⁺ T cell recognition and localization to tumors [35, 172]. Pharmacological inhibition of cathepsin B enhances MHC-I antigen presentation and subsequent CD8⁺ T cell killing, and the combination of the cathepsin B inhibitor with PD-1 antibody further boosted antitumor efficacy in xenograft mice and colorectal cancer organoids [44]. Furthermore, as described previously, M2 polarization is one of the well-investigated functions of cathepsins for immune evasion. A clinical trial of ICIs on prostate cancer revealed high infiltration of M2 TAMs was associated with a poor outcome [173]. Increased expression of immune checkpoints on the cell surface, impaired antigen presentation, and the release of anti-inflammatory cytokines, including TGF-β, IL-4, IL-13, IL-10, and IL-1RA, may collectively explain the immunosuppressive capacity of these cells to resist ICI treatment [174].

Finally, an intriguing pre-clinical study suggested the potential of cysteine cathepsins in degrading chemotherapeutic agents. The authors revealed that the combination of bleomycin and a pan-cathepsin inhibitor exhibited increased cytotoxicity on bladder cancer cells by blocking the degradation of bleomycin [175]. Owing to the broad scope of cathepsin activities, it is crucial to consider the interaction of cathepsins with chemotherapy consistently.

4. CATHEPSINS AS A PROMISING TARGET FOR FUTURE CANCER TREATMENT

Cathepsins exist in lysosomes, cytosol, nucleus, and extracellular space, demonstrating a variety of function in the TME and sometimes even systemically. As they are up-regulated in both cancer cells and the surrounding cells in the TME, there is no room for argument about the importance of blocking their function to develop innovative anti-cancer treatment strategies. However, cathepsins play a part in a wide range of crucial inter- and extra-cellular molecular cascades, often promoting, and sometimes inhibiting, cancer progression, facilitated by complicated interactions with other cells in the TME. The most important challenge lies in formulating a treatment strategy to control the cathepsin family properly.

JPM-OEt and its natural product, E-64, are widely used as pan-cathepsin inhibitors. Earlier research revealed potent anti-tumor activity of JPM-OEt in suppressing tumorigenesis, angiogenesis, and tumor invasion, therefore resulting in the significant tumor reduction in RIP1-Tag2 mice, a pancreatic islet cell carcinogenesis model, and K14-HPV16 transgenic mice, a 17 β -estradiol (E2)-induced cervical carcinogenesis model [176]. In a recent clinical study, E-64 was found to have a sufficient inhibitory effect on circulating cathepsins in breast cancer patients. In this cohort, E-64 demonstrated efficacy in inhibiting cysteine peptidase-like activity in 64% of the patients, whereas chicken egg white cysteine was effective in only 34% of the patients [177]. However, numerous other studies concluded that E-64 monotherapy had no impact on tumor growth in in vivo models. Olson et al. pointed out issues related to drug delivery as one of the potential reasons [5]. As another reason, inhibitor-induced compensatory mechanism in cathepsin family has been indicated. Specifically, E-64 and cystatin C promoted cathepsin S activation while suppressing cathepsin L activity [178].

Fsn5030 is a humanized monoclonal antibody which has a strong affinity with extracellular cathepsin S. It has been demonstrated to curb tumor growth and neovascularization by inhibiting the function of cathepsin S, thereby blocking endothelial invasion and tubal formation in cell-based assays and xenograft mouse models [21, 179]. In combination with chemotherapy (irinotecan), Fsn5030 showed a synergic anti-tumor activity on colorectal cancers [180]. Another study showed that Fsn5030-mediated targeting of cathepsin S triggers natural killer cell-based killing of colorectal cancer cells [181]. These studies strongly suggest that Fsn5030 could be a promising drug warranting clinical trials, but associated studies have been suspended in this decade.

The challenge lies in the fact that there are currently no robust small molecules available to selectively inhibit individual cathepsins. A few studies have reported the effectiveness of innovative selective cathepsin inhibitor in basic studies (Table. 2). A selective cathepsin C inhibitor, AZD7986, effectively suppressed lung metastasis of breast cancer in an orthotopic implantation mouse model by inhibiting NET formation [74]. Another selective cathepsin L and S inhibitor, ASPER-29, also successfully inhibited lung and liver metastases of pancreatic cancer in in-vivo models [182]. GB11-NH2 is a small molecule inhibitor against cathepsin B, L, and S. It reduces the viability of bone marrow-derived macrophages by causing autophagic imbalance and increased oxidative stress. Interestingly, GB11-NH2-induced TAM apoptosis leads to further cell death of adjacent mammary tumor [152, 183]. Other studies have discovered novel selective inhibitors against cathepsins, paving the way for cathepsin-targeted strategies [184, 185]. These potential treatments should be rapidly, yet cautiously, subjected to further translational and clinical trials.

Table 2.

Innovative cathepsin inhibitors targeting the tumor microenvironment.

Drug Name	Target Cathepsins	In Vitro Study	In Vivo Study	Clinical Study	References
ASPER-29	L and S	Inhibits migration and invasion of pancreatic cancer cells.	Inhibits liver and lung metastasis of pancreatic cancer cells in xenotransplantation mouse models.	Not applicable	[182]
AZD7986 (Brensocatib)	C	Not applicable	Suppresses the circulating NETs and alleviates lung metastasis of breast cancer in mouse models.	Phase 3 (bronchiectasis, COVID-19)	[74]
Cycloastragenol (CAG)	B	Not applicable	Synergically reduces the tumor burden by enhancing CD8+ infiltration and cytotoxic activity with anti-PD-1 antibody.	Not applicable	[44]
CLIK-148	L	Inhibits differentiation of functional CD14+ MDSCs.	Reduces tumor growth of breast cancer cells.	Not applicable	[76]
Odanacatib	K	Increases osteoclast fusion and bone resorption.	Synergically reduces tumor size with oxaliplatin in xenograft mouse models of colon cancer cells.	Phase 2 (bone metastasis of breast cancer) Phase 3 (osteoporosis)	[150, 164]
LY3000328	S	Inhibits proliferation of Tregs and increases their apoptosis.	Reduces splenic and intra-tumoral Tregs in mice.	Phase 1	[77]
1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG)	B	Suppresses proliferation and colony formation of colon cancer cells.	Suppresses the ECM remodeling and regulates the populations of T cells, macrophages, and MDSCs, inhibiting liver and lung metastasis in colon cancer-implanted mouse models.	Phase 2 (advanced non-Hodgkin lymphoma, metastatic triple negative breast cancer)	[11]

In summary, numerous preclinical studies suggest the potential benefits of targeting cathepsins to advance cancer treatment. However, concerns remain regarding the clinical application of these drugs, particularly due to the broad distribution and multifunctionality of the cathepsin family, which may lead to adverse effects associated with pan-cathepsin inhibitors. Additionally, cathepsin inhibition may exhibit both beneficial and harmful effects, albeit predominantly beneficial, since the TME involves complex interactions among various cell types. This may explain the reason pan-cathepsin inhibitors were not suitable in human trials. Selective inhibitors, particularly those targeting cathepsins B, K, and S, appear more promising as they may minimize unnecessary adverse effects by selectively blocking specific tumor-promoting functions of cathepsins. Nonetheless, a significant limitation of these inhibitors lies in the scarcity of preclinical and clinical data, largely due to their limited accessibility, mandating further publications by multiple research groups and in diverse experimental settings. Furthermore, the compensatory mechanisms among cathepsins, which share overlapping roles in intracellular signaling processes, have been identified as another challenge to selective cathepsin inhibitions [178]. Although our current understanding of the clinical responsiveness of cancers to cathepsin inhibitors remains limited and requires further validation, the development of optimal cathepsin-targeted strategies, encompassing precise adjustments to administration routes, dosages, treatment durations, and timing, holds significant promise for future cancer treatments.

5. CLINICAL TRIALS FOCUSING ON CATHEPSIN INHIBITION IN DISEASES

As stated above, cathepsins are increasingly recognized as promising targets for cancer therapy due to their dual role in facilitating cancer progression and optimizing the TME. Unfortunately, however, no therapeutic reagent to block cathepsins has successfully shown a clinical benefit to prolong the survival in cancer patients. This can be attributed to the limited number of cathepsin inhibitors have progressed to clinical trials despite numerous studies showing their potential in suppressing cancer progression or modifying the TME in vitro or in vivo. Here, we introduce ongoing and previous clinical trials and potentially beneficial cathepsin inhibitors which demonstrated positive outcomes in basic research, specifically through neutralizing the TME.

The sole clinical trial involving a cathepsin K inhibitor, odanacatib (MK-0822), was performed with cancer patients to cure bone resorption by bone metastasis of breast cancer [186]. In this study, however, only the bone resorption marker, urinary N-telopeptide of type I collagen normalized to creatinine, was measured as the primary endpoint, without evaluating the progression of bone metastasis. Odanacatib is a selective cathepsin K inhibitor for humans. It is a long-lasting drug with a long half-life and a low clearance in vivo, thus has been tested in numerous clinical trials, primarily to treat osteoporosis, a pathological bone density loss [187–190]. In a double-blind, randomized control study, a 4-week treatment of ondanacatib suppressed the bone resorption similarly to zoledronic acid, a standard therapy for bone resorption, and caused only minor side effects, suggesting its safety and well-tolerance [186]. A supporting study revealed that odanacatib suppressed mRNA expressions of secreted pro-osteoclast factors, including parathyroid hormone-related protein, CXCR-4, and TNF-α, as well as protein expressions of cathepsin K, IL-6, IL-1β, and phosphorylated NF-κB [191]. However, an increased risk of stroke due to unknow mechanism was reported with ondanacatib use, indicating the need for cautious consideration in its clinical application [192].

A multi-centric randomized control trial of AZD7986, or Brensocatib, a potent inhibitor of cathepsin C and G, was conducted for patients with COVID-19, showing compatible adverse events in the treatment group to the placebo group [193]. In another large-phase 2 study for patients with bronchiectasis, a slightly higher incidence of adverse effects, including significantly more headache, dyspnea, and skin events, was observed by Brensocatib treatment than by placebo. In contrast, the incidence of severe adverse effects in both treatments was comparable [194]. Given the promising impact of Brensocatib in suppressing breast cancer lung metastasis, this drug should be rising as the next candidate to be applied to clinical trials for cancer patients [74].

There are some other cathepsin inhibitors that were tested in phase 2 clinical trials for patients with rheumatoid arthritis (NCT00425321; RWJ-445380, cathepsin S inhibitor), osteoporosis (NCT00112437, NCT00170911; odanacatib and AAE581, cathepsin K inhibitors)[195], or bronchiectasis (NCT05238675; BI 1291583, cathepsin C inhibitor)[196]. Additionally, phase 1 studies have shown the tolerability of cathepsin inhibitors, including VBY-036 (cathepsin S inhibitor; NCT01911637, NCT01892891), VBY-891 (cathepsin S inhibitor; NCT01947738), RO5459072 (cathepsin S inhibitor; NCT02679014), LY3000328 (cathepsin S inhibitor; NCT01515358), SLV213 (cathepsin L inhibitor; NCT06146374), and GSK2793660 (cathepsin C inhibitor; NCT02058407). However, at the moment, no well-designed prospective clinical trial with cathepsin inhibitors has been conducted. These trials suggest a satisfactory level of safety, with a low incidence of adverse events associated with selective and pan-cathepsin inhibitors, whereas some mild to moderate unfavorable events, including skin rash, were observed [194, 197]. Except for an increased risk of stroke by odanacatib [189], no drug-induced death or significantly higher rates of study discontinuation due to cathepsin inhibitors were reported in the clinical studies of brensocatib [193, 194], LY3000328 [198], BI 1291583 [199], or GSK2793660 [197], expecting an acceptable safety and feasibility for the clinical use of selective cathepsin inhibitors.

6. CONCLUSION

Cathepsins, involved in various processes within the TME and systemic homeostasis, are increasingly recognized as promising targets for cancer therapy. Their roles in cancer progression, metastasis, and chemoradioresistance underpin their therapeutic potential. However, despite encouraging preclinical data, there is a gap in translating these findings to the clinic, with few cathepsin inhibitors having made it to clinical trials and none demonstrating a significant survival benefit. Furthermore, the wide range of cathepsin activities also raises concerns of potential off-target toxicities. Therefore, it is crucial to conduct more clinically relevant studies to explore the therapeutic potential and safety of targeting cathepsins in the context of cancer treatment.

Abbreviations

BCG

Bacillus Calmette–Guérin

cFLIP

cellular FLICE-like inhibitory protein

EC

endothelial cell

ECM

extracellular matrix

EMT

epithelial-mesenchymal transition

FAK

focal adhesion kinase

FGF

fibroblast growth factor

HSC

hematopoietic stem cell

ICI

immune checkpoint inhibitor

IFN-γ

interferon γ

IRF1

interferon regulatory factor 1

JAM-B

junctional adhesion molecule-B

LDHA

lactate hydrogenase A

LFA-1

lymphocyte function-associated antigen 1

LRP1

LDL receptor-related protein 1

Mac-1

macrophage antigen-1

MDSC

myeloid-derived suppressor cell

MHC

major histocompatibility complex

MMP-9

matrix metalloproteinase 9

NET

neutrophil extracellular trap

NF-κB

nuclear factor kappa B

NLRP3

NOD-like receptor family, pyrin domain containing-3 protein

OGT

O-GlcNAc transferase

PAR4

protease-activated receptors 4

PGG

1,2,3,4,6-Penta-O-galloyl-β-D-glucose

PR3

proteinase 3

RANKL

receptor activator of nuclear factor kappa-B ligand

SCC1

squamous cell carcinoma antigen 1

SPARC

secreted protein acidic and rich in cysteine

TAM

tumor-associated macrophage

TFEB

transcription factor EB

TLR

toll-like receptor

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand

Treg

regulatory T cell

TSP-1

thrombospondin-1

Usp27x

ubiquitin-specific protease 27x

VEGF

vascular endothelial growth factor

Availability of data and material:

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.