Abstract

   Treatment of glioblastoma (GBM) remains very challenging, and it is
   particularly important to find sensitive and specific molecular
   targets. In this work, we reveal the relationship between the
   expression of cathepsins and radioresistance in GBM. We analyzed
   cathepsins (cathepsin B, cathepsin D, cathepsin L, and cathepsin Z/X),
   which are highly associated with the radioresistance of GBM by
   regulating different types of cell death. Cathepsins could be potential
   targets for GBM treatment.

   Keywords: glioblastoma, cathepsins, radiotherapy, radiosensitivity,
   cell death

1. Introduction

   Glioblastoma (GBM) is extremely aggressive with a poor prognosis, and
   its exact etiology is unknown [[32]1]. The survival and prognosis of
   GBM are related to malignancy and treatment sensitivity. The treatment
   for GBM consists of surgical resection combined with chemoradiotherapy.
   Radiotherapy (RT) remains a critical component of the therapy for GBM
   and other malignancies. Ionizing radiation directly or indirectly
   causes oxidative injury at the local or abscopal site [[33]2] including
   DNA damage, cell cycle arrest, apoptosis, etc.

   Cathepsins, mainly lysosomal proteases, may degrade proteins under
   acidic pH conditions. According to their catalytic amino acids,
   cathepsins are classified into cysteine proteases (cathepsin B, C, F,
   H, L, K, O, S, W, and Z (also known as CTSX)), serine proteases
   (cathepsin A and G), and aspartic amino acid proteases (cathepsins D
   and E). Structurally, these enzymes have not only rather short active
   site cleavage, including three well-binding substrate-binding subsites
   (S2, S1, and S1′), but also rather extensive binding regions (S4, S3,
   S2′ and, S3′). This geometry distinguishes them from other protease
   classes, such as serine and aspartic proteases, with six and eight
   substrate binding sites. Exopeptidases (cathepsins B, C, H, and X)
   compared to endopeptidases (such as cathepsins L, S, V, and F) have
   structural features that facilitate the binding of N- and C-terminal
   groups at the active site of a substrate in cracks [[34]3]. The
   activities of cathepsins are tightly controlled at the transcriptional
   and post-transcriptional levels by various regulators under
   physiological conditions. First, a cathepsin is synthesized as a
   preproenzyme, followed by the removal of the pro-peptide (also a signal
   peptide) from the endoplasmic reticulum, glycosylation in the Golgi
   apparatus, transport to the lysosome, and activation by controlled
   protein hydrolysis [[35]4]. Secondly, it is worth noting that the
   absence of one cathepsin can be compensated by the others in a complex
   protein regulatory network. For example, in transgenic mouse models of
   pancreatic duct adenocarcinoma, breast cancer cells, and
   myeloid-derived suppressor cells, reduced expression of cathepsin B was
   compensated by cathepsin X/Z in breast cancer cells [[36]5]. Finally,
   many studies have suggested a causal relationship between cathepsins
   and GBM. For example, the expression of CTSB (an exopeptidase, acting
   as an exo- or endopeptidase) and its enzymatic activity are associated
   with high tumor aggressiveness and grade in GBM [[37]6]. Likewise,
   cathepsin D (CTSD) remains a normal and major component of lysosomes in
   glioma cells compared to normal astrocytes. In addition, cathepsin S
   (CTSS) is unexpressed in normal glial, neuronal, or endothelial cells,
   but is expressed in GBM cells [[38]7].

   Cathepsins are involved in cell death, invasion, and metastasis
   [[39]8,[40]9]. Extracellular secreted cathepsins can alter the tumor
   microenvironment by degrading the extracellular matrix (ECM) and
   activating, processing, or degrading various growth factors, cytokines,
   and chemokines. Cathepsins also disrupt cell–cell adhesion molecules
   that contribute to tissue invasion and metastasis, and thus represent
   an important class of proteins that contribute to cancer progression
   and spread [[41]10]. In addition, it has been shown that proteolytic
   enzymes, including lysosomal endopeptidases (cathepsins), are
   inseparable from extracellular matrix components, and essential for the
   invasiveness of GBM [[42]11]. A previous study found that CTSL is
   involved in X-ray-induced invasion and migration of human GBM U251
   cells, and inhibiting the expression of CTSL can weaken the invasive
   ability of U251 cells [[43]12]. In addition, X-ray significantly
   increased the levels of CTSL in cytoplasmic and nuclear of U251 cells,
   and the inhibition of CTSL increased radiation-induced changes
   [[44]13]. It is inferred that CTSL may be involved in regulating the
   radiosensitivity of GBM in vitro.

   From the above, our results will elucidate the roles of cathepsins in
   regulating the radioresistance of GBM and provide a theoretical basis
   for fulfilling GBM treatment strategies.

2. Materials and Methods

   The correlation analysis between cathepsins and GBM prognosis was
   obtained from GeneExpression Profiling Interactive Analysis (GEPIA).
   The protein–protein interaction (PPI) analysis was based on the STRING
   public database. The functional heterogeneity of cancer cells was
   explored by ScRNA-seq and distinct functional states of cancer cells
   were detected by CancerSEA Online. Venn Diagrams were drawn for
   interaction protein intersection analysis. GBM ([45]GSE153982, HK-374
   cells) radiotherapy data were obtained from the Gene Expression Omnibus
   database (GEO), and differential gene expression analysis was performed
   using the NetworkAnalyst online tool. The KEGG enrichment analysis was
   performed using the DAVID and KOBAS online tools. The survival curve
   was plotted by the Kaplan–Meier method, and the difference between
   groups was tested by log-rank. The co-expressed genes were analyzed by
   the Pearson method; p < 0.05 was considered statistically significant.
   The websites used in this study were as follows:

   GEPIA: [46]http://gepia.cancer-pku.cn, (accessed on 30 December 2021)

   CancerSEA: a cancer single-cell state atlas, (accessed on 20 November
   2022)

   STRING: [47]https://cn.string-db.org, (accessed on 30 December 2021)

   GEO: [48]https://www.ncbi.nlm.nih.gov/geo/,(accessed on 10 March 2022)

   NetworkAnalyst: [49]https://www.networkanalyst.ca, (accessed on 30 June
   2022)

   DAVID: [50]https://david.ncifcrf.gov/, (accessed on 10 June 2022)

   KOBAS: [51]http://kobas.cbi.pku.edu.cn/, (accessed on 10 June 2022)

3. Results

3.1. Cathepsins and Clinical Prognosis in GBM

   The involvement of cathepsins in the progression of malignancies is
   frequently associated with aberrant proteolytic regulatory mechanisms.
   We analyzed the relationship between cathepsins and GBM based on the
   GEPIA database and found that CTSS, cathepsin C (CTSC), cathepsin K
   (CTSK), cathepsin O (CTSO), CTSB, CTSL, CTSZ/X, and CTSD were highly
   expressed in GBM ([52]Figure 1), while only CTSB, CTSD, CTSL, and
   CTSZ/X (p < 0.05) were associated with overall survival (OS)
   ([53]Figure 2). To better understand the role of cathepsins in GBM, the
   relationship between cathepsins and the different functional states of
   GBM was explored based on the CancerSEA database (CancerSEA: a cancer
   single-cell state atlas). The results showed that only CTSB and CTSZ/X
   were significantly associated with GBM functional status ([54]Figure
   S1). Combined with the GEPIA analyses, we finally decided to explore
   the potential biological relationship between CTSB, CTSD, CTSL, CTSZ/X,
   and GBM.

Figure 1.

   [55]Figure 1
   [56]Open in a new tab

   Cathepsin mRNA expression is upregulated in GBM patients. (A–H)
   Compared with normal tissues, CTSS, CTSC, CTSK, CTSO, CTSB, CTSD, CTSL,
   and CTSZ/X are highly expressed in tumors. Number of tumor samples (n =
   163); number of tissue samples (n = 207). The red boxplot represents
   the tumor, and the grey boxplot represents the normal tissue. The data
   are presented as the mean ± SD, * p < 0.05.

Figure 2.

   [57]Figure 2
   [58]Open in a new tab

   Cathepsin mRNA expression level in the OS of GBM patients. (A–D) The
   expressions of CTSS, CTSC, CTSK, and CTSO were not significantly
   associated with shortened overall survival in GBM patients (p > 0.05).
   (E–H) The expressions of CTSB, CTSD, CTSL, and CTSZ/X were
   significantly associated with shorter overall survival in GBM patients
   (p > 0.05). Number of tumor samples (n = 162).

3.2. Predicted PPIs of Cathepsins

   To further investigate the specific mechanism by which cathepsins are
   involved in the regulation of the prognosis in GBM, we screened the
   PPIs of CTSB, CTSL, CTSZ/X (cysteine protease), and CTSD (aspartic
   protease) according to the public database STRING
   ([59]https://cn.string-db.org, accessed on 30 December 2021) to figure
   out the potential downstream targets. ([60]Figure 3; [61]Table S1,
   Supporting Information), and then we performed an overlapping analysis
   of the PPIs of CTSB, CTSL, CTSZ/X, and CTSD by using Jvenn mapping. The
   results showed only 13 identical interacting proteins ([62]Table S2,
   Supporting Information), verifying that although the two protease
   families CTSB, CTSL, and CTSZ/X (cysteine protease) and CTSD (aspartic
   protease) have similar structural elements and share the same
   proteolytic machinery, their conformations and catalytic activities,
   tissue and cellular distribution patterns, and physiological roles are
   different.

Figure 3.

   [63]Figure 3
   [64]Open in a new tab

   Top 50 PPIs of CTSB (A); CTSD (B); CTSL (C); CTSZ/X (D); Overlapping
   interacting proteins of four cathepsins (E).

3.3. Cathepsins and Cell Death

   Cathepsins are lysosomal proteolytic enzymes and might perform distinct
   roles in cancer progression, including invasion and apoptosis
   [[65]10,[66]14,[67]15]. Additionally, cathepsins may equally possess
   tumor-suppressive roles [[68]16], depending on the cellular context.
   Therefore, it is important to perform in vivo analysis to understand
   the functions of cathepsins in the pathobiology of GBM [[69]17]. We
   first analyzed the interacting proteins of the four OS-related
   cathepsins ([70]Table S1, Supporting Information), secondly, we
   selected a set of cell death-related genes with a good correlation
   [[71]18] ([72]Table S3, Supporting Information), and finally we
   analyzed and screened specifically for cell death-related interacting
   proteins ([73]Figure S2, Intersection of interacting proteins of CTSB,
   CTSD, CTSL, and CTSZ/X with cell death-related genes; [74]Table S4,
   Supporting Information). Cell death-related genes were obtained from
   the GO ([75]http://geneontology.org, accessed on 10 October 2022) and
   KEGG ([76]https://www.kegg.jp, accessed on 10 October 2022) databases
   [[77]18]. Cell death-related interacting proteins were obtained based
   on the intersection of cathepsin-related interacting proteins and cell
   death-related genes.

3.3.1. CTSB and Cell Death

   CTSB belongs to the MEROPS peptidase subfamily C1A, also known as thiol
   protease. It contains the propeptide-C1 domain and pept-C1 domain. The
   propeptide-C1 domain is found at the N-terminal of CTSB and CTSB -like
   peptidases. They are synthesized as inactive zymogens. Activation of
   the peptidases occurs with the removal of the propeptide. The Pept-C1
   domain can catalyze the hydrolysis of peptide bonds in the polypeptide
   chain through the mechanism of the sulfhydryl group of the cysteine
   residue in the active center acting as a nucleophile. CTSB and its
   interacting proteins most likely act through peptide bond hydrolysis
   [[78]19,[79]20]. CTSB can be involved in the regulation of apoptosis,
   necroptosis, autophagy, pyroptosis, and ferroptosis by interacting
   proteins ([80]Figure 4, Blue arrows). However, the exact mechanism
   remains to be explored.

Figure 4.

   [81]Figure 4
   [82]Open in a new tab

   The involvement of CTSB/CTSD/CTSL/CTSZ/X-interacting proteins in cell
   death. Yellow circles indicate cathepsin-interacting proteins
   associated with cell death. Blue arrows represent the interacting
   proteins of CTSB involved in cell death; red arrows represent the
   interacting proteins of CTSD involved in cell death; green arrows
   represent the interacting proteins of CTSL involved in cell death;
   purple arrows represent the interacting proteins of CTSZ/X involved in
   cell death. 1. The process of apoptosis and necroptosis. 2. The process
   of autophagy. 3. Pyroptosis process. 4. Ferroptosis process.

   Firstly, two distinct pathways of apoptosis are involved here, the
   death receptor pathway and the mitochondrial pathway. The death
   receptor pathway is initiated by death receptors and related ligands on
   the cell surface, and CTSB leads to apoptosis by activating caspases 3,
   7, and 8 to regulate intracellular structural and functional proteins.
   The mitochondrial pathway involves many conserved signaling proteins
   and depends on mitochondrial integrity. This process is regulated by
   the BCL-2 family consisting of pro-apoptotic (BAX, BAK, BAD, BID, PUMA,
   BIM, and PYCARD) and anti-apoptotic (BCL-2, BCL-xL, BCL-w, and Mcl-1)
   factors that tightly regulate the balance between protein activities.
   CTSB can act on anti-apoptotic or pro-apoptotic factors, directly or
   indirectly regulating the release of cytochrome c from mitochondria to
   regulate apoptosis. Previous studies have also indicated that CTSB
   downregulation can reduce TNF-α-mediated apoptosis and autophagy, and
   that CTSB, CTSH, CTSL, and CTSS can cleave canonical caspase substrates
   such as procaspase-1, -3, and -8 [[83]21,[84]22] and release the
   pro-apoptotic mitochondrial enzyme, cytochrome c [[85]23], which
   activates caspases and apoptosis. EGFR as a predicted interacting
   protein of CTSB and its inhibition in a mouse lung tumor model showed
   that it can participate in apoptosis by activating BIM and PUMA
   [[86]24].

   Secondly, CTSB is also involved in the regulation of TNF signaling
   pathway-mediated necroptosis. TNF binds to its receptor TNFR1, which
   consists of the TNFR1-related death domain proteins TRADD, TRAF2, and
   RIPK1, and an inhibitor of apoptosis (cIAP1 or cIAP2). CTSB may affect
   RIP1 ubiquitination by cleaving cIAPs, thereby regulating necroptosis.
   The necroptotic process is dominated by stimulated poly (ADP-ribose)
   polymerase 1 (PARP-1), Ca^2+-dependent calpain (Calpain), and the
   proapoptotic BCL-2 member BAX. Then, the truncated AIF (tAIF) is
   released from the mitochondria, thereby redistributing tAIF from the
   cytoplasm into the nuclear compartment, which in turn promotes
   chromatin disassembly and loss of cell viability. The anti-apoptotic
   protein BCL-2 inhibits this release. The roles of calpain, CTSB, and
   PARP in the nuclear translocation of AIF have been studied in the
   pathology of cerebral ischemia [[87]25], and the activation of calpain
   and CTSB favors the release of AIF [[88]26]. Studies have shown that in
   long-term liver injury and fibrosis, elevated levels of cytoplasmic
   CTSB can activate NOD-like receptors of the pyrin domain 3 (NLRP3)
   inflammasome [[89]27]. Inhibition of Hsp90, an interacting protein of
   CTSB, attenuated TNF-α-induced necroptosis cell death by inhibiting the
   activation of the RIP1–RIP3–MLKL pathway [[90]28].

   Thirdly, CTSB can participate in the regulation of autophagy through
   interacting proteins in three stages (induction, nucleation, and
   elongation; [91]Figure 4). The process of autophagy following nutrient
   or growth factor deprivation, activation of AMPK, and/or inhibition of
   mTOR leads to the activation of ULK, which phosphorylates Beclin1,
   leading to the activation of VPS34 and formation of phagocytes. CTSB
   may participate in this process by interacting with beclin1. Phagocyte
   nucleation occurs at the onset of the autophagy cascade through the
   phosphorylation of phosphatidylinositol 3-kinase class III (PI3KC3)
   complex I, which then interacts with PI3P to attract the
   ATG12~ATG5–ATG16L1 complex, which contributes to the addition of
   signature Protein LC3 (microtubule-associated protein light chain 3) to
   the extended isolation membrane. LC3-I is then converted to LC3-II,
   which is the fulcrum for phagocyte membrane expansion and closure.
   ATG5, 7, 12, and LC3, the key functional proteins of autophagy, are the
   interacting proteins of CTSB and may be cleaved by CTSB. Therefore,
   CTSB may play an important role in the regulation of autophagy
   nucleation and elongation. Additionally, the downregulation of CTSB
   resulted in a significant decrease in the mRNA of MAP1LC3-1 and ATG5 in
   granulosa cells [[92]29]. In addition, CTSB downregulation resulted in
   a significant decrease in MAP1LC3-1 and ATG5 mRNA in granulosa cells,
   suggesting multiple regulatory mechanisms for both by CTSB.

   Fourthly, CTSB induces pyroptosis by cleaving the inflammasome complex
   (NLRP1/NLRP3/AIM2) and activating caspase1. CTSB may aggravate acute
   pancreatitis (AP) by activating the NLRP3 inflammasome and promoting
   caspase-1-induced pyroptosis [[93]30]. In addition, NLRP4, one of the
   CTSB-interacting proteins, has been demonstrated to induce a
   GSDMD-dependent pyroptosis [[94]31,[95]32].

   Finally, CTSB also regulates ferroptosis by cleaving CP
   (Ceruloplasmin). CP assists ferroportin (FPN) in exporting Fe^2+,
   thereby further participating in the regulation of ferroptosis
   [[96]33]. CTSB can also indirectly induce ferroptosis by increasing
   unstable cellular iron content through interacting proteins ATG5 and
   ATG7 [[97]34]. Evidence of ferroptosis in diseases ranging from
   neurotrauma to cancer underscores the importance of its identification
   for clinical application, which is further reinforced when
   CTSB-knockout primary fibroblasts do not respond to various inducers of
   ferroptosis [[98]35]

3.3.2. CTSD and Cell Death

   CTSD belongs to the family of aspartic proteases, different from the
   family of cysteine proteases, containing the A1-propeptide, TAXi-N
   (Triticum aestivum endoxylanase inhibitor-N-termini), and TAXi-C
   (Triticum aestivum endoxylanase inhibitor-C-termini) domains
   [[99]36,[100]37,[101]38] ([102]https://smart.embl.de/smart/, accessed
   on 30 December 2021). Most eukaryotic endopeptidases (Merops family A1)
   are synthesized with the signal peptides and propeptides. The
   propeptide contains two helices that block the active site cleft,
   specifically the Asp11 residue conserved in pepsin, which forms
   hydrogen bonds with the conserved Arg residue in the propeptide. This
   hydrogen bond stabilizes the propeptide conformation and may be
   responsible for triggering the conversion of pepsinogen to pepsin under
   acidic conditions [[103]39,[104]40]. The domains TAXi-N and TAXi-C are
   initially found in aspartic protease and xylanase inhibitors. The N-
   and C-termini of members of this family provide the catalytic pockets
   necessary for proteolysis. CTSD can exert a proteolytic function
   through the domains TAXi-N and TAXi-C. Increasing evidence shows that
   CTSD is actively involved in apoptosis, necroptosis, autophagy, and
   ferroptosis through interacting proteins ([105]Figure 4, red arrows).

   Firstly, intracellular CTSD is a key mediator in the activation of pro-
   and anti-apoptotic proteins or nuclear proteins involved in apoptosis.
   It can trigger BAX insertion into the mitochondrial membrane directly
   or indirectly through Bid lysis [[106]41,[107]42], causing both the
   release of mitochondrial cytochrome c and activation of caspase 3,
   leading to apoptosis [[108]43,[109]44]. It can also regulate apoptosis
   by disrupting the balance between BH3/BCL-2 [[110]44,[111]45]. By
   analyzing the interacting proteins of CTSD, we hypothesized that it
   could act on IAP to deregulate the inhibition of caspase 3/9 and cleave
   pro-apoptosis-related factors (e.g., caspase 3/9, TNF, TP53, cytochrome
   c, AKT, etc.) to regulate apoptosis.

   Secondly, CTSD cleaves caspase 8 and dissociates it from mitochondria.
   Caspase 8 dissociation may lead to elevated levels of RIP-1
   ubiquitination and the induction of necroptosis [[112]46]. In addition,
   we proposed that CTSD could block TNF-mediated signaling as well as act
   on Calpain or Bid to regulate necroptosis.

   Thirdly, CTSD can regulate autophagy. CTSD is one of the major
   lysosomal proteases indispensable for the maintenance of cellular
   proteostasis by turning over substrates of endocytosis, phagocytosis,
   and autophagy. CTSD-deficient mouse models show lysosomal hypertrophy
   and impaired autophagic flux in the visceral and central nervous
   systems, and these phenotypes can be ameliorated by recombinant human
   CTSD [[113]47]. Another study showed that CTSD deficiency or dual
   deficiency of CTSB and CTSL resulted in the severe blockage of
   autophagic flux, demonstrating that these proteases are essential for
   lysosomal proteolysis [[114]48]. Some researchers have also proposed
   that when the expression of CTSD is significantly reduced, the levels
   of autophagy in the late stage are reduced accordingly [[115]49].
   Ferroptosis is an autophagic cell death process
   [[116]34,[117]50,[118]51]. However, in the process of autophagy,
   establishing whether CTSD can regulate autophagy or ferroptosis by
   acting on autophagy-related proteins (Beclin1, ATG5, and ATG7) needs
   further experiments.

3.3.3. CTSL and Cell Death

   CTSL contains the domains Inhibitor-I29 and Pept-C1 [[119]52,[120]53]
   ([121]https://smart.embl.de/smart/, accessed on 30 December 2021).
   Inhibitor-I29 is the cathepsin propeptide inhibitor domain. This domain
   is located at the N-terminus of some C1 peptidases and acts as a
   propeptide, which prevents substrates from entering the active site.
   CTSL can be activated by interaction with a second peptidase or by
   autocatalytic cleavage to remove the N-terminal inhibitory domain. Both
   CTSB and CTSL belong to the papain family of cysteine proteases and, in
   addition, because CTSL contains the peptidase-C1 structural domain, it
   also belongs to the cysteine peptidase family C1, subfamily C1A (papain
   family, CA family) [[122]54]. CTSB and CTSL play an important role in
   apoptosis and necroptosis. However, there are differences between the
   interacting proteins of these two cathepsins and the specific
   mechanisms involved in cell death.

   CTSL is responsible for the degradation of multiple proteins and is
   known to be involved in neuronal apoptosis associated with abnormal
   cell cycles [[123]55]. Cleavage of IAP by CTSL relieved the inhibition
   of caspase 3/9, and CTSL disrupted the balance between BH3/BCL-2 to
   regulate apoptosis. We propose a hypothesis based on the interacting
   proteins of CTSL: CTSL may participate in necroptosis either through
   interacting proteins or by activating apoptosis and then participating
   in necroptosis.

3.3.4. CTSZ/X and Cell Death

   CTSZ/X contains the domain of peptidase-C1, also belongs to cysteine
   peptidase family C1, subfamily C1A, exhibits carboxymonopeptidase and
   carboxydipeptidase activities, and is involved in maintaining
   homeostasis and immune cell function [[124]56,[125]57]
   ([126]https://smart.embl.de/smart/, accessed on 30 December 2021).
   CTSZ/X is involved in cell death via interacting proteins ([127]Figure
   4, purple arrows). CTXZ/X plays a critical role in apoptosis,
   necroptosis, ferroptosis, and autophagy.

   It has been shown that CTSZ/X cleaves IAP to deregulate caspase 3/9
   inhibition and disrupts the balance between BH3/BCL-2 to regulate
   apoptosis. CTSZ/X also mediates the EGFR/PI3K/AKT signaling pathway
   [[128]24,[129]58], P53/AKT signaling pathway [[130]59], PARP/CAPN1
   signaling pathway [[131]60], and IGF2R/caspase3 signaling pathways
   [[132]61] which are involved in apoptosis ([133]Figure 4, purple
   arrow). Strong upregulation of CTSX/Z caused by H. pylori (Helicobacter
   pylori) infection has been reported to be strongly associated with
   gastric carcinogenesis, while knockdown of CTSX/Z leads to G1 phase
   arrest and apoptosis [[134]62]. Knockdown of CTSX/Z expression or
   treatment with specific CTSX/Z inhibitors protected the
   pheochromocytoma cell line PC12 and neuroblastoma cell line SH-SY5Y
   from 6-hydroxydopamine (6-OHDA) toxicity and reduced apoptosis
   [[135]63]. In H9c2 cardiomyocytes, hypoxia induces activation of
   CTSB-interacting protein IGF2R and activation of caspase 3, leading to
   apoptosis [[136]61]. Additionally, CTSZ/X regulates necroptosis,
   probably by cleaving IAPs during necroptosis.

   Finally, we used CTSZ/X and its interacting proteins to identify
   potential mechanisms that may be involved in autophagy, necroptosis,
   and ferroptosis ([137]Figure 4, purple arrows), but no evidence of
   CTSZ/X involvement in autophagy, necroptosis, and ferroptosis has been
   reported. Whether CTSZ/X can be involved in cell death through cleavage
   of interacting proteins requires further confirmation.

3.4. Cathepsin-Mediated Radioresistance in GBM

   RT induces changes in cathepsin expression, which might be associated
   with the radioresistance of GBM. To investigate the specific mechanism
   of radioresistance, we performed differential gene expression analysis
   using the GEO database and found that CTSB, CTSL, and CTSD increased in
   GBM after radiotherapy ([138]GSE153982). We combined the changes in
   interacting proteins of CTSB, CTSL, and CTSD after radiotherapy
   ([139]Table S5, Supporting Information) to infer the possibility that
   CTSB, CTSL, and CTSD are involved in apoptosis, necroptosis, and
   autophagy. The increase in CTSB, CTSL, and CTSD after radiotherapy
   first affects mitochondrial permeability, participates in apoptosis and
   necroptosis through the p53 signaling pathway, or directly participates
   in apoptosis through the mitochondrial pathway; secondly, mitochondria
   permeability through DNA damage or activation of the p53 signaling
   pathway affected by calpain is involved in necroptosis; finally, it is
   involved in necroptosis by mediating the classical pathway
   (TNF/RIP1/RIP3 signaling pathway) ([140]Figure 5). We also performed a
   functional enrichment analysis using proteins that were differentially
   expressed after IR ([141]Figure 6, [142]Table S6, Supporting
   Information). KEGG enrichment analysis results revealed that the top
   six enriched pathways for differential genes were the cell cycle, p53
   signaling pathway, DNA replication, nucleocytoplasmic transport,
   lysosome, etc. The p53 signaling pathway is involved in apoptosis and
   senescence and inhibits tumorigenesis by preventing the growth and
   division of damaged and potentially precancerous cells. Studies in
   model systems have shown that enhancing or reactivating p53 activity in
   cancer cells, either in combination with stress-inducing therapy or
   alone, can improve the efficiency of cancer therapy. The p53 signaling
   pathway is crucially meaningful in regulating the apoptosis of human
   GBM cells. Knockdown of CTSK could promote p53-dependent BAX
   upregulation to enhance oxaliplatin-induced apoptosis [[143]64]. The
   expression of CTSL was significantly increased in p53-mutated human
   non-small cell lung cancer tissues, suggesting that there may also be
   some regulation of CTSL expression by p53 [[144]65]. Further studies
   have suggested that it would be a brilliant idea to identify the
   p53–cathepsin axis as the canonical framework for cell death. As
   mentioned above, combined with the results of our differential gene
   analysis before and after RT, we believe that an increase in CTSB,
   CTSL, and CTSD after radiotherapy can directly or indirectly activate
   or inhibit the occurrence of apoptosis, necroptosis, or autophagy, and
   the specific mechanism requires further experiments to verify.

Figure 5.

   [145]Figure 5
   [146]Open in a new tab

   Differentially expressed CTSB, CTSL, and CTSD-interacting proteins
   after RT are involved in cell death. The orange circles represent the
   differentially expressed CTSB, CTSL, and CTSD-interacting proteins
   after RT.

Figure 6.

   [147]Figure 6
   [148]Open in a new tab

   KEGG pathway enrichment analysis of differential genes after
   radiotherapy in GBM. In the GEO database, 12 samples
   (radiotherapy-treated in GBM cells) were analyzed. Differential genes
   after RT were analyzed using DAVID and KOBAS.

4. Discussion

   GBM is the most common primary brain tumor in adults. Conventional
   treatment consists of surgical resection followed by 6 weeks of
   radiation therapy and concurrent temozolomide chemotherapy. Despite
   this treatment, the prognosis is poor, with a median survival of 16
   months. Hypofractionation and stereotactic radiosurgery for radiation
   therapy dose intensification is a promising strategy that has been
   explored to address these challenges [[149]66]. In this study, we found
   that cathepsin (CTSB, CTSL, and CTSD) expression is upregulated after
   IR treatment in GBM, and cathepsins can regulate cell death in various
   ways through their interacting proteins. Cathepsins are promising
   targets for the radiosensitization of GBM.

   Cathepsins belong to a broad family of peptidases that are normally
   active primarily in endosomes and lysosomes and are involved in the
   lysosomal death pathway [[150]67]. The main feature of the lysosomal
   death pathway is lysosomal membrane permeation (LMP). During LMP,
   lysosomal membrane integrity is lost, luminal contents such as
   cathepsins are released into the cytoplasm, and cathepsins cause damage
   to organelles and induce cell death such as apoptosis, pyroptosis, or
   necroptosis [[151]68,[152]69]. Further studies have shown that LMP is
   associated with radiosensitivity. Irradiation-induced lysosomal
   biogenesis leads to the release of more lysosomal hydrolysate into the
   cytoplasm, induces more cell death, and achieves radiosensitization
   [[153]70]. The specific effect of cathepsins on cell death depends on
   whether their cleavage of the substrate is activating or inactivating.
   For example, in glioma stem cells, CTSK is responsible for the cleavage
   and inactivation of stromal-derived factor-1a, and this inactivation
   promotes stemness loss and increased sensitivity to chemoradiotherapy
   in glioma stem cells [[154]71]. By contrast, CTSB processes BID into
   active tBID excessively, which induces the release of cytochrome C from
   the mitochondria. Second, we should also consider that cathepsins are
   affected by upstream signaling factors. For example, cervical cancer
   cells with knockdown of the cystine protease inhibitor SERPINB3
   (squamous cell carcinoma antigen 1, SCCA1) are more sensitive to
   ionizing radiation, mainly due to lysosomal-like death caused by
   enhanced CTSL activity [[155]72,[156]73]. Thirdly, several studies on
   cathepsin-mediated radiosensitivity of GBM have been reported. For
   example, the knockdown of CTSB resulted in cell cycle arrest in the
   G0/G1 phase, where the efficiency of homologous recombination was
   impaired [[157]74]. In addition, CTSD was found to be more
   significantly upregulated in radiation-resistant clones than in
   parental U251 cells, and the knockdown of CTSD improved the
   radiosensitivity of U251 cells. Subsequent studies found that CTSD
   modulates the radiosensitivity of GBM by affecting the fusion of
   autophagosomes and lysosomes [[158]75]. Similarly, CTSL is involved in
   regulating the radiosensitivity of glioma cells by acting as an
   upstream regulator of NF-κB activation, and inhibition of CTSL
   sensitizes glioma cells to radiation [[159]76]. In sum, it is important
   to understand the complete biology of cathepsins in GBM, including
   their impact on cell death, invasion, and the tumor microenvironment.
   This will improve the benefits of clinical treatments for GBM.

   The field of cysteine cathepsins has undergone major changes in recent
   years, with CTSL, CTSB, and CTSD as possible targets for GBM therapy.
   For example, KGP94, a small molecule inhibitor of CTSL, has shown good
   therapeutic effects in vitro against the breast and prostate cell lines
   [[160]77], and we speculate that inhibition of CTSL in GBM may likewise
   be an effective targeted therapeutic approach. Inhibition of CTSB has
   shown promising results in GBM studies. Caffeine reduces mRNA, protein
   expression, and activity of CTSB via Rho-associated protein kinase
   (ROCK), which in turn reduces glioma cell invasion and tumor growth in
   an orthotopic xenograft animal model via the FAK/ERK signaling pathway
   [[161]78]. Furthermore, CTSB is shown to be a direct target of miR-140,
   and inhibition by miR-140 results in reduced temozolomide resistance
   and cell migration [[162]79]. Since cathepsins are ubiquitous proteases
   and distributed in various tissues and organs, targeting them could
   cause corresponding side effects in different organs, such as lung
   fibrosis [[163]80], morphea-like skin lesions [[164]81,[165]82],
   cardiovascular complications [[166]83], etc. Therefore, targeting
   cathepsin presents significant challenges. It is important to fully
   understand the role of cathepsin in the physiological and pathological
   processes of various tissues and organs, and balance the clinical
   benefits and organ toxicity. The specific mechanism of cathepsins
   involved in cell death studied in this paper can provide ideas for
   designing small molecule drugs and minimizing side effects. It should
   also be noted that CTSL have tumor-promoting or tumor-suppressing
   functions in different preclinical models [[167]84,[168]85]. Targeting
   one of the cathepsins will cause the compensation of its diminished
   activity by the others and the impact on an illness would be reduced.
   The activities of other cathepsins will compensate for the reduced
   activity of the targeted cathepsin, which means that their activities
   will not diminish; e.g., the constitutive loss of CTSH can be partially
   functionally compensated by other cysteine- or aspartate-type proteases
   of type II pneumocytes, such as CTSC and CTSE [[169]86]. CTSL was found
   to be upregulated in the thyroid glands of mice deficient in CTSK or
   CTSB, suggesting that cathepsin L compensates for cathepsin K
   deficiency [[170]87]. Therefore, targeting one of the cathepsins may
   not achieve the desired inhibitory effect, but the compensatory
   mechanism of cathepsins may be a promising research direction, and
   targeting the sensors of the compensatory mechanism or rational use of
   pan-cysteine cathepsin inhibitors targeting multiple family members
   (pan-cathepsin inhibitor JPM-OEt) [[171]88] may provide an idea for
   targeted therapy. Therefore, understanding the complete biological
   properties of these cathepsins will allow us to obtain the highest
   clinical benefit. There are still some limitations in our study, as
   cysteine cathepsins are expressed in inactive performance and their
   activation and activities are dependent on many factors, such as mRNA
   and protein expression, tumor epigenetics, etc. The study would have
   much higher relevance if protein levels would be included. At the same
   time, based on the results provided by ScRNA-seq, we also found that
   cathepsins have significant relationships with different functional
   states of GBM (especially CTSB and CTSZ/X). Finally, cathepsins,
   especially extracellular cathepsins, are now also considered suitable
   targets for targeted drug delivery. In addition, the application of
   cathepsins, especially CTSB, in some prodrugs and antibody–drug
   conjugates can successfully activate and release drugs [[172]89]. This
   trend may even increase in the future, showing that cathepsin holds
   some promise for future drug discovery.

5. Conclusions

   In conclusion, this paper elucidates the role of cathepsins in the
   regulation of cell death and cathepsins are potential targets for GBM
   radiosensitization.

Supplementary Materials

   The following supporting information can be downloaded at
   [173]https://www.mdpi.com/article/10.3390/cells11244108/s1, Figure S1:
   The CancerSEA tool explores the relationship between cathepsin
   expression and different functional states in tumors; * p < 0.05; ** p
   < 0.01; *** p < 0.001. Bar graphs show the number of datasets where
   cathepsins are significantly associated with the corresponding state.
   Red indicates a positive correlation; blue indicates a negative
   correlation. Figure S2: The interacting proteins of CTSB, CTSD, CTSL,
   and CTSZ/X intersect with five cell death-related genes. Table S1: The
   interacting proteins of CTSB, CTSD, CTSL, and CTSZ/X; Table S2:
   Overlapping interacting proteins of CTSB, CTSD, CTSL, and CTSZ/X. Table
   S3: Gene list of five cell death modes. Table S4: Interacting proteins
   of four cathepsins involved in cell death. Table S5: After
   radiotherapy, the absolute value of log2FC of differential genes was
   greater than 0.5 to obtain the gene set, which was intersected with
   cathepsin cell death-related proteins. Table S6: Post-radiotherapy
   differential genes of HK-374 cells were obtained from the GEO database.
   ([174]GSE153982) (p < 0.05).
   [175]Click here for additional data file.^ (699.6KB, zip)

Author Contributions

   Conceptualization, project administration, X.L.; manuscript writing,
   X.D.; formal analysis and investigation, X.D., H.C., M.R. and X.L.;
   C.Z. made constructive suggestions for revision and participated in the
   revision of the article, supplementing the framework and content of the
   article. All authors have read and agreed to the published version of
   the manuscript.

Institutional Review Board Statement

   Not applicable (no human subjects).

Informed Consent Statement

   Not applicable (no human subjects).

Data Availability Statement

   GEO public database: [176]https://www.ncbi.nlm.nih.gov/, accessed on 10
   March 2022; String public database: [177]https://cn.string-db.org/,
   accessed on 30 December 2021; GEPIA public database:
   [178]http://gepia.cancer-pku.cn/, accessed on 30 December 2021;
   CancerSEA: a cancer single-cell state atlas.

Conflicts of Interest

   The authors declare no conflict of interest. The funders had no role in
   the design of the study; in the collection, analyses, or interpretation
   of data; in the writing of the manuscript, or in the decision to
   publish the results.

Funding Statement

   This work was supported by the National Natural Science Foundation of
   China (81972969, 81872558, 81773363, and 81673092).

Footnotes

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   claims in published maps and institutional affiliations.

References