Dysregulation of Type II Transmembrane Serine Proteases and Ligand-Dependent Activation of MET in Urological Cancers.

Unlike in normal epithelium, dysregulated overactivation of various proteases have been reported in cancers. Degradation of pericancerous extracellular matrix leading to cancer cell invasion by matrix metalloproteases is well known evidence. On the other hand, several cell-surface proteases, including type II transmembrane serine proteases (TTSPs), also induce progression through activation of growth factors, protease activating receptors and other proteases. Hepatocyte growth factor (HGF) known as a multifunctional growth factor that upregulates cancer cell motility, invasiveness, proliferative, and anti-apoptotic activities through phosphorylation of MET (a specific receptor of HGF). HGF secreted as inactive zymogen (pro-HGF) from cancer associated stromal fibroblasts, and the proteolytic activation by several TTSPs including matriptase and hepsin is required. The activation is strictly regulated by HGF activator inhibitors (HAIs) in physiological condition. However, downregulation is frequently observed in cancers. Indeed, overactivation of MET by upregulation of matriptase and hepsin accompanied by the downregulation of HAIs in urological cancers (prostate cancer, renal cell carcinoma, and bladder cancer) are also reported, a phenomenon observed in cancer cells with malignant phenotype, and correlated with poor prognosis. In this review, we summarized current reports focusing on TTSPs, HAIs, and MET signaling axis in urological cancers.


Introduction
The interaction between cancer cells and stromal cells in the pericellular microenvironment has been fully discussed in the literature [1,2]. On the cancer cell surface, a variety of receptors for cytokines, growth factors, and protease activating-type receptor are widely expressed. The corresponding ligands (such as cytokines and growth factors) are provided from blood cells, stromal cells, and cancer cells in various situations. Among these, several growth factors must be proteolytically activated to achieve the biological activities. Recently, the activating process has been clarified. For example, hepatocyte growth factor (HGF) is secreted as an inactive zymogen (pro-HGF) by stromal fibroblasts and converted to active mature form by proteolytic cleavage [2][3][4]. HGF activator (HGFA) was initially identified and considered to be the single major activator in serum [3][4][5][6][7]. Indeed, coagulation cascade is initiated in pericancerous lesion, and then HGFA changes to the active form by thrombin-induced proteolytic 2 of 19 activation [3][4][5][6][7]. Subsequently, several cell-surface transmembrane-type activators, including hepsin and matriptase, were identified [8][9][10][11][12]. These proteases may also be candidates for major activators considering HGF behavior (the so-called sticky protein) and the functional location (MET is located on the cell membrane). In physiological condition, the activation is tightly regulated by specific cell-surface protease inhibitors such as HGFA inhibitors (HAIs). However, dysregulated overactivation of pro-HGF has been reported to aggressively induce cancer invasion and metastasis through increased phosphorylation of MET [10][11][12]. This review focuses on the cell-surface pro-HGF activation system in urological cancers, including prostate cancer (PC), renal cell carcinoma (RCC), and urothelial carcinoma (UC) of bladder, and summarizes its significance in the progression of cancer.

HGF/MET Signaling Axis
MET, encoded by Met proto-oncogene located on chromosome 7q31, is a tyrosine kinase-type specific receptor of HGF, which forms disulfide-inked heterodimer consisting of an extracellular alpha chain and single-pass transmembrane beta chain [3,[13][14][15]. As shown in Figure 1, the intracellular domain of the beta chain comprises a juxtamembrane domain and catalytic kinase domain containing an activation loop and carboxy-terminal multifunctional docking site. The juxtamembrane domain downregulates the kinase activity by phosphorylation of Ser975, while the catalytic kinase domain upregulates the activity by phosphorylation of Tyr1234 and Tyr1235. The multifunctional docking sites contain Tyr1349 and Tyr1356, which lead to downstream signaling through several intracellular adaptor proteins [3,[13][14][15][16]. Increased expression of MET with worse prognosis has been reported in various cancer cells, and phosphorylation (activation) potently promotes invasion and metastasis [16][17][18][19]. Activation of HGF/MET signaling axis in cancer cells also plays a significant role in proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), and drug resistance [3,13,14]. Activation is introduced by: 1) ligand (HGF)-dependent activation, 2) reciprocal activation by overexpression-induced MET oligomerization, 3) activating point mutation of tyrosine kinase domain, and 4) transactivation by heterodimerization with another receptor tyrosine kinase [3,13,14]. In the ligand-dependent activation, proteolytic activation of pro-HGF is necessary. As mentioned above, two major activating protease families were reported: 1) a serum serine protease, HGFA; and 2) type II transmembrane serine proteases (TTSPs) such as matriptase, hepsin, and transmembrane protease/serine (TMPRSS) 2 [3,[10][11][12]. Although these pro-HGF activating proteases are tightly regulated by two transmembrane serine protease inhibitors, HAI-1 and HAI-2, downregulation of HAIs has been observed in several cancers and has been shown to induce progression [11,12]. Int

Type-II Transmembrane Serine Proteases (TTSP) in Cancers
The TTSP family in humans consists of 17 serine proteases [3,10,17]. The structures are specified as a single-pass hydrophobic transmembrane domain near the N-terminus with a short intracellular domain and a large extracellular portion including a carboxy-terminal serine protease domain [3,10,17]. All TTSPs are divided into the four subfamilies of hepsin, matriptase, human airway trypsin-like protease (HAT) and corin (Table 1) [3,10,17]. All TTSPs belong to the S1 peptidase family (noted in MEROPS as clan PA, family S1), and a catalytic triad consists of serine, aspartate, and histidine residues, as shown in Figure 2 [20,21]. Hepsin, matriptase and TMPRSS2 shows a strong cleavage preference for substrate with arginine in the P1 position [20,21]. In urogenital cancers, the expression of matriptase, hepsin and TMPRSS2 has been reported ( Figure 2). Therefore, we focused on these TTSPs in this review.

Type-II Transmembrane Serine Proteases (TTSP) in Cancers
The TTSP family in humans consists of 17 serine proteases [3,10,17]. The structures are specified as a single-pass hydrophobic transmembrane domain near the N-terminus with a short intracellular domain and a large extracellular portion including a carboxy-terminal serine protease domain [3,10,17]. All TTSPs are divided into the four subfamilies of hepsin, matriptase, human airway trypsin-like protease (HAT) and corin (Table 1) [3,10,17]. All TTSPs belong to the S1 peptidase family (noted in MEROPS as clan PA, family S1), and a catalytic triad consists of serine, aspartate, and histidine residues, as shown in Figure 2 [20][21][22]. Hepsin, matriptase and TMPRSS2 shows a strong cleavage preference for substrate with arginine in the P1 position [20][21][22]. In urogenital cancers, the expression of matriptase, hepsin and TMPRSS2 has been reported ( Figure 2). Therefore, we focused on these TTSPs in this review.
(including matriptase), which induces HGF/MET signaling axis, has been considered a major suppressive function in cancer progression [11,12]. Additionally, an alternative function such as the activation of caspase 3 in esophageal squamous cell carcinoma leading to the promotion of apoptosis and inhibition of proliferation was also reported [48,51]. However, HAI-2 has also been reported to be required for invasive growth in oral squamous cell carcinoma, which suggests that the role of HAI-2 may be tissue or cell-type specific and dependent on targeting TTSPs [52]. HAIs show single-pass transmembrane protein with intracellular carboxy-terminus and extracellular specific protease inhibitor domains, the so-called Kunitz domain (KD). HAI-1 is composed of two KDs, L domain, and motif at N terminus with seven cysteines (MANSC) domains. There are two isoforms in HAI-2. Similar to HAI1, HAI-2 isoform A has two KDs, whereas isoform B has a single KD. Figure 4)

HGF/MET Signaling and Hepsin
Increased expression of MET and phosphorylation in proliferative inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN), which are known precancerous findings, have been reported [53,54]. Regarding carcinogenesis, two interesting phenomena were reported in mouse models: 1) conditional MET overexpression in prostatic luminal cells revealed development of PIN by administration of HGF, however, the conditional transgenic mouse did not produce PC [55] and 2) Conditional overexpression of hepsin in prostate epithelium revealed disruption of basement membrane in vivo [56]. These results suggested that the single expression of an HGF-related molecule may be insufficient for carcinogenesis because co-existence of MET with PTEN loss or expression of hepsin with Myc in normal prostate epithelium revealed development of cancer [55][56][57]. However, expression of MET in PC induced aggressive metastatic phenotype with sarcomatoid features [55], and expression of hepsin in localized cancer promoted metastatic cancer with differentiation to neuroendocrine phenotype [56]. Therefore, HGF/MET signaling and hepsin may have a significant role in the progression of PC [58][59][60]. Indeed, upregulation of MET was observed in metastatic lesion compared with primary site [61]. In addition, overexpression of MET is reported in patients with HAIs show single-pass transmembrane protein with intracellular carboxy-terminus and extracellular specific protease inhibitor domains, the so-called Kunitz domain (KD). HAI-1 is composed of two KDs, L domain, and motif at N terminus with seven cysteines (MANSC) domains. There are two isoforms in HAI-2. Similar to HAI1, HAI-2 isoform A has two KDs, whereas isoform B has a single KD.
HAI-2 is ubiquitously expressed in normal cells, including epithelial, mesenchymal, blood cells and trophoblasts [12]. HAI-2 is reported to maintain the integrity of intestinal epithelium through regulation of matriptase-induced epithelial cell adhesion molecule (EpCAM) cleavage [44]. Downregulation by hypermethylation of SPINT2 gene has been reported in several cancers, including hepatocellular carcinoma, RCC, melanoma, gastric carcinoma, and esophageal squamous cell carcinoma [45][46][47][48]. Expression of HAI-2 is also decreased in PC. However, no apparent SPINT2 promoter methylation has been observed in either clinical samples or cell lines [49]. In this report, the authors suggest that posttranslational regulation of HAI-2 expression is essential in prostate cancer. The regulatory role of HAI-2 in the activation of pro-HGF by inhibiting the activating proteases (including matriptase), which induces HGF/MET signaling axis, has been considered a major suppressive function in cancer progression [11,12]. Additionally, an alternative function such as the activation of caspase 3 in esophageal squamous cell carcinoma leading to the promotion of apoptosis and inhibition of proliferation was also reported [47,50]. However, HAI-2 has also been reported to be required for invasive growth in oral squamous cell carcinoma, which suggests that the role of HAI-2 may be tissue or cell-type specific and dependent on targeting TTSPs [51].

HGF/MET Signaling and Hepsin
Increased expression of MET and phosphorylation in proliferative inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN), which are known precancerous findings, have been reported [52,53]. Regarding carcinogenesis, two interesting phenomena were reported in mouse models: 1) conditional MET overexpression in prostatic luminal cells revealed development of PIN by administration of HGF, however, the conditional transgenic mouse did not produce PC [54] and 2) Conditional overexpression of hepsin in prostate epithelium revealed disruption of basement membrane in vivo [55]. These results suggested that the single expression of an HGF-related molecule may be insufficient for carcinogenesis because co-existence of MET with PTEN loss or expression of hepsin with Myc in normal prostate epithelium revealed development of cancer [54][55][56]. However, expression of MET in PC induced aggressive metastatic phenotype with sarcomatoid features [54], and expression of hepsin in localized cancer promoted metastatic cancer with differentiation to neuroendocrine phenotype [55]. Therefore, HGF/MET signaling and hepsin may have a significant role in the progression of PC [57][58][59]. Indeed, upregulation of MET was observed in metastatic lesion compared with primary site [60]. In addition, overexpression of MET is reported in patients with metastatic PC and positively correlated with progression [3,53,61]. Furthermore, inhibition of hepsin by small molecule inhibitor has been reported to reduce bone metastasis of PC [58].
In the normal prostate, expression of MET is observed in basal cells, but not in luminal cells [52]. Androgen receptor (AR) is, however, expressed in luminal cells [52]. Of interest, the inverse expression of MET and AR has also been reported in PC [62,63]. For example, MET is highly expressed in AR-negative prostate cancer cell lines such as PC3 and DU145, however, it is downregulated in AR-positive cell lines (LNCaP, LAPC-4, CWR22, and LuCaP) [62,63]. In addition, the degree of MET expression in CWR22 cell line is reported to continue increasing during the process to become androgen independent phenotype (CWR22R), and downregulation of AR induced upregulation of MET with the signaling system is observed [62,63].
Despite this, AR signaling remains in the majority of CRPC. This is explained by the clinical efficacy of AR signaling-targeted agents (abiraterone and enzalutamide) for CRPC, and a recent clinical phase III trial of cabozantinib (dual inhibitor of MET and vascular endothelial growth factor receptor for CRPC revealed insufficient results [64]. The reported efficacy of combined therapy targeting both AR and MET suggests the coexistence of AR-positive cells and MET-positive cells in CRPC [65].

Matriptase and HAIs
Inhibition of matriptase by synthetic small molecule inhibitor and analysis of downregulation using hammerhead ribozyme revealed reduced invasiveness and motility of PC cell lines [66]. The significant role of matriptase in cancers has been discussed along with the ratio of HAIs activity. Several studies revealed that overexpression and functional upregulation of matriptase rather than HAIs leads to cancer progression [67,68]. In PC, increased expression of matriptase with concomitant loss of HAI-1 was observed [67]. In addition, a correlation between downregulation of HAI-2 with upregulation of matriptase and increasing PC tumor grade has been reported [49,69]. However, the following evidence suggests that HAI-2 is a main regulator of matriptase [49,50,[68][69][70]. The role of HAI-2 in invasiveness and metastatic potential of PC cells was analyzed using human prostate cancer progression model cell lines and the xenograft model [49,68,69]. As a result, the expression of HAI-2 but not HAI-1 in cancer cells was gradually downregulated depending on increasing invasiveness accompanying matriptase upregulation. In additional analyses, the authors confirmed that matriptase induced upregulation of PC cell motility as well as the fact that invasive and metastatic capabilities were negatively regulated by HAI-2. This phenomenon was also confirmed by the inhibition of matriptase using recombinant protein consisting of HAI-2-Kunitz domains [68,69].

TMPRSS2 in PC
As noted above, TMPRSS2 is a member of the TTSP family. In the pathogenesis of viral infection, TMPRSS2 is known to proteolytically activate spike glycoprotein of emerging coronaviruses, including severe acute respiratory syndrome (SARS) coronavirus (CoV), SARS-CoV-2 (COVID-19), human CoV-229E, and Middle East Respiratory Syndrome CoV (CoV-EMC), which is required for virus-cell membrane fusions [71][72][73]. The proteolytic cleavage and activation of hemagglutinin protein are also essential for the viral infectivity of influenza A virus (strains of H1N1, H3N2, and H7N9) [73,74]. Recent studies revealed that a gene fusion of TMPRSS2 to erythroblast transformation-specific (ETS) transcription factors is commonly observed in prostate cancer tissues [75,76]. Among these, fusion of TMPRSS2 and ERG is the most common (approximately 50%) chromosomal rearrangement [75,76]. However, no apparent proteolytic activity in this fusion protein has been reported (the protein mainly acts as a transcription factor). Expression of TMPRSS2 is positively regulated by androgens and expressed in both primary and metastatic sites of hormone sensitive PC [77,78]. Expression is reported to be increased in higher Gleason pattern [77,78]. As substrates, components of extracellular matrix (nidogen-1, laminin β1), matriptase zymogen, and pro-HGF were reported [78]. Ko et al. reported that androgen-induced TMPRSS2 can initiate a pericellular proteolytic cascade to activate matriptase, which promotes cancer cell invasion [79]. In addition, the correlation between increased expression of TMPRSS2 and increased matriptase activation in PC tissue was also confirmed. They concluded that TMPRSS2 can promote PC progression through increased matriptase activation and degradation of nidogen-1 and laminin β1 [79].
The evidences of Sections 3.1.1-3.1.3 are summarized in Figure 4. tissue was also confirmed. They concluded that TMPRSS2 can promote PC progression through increased matriptase activation and degradation of nidogen-1 and laminin β1 [80].  Figure 5)

HGF/MET Signaling
The most common histological subtype is clear cell RCC (approximately 80%-90% of RCC), followed by papillary RCC (10%-15%) [81]. Since expression of MET is observed in all subtypes, activating germline point mutations of MET are well known as the major cause of hereditary papillary RCC (PRCC) type-1 [15,16]. The mutation occurs at the tyrosine kinase (TK) domain and induces continuous ligand-independent phosphorylation of MET [15,16]. Although the mutation is observed only in 10%-46% of sporadic PRCC, overexpression including amplification of MET is common [82]. Of interest, phosphorylation of MET with auto-activating mutation was enhanced to a significant degree by HGF stimulation [83].
Inactivation of von Hippel-Lindau (VHL) tumor suppressor gene is the most common genetic abnormality of clear cell RCC [84,85]. Inactivation occurs not only in VHL patients (germline mutation) but also in patients with sporadic clear cell RCC (somatic abnormality) by deletion, mutation and epigenetic hypermethylation [84,85]. Deficiency of VHL protein in clear cell RCC causes progression of angiogenesis through activation of hypoxia inducible factor (HIF) 1α and 2α, which are major transcription factors of vascular endothelial growth factor (VEGF) and plateletderived growth factor (PDGF) [84]. Of interest, HIF-1α is also reported to activate transcription of MET [3,85]. Indeed, overexpression of MET with worsening prognosis has been reported in patients with cc RCC. In addition, a significant body of evidence has revealed that activation of MET in RCC cells promotes proliferation, invasive activity, motility, antiapoptotic activity, and angiogenesis [3,43,[85][86][87][88][89]. In addition, CD105+/CD24− cancer stem cells of RCC also expressed MET, and inhibition reduced development of bone metastasis [90].
Phase III METEOR(NCT01865747) trial revealed that cavozantinib, a novel TK inhibitor of VEGF receptor and MET, has demonstrated favorable efficacy for progression-free survival (PFS),

HGF/MET Signaling
The most common histological subtype is clear cell RCC (approximately 80%-90% of RCC), followed by papillary RCC (10%-15%) [80]. Since expression of MET is observed in all subtypes, activating germline point mutations of MET are well known as the major cause of hereditary papillary RCC (PRCC) type-1 [15,16]. The mutation occurs at the tyrosine kinase (TK) domain and induces continuous ligand-independent phosphorylation of MET [15,16]. Although the mutation is observed only in 10%-46% of sporadic PRCC, overexpression including amplification of MET is common [81]. Of interest, phosphorylation of MET with auto-activating mutation was enhanced to a significant degree by HGF stimulation [82].
Inactivation of von Hippel-Lindau (VHL) tumor suppressor gene is the most common genetic abnormality of clear cell RCC [83,84]. Inactivation occurs not only in VHL patients (germline mutation) but also in patients with sporadic clear cell RCC (somatic abnormality) by deletion, mutation and epigenetic hypermethylation [83,84]. Deficiency of VHL protein in clear cell RCC causes progression of angiogenesis through activation of hypoxia inducible factor (HIF) 1α and 2α, which are major transcription factors of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) [83]. Of interest, HIF-1α is also reported to activate transcription of MET [3,84]. Indeed, overexpression of MET with worsening prognosis has been reported in patients with cc RCC. In addition, a significant body of evidence has revealed that activation of MET in RCC cells promotes proliferation, invasive activity, motility, antiapoptotic activity, and angiogenesis [3,42,[84][85][86][87][88]. In addition, CD105+/CD24− cancer stem cells of RCC also expressed MET, and inhibition reduced development of bone metastasis [89].
Phase III METEOR(NCT01865747) trial revealed that cavozantinib, a novel TK inhibitor of VEGF receptor and MET, has demonstrated favorable efficacy for progression-free survival (PFS), overall survival (OS), and objective response rate (ORR) in patients with metastatic RCC in a second-or third-line treatment setting [90]. In addition, subgroup analysis of the METEOR trial demonstrated that treatment has clinical benefits in PFS, OS, and ORR for RCC patients with bone metastases associated with poor prognosis [91].

HAIs and TTSPs in RCC
Increased expression of matriptase and hepsin was reported to be correlated with poor prognosis [3,42,92]. In addition, decreased expression of HAI-1 and HAI-2 also revealed worse prognosis in patients with RCC [3,42,45,93]. As mentioned above, hypermethylation of SPINT2 gene in RCC has been reported [45]. Morris et al. reported that SPINT2 promoter methylation was observed in approximately 30% of ccRCC and 40% of pRCC patients with sporadic type, and they concluded that epigenetic inactivation caused loss of tumor suppressor activities in RCC [45].

MET and Matriptase/HAI-2 in Bone Metastasis
Approximately 30% of advanced RCC patients have bone metastasis [91,94]. Osteolytic metastasis affects patient quality of life through skeletal-related events. As mentioned above, bone metastasis is reported to be a negative prognostic factor and shows resistance to multimodal treatment, including surgery, radiation, and recent VEGFR-targeted medicines [91,94]. Interestingly, immunohistochemical analysis has revealed a significant increase in the expression of MET and matriptase in bone metastasis compared with primary site [87]. In analysis of mouse model of bone metastasis using human RCC cell line (786-O), expression of HGF, matriptase and decreased expression of HAI-2 were observed in bone metastasis compared with control [88]. Furthermore, increased phosphorylation of MET was also observed in bone metastasis [88]. These reports suggested that the HGF-dependent MET signaling axis plays a significant role in RCC bone metastasis.
The evidences of Sections 3.2.1-3.2.3 are summarized in Figure 5. overall survival (OS), and objective response rate (ORR) in patients with metastatic RCC in a secondor third-line treatment setting [91]. In addition, subgroup analysis of the METEOR trial demonstrated that treatment has clinical benefits in PFS, OS, and ORR for RCC patients with bone metastases associated with poor prognosis [92].

HAIs and TTSPs in RCC
Increased expression of matriptase and hepsin was reported to be correlated with poor prognosis [3,43,93]. In addition, decreased expression of HAI-1 and HAI-2 also revealed worse prognosis in patients with RCC [3,43,46,94]. As mentioned above, hypermethylation of SPINT2 gene in RCC has been reported [46]. Morris et al. reported that SPINT2 promoter methylation was observed in approximately 30% of ccRCC and 40% of pRCC patients with sporadic type, and they concluded that epigenetic inactivation caused loss of tumor suppressor activities in RCC [46].

MET and Matriptase/HAI-2 in Bone Metastasis
Approximately 30% of advanced RCC patients have bone metastasis [92,95]. Osteolytic metastasis affects patient quality of life through skeletal-related events. As mentioned above, bone metastasis is reported to be a negative prognostic factor and shows resistance to multimodal treatment, including surgery, radiation, and recent VEGFR-targeted medicines [92,95]. Interestingly, immunohistochemical analysis has revealed a significant increase in the expression of MET and matriptase in bone metastasis compared with primary site [88]. In analysis of mouse model of bone metastasis using human RCC cell line (786-O), expression of HGF, matriptase and decreased expression of HAI-2 were observed in bone metastasis compared with control [89]. Furthermore, increased phosphorylation of MET was also observed in bone metastasis [89]. These reports suggested that the HGF-dependent MET signaling axis plays a significant role in RCC bone metastasis.

Significance of HGF-dependent MET Activation in Muscle Invasive Bladder Cancer
Pathological analysis shows that approximately 90% of bladder cancers are UC and that 5% are squamous cell carcinoma [95]. Several studies reported a positive correlation between increased expression of MET and high tumor grade, pathological staging, and survival [96][97][98][99][100]. Phosphorylation was also analyzed by immunohistochemistry [97,98]. Increased phosphorylation of Tyr1234/1235 (at activation loop) and Tyr1349 (at multi-docking site) was positively correlated with pT stage, and multivariate analysis prompted the authors to conclude that phosphorylated Tyr1349 was a significant predictive factor of pT stage only. In addition, phosphorylated Tyr1349 was correlated with expression of matrix metalloprotease (MMP)-2, MMP-7, and E-cadherin [97]. Furthermore, the relationship between increased phosphorylation of Tyr 1235 in MET and worse cancer-specific survival in patients with muscle invasive bladder cancer (MIBC) was reported by Yamasaki et al. In this study, increased expression of matriptase and decreased expression of HAI-1 were also correlated with poor prognosis [98]. Interestingly, the area of HAI expression and MET phosphorylation presented reciprocally in spite of the strong expression of both total MET and matriptase (Figure 6b) [98]. These findings suggested that HAI-1 may regulate the ligand-dependent activation of MET in patients with MIBC. As a biomarker, the utility of urinary soluble MET concentration to detect bladder cancer and MIBC was reported [101,102]. In vitro analysis, efficient suppression of invasion and proliferation by cabozantinib (dual inhibitor of MET and VEGFR) was observed in invasive UC cell lines (5637, T24) [103]. HGF/MET signaling-induced expression of MMP-1 was also suppressed by cabozantinib in the same cell lines. HGF/MET signaling-induced EMT is a major phenomenon in various cancer cells [103]. Recently, it has been reported that HGF/MET signaling-induced inhibition of SMURF2 yielded stabilization of TGF-β receptor, and that upregulation of TGF-β signaling axis leads to EMT of UC cell line [104]. However, compared with PC and RCC, the function of TTSPs and HAIs in UC is not well known. Therefore, further analysis is required.  Figure 6a)

Significance of HGF-dependent MET Activation in Muscle Invasive Bladder Cancer
Pathological analysis shows that approximately 90% of bladder cancers are UC and that 5% are squamous cell carcinoma [96]. Several studies reported a positive correlation between increased expression of MET and high tumor grade, pathological staging, and survival [97][98][99][100][101]. Phosphorylation was also analyzed by immunohistochemistry [98,99]. Increased phosphorylation of Tyr1234/1235 (at activation loop) and Tyr1349 (at multi-docking site) was positively correlated with pT stage, and multivariate analysis prompted the authors to conclude that phosphorylated Tyr1349 was a significant predictive factor of pT stage only. In addition, phosphorylated Tyr1349 was correlated with expression of matrix metalloprotease (MMP)-2, MMP-7, and E-cadherin [98]. Furthermore, the relationship between increased phosphorylation of Tyr 1235 in MET and worse cancer-specific survival in patients with muscle invasive bladder cancer (MIBC) was reported by Yamasaki et al. In this study, increased expression of matriptase and decreased expression of HAI-1 were also correlated with poor prognosis [99]. Interestingly, the area of HAI expression and MET phosphorylation presented reciprocally in spite of the strong expression of both total MET and matriptase (Figure 6b) [99]. These findings suggested that HAI-1 may regulate the ligand-dependent activation of MET in patients with MIBC. As a biomarker, the utility of urinary soluble MET concentration to detect bladder cancer and MIBC was reported [102,103]. In vitro analysis, efficient suppression of invasion and proliferation by cabozantinib (dual inhibitor of MET and VEGFR) was observed in invasive UC cell lines (5637, T24) [104]. HGF/MET signaling-induced expression of MMP-1 was also suppressed by cabozantinib in the same cell lines. HGF/MET signaling-induced EMT is a major phenomenon in various cancer cells [104]. Recently, it has been reported that HGF/MET signaling-induced inhibition of SMURF2 yielded stabilization of TGF-β receptor, and that upregulation of TGF-β signaling axis leads to EMT of UC cell line [105]. However, compared with PC and RCC, the function of TTSPs and HAIs in UC is not well known. Therefore, further analysis is required. (a)

Conclusions
In this review, we summarized the significance of pericellular pro-HGF activation in PC, RCC, and bladder cancer. Overactivation (induced by increased HGF-activating TTSPs or decreased HAIs) revealed cancer progression through MET phosphorylation.

Conclusions
In this review, we summarized the significance of pericellular pro-HGF activation in PC, RCC, and bladder cancer. Overactivation (induced by increased HGF-activating TTSPs or decreased HAIs) revealed cancer progression through MET phosphorylation.
Amplification of MET in lung cancer is a well-known mechanism of resistance to epidermal growth factor receptor-targeted tyrosine kinase inhibitors (EGFR-TKIs) [105][106][107]. In addition, overexpression of HGF is observed in approximately 61% of patients with EGFR-TKIs-resistant lung cancer and is known as a cause of acquired resistance [106,107]. The MET-signaling pathway is a major target in these patients. However, resistance to anti-MET therapy is also promoted by high amounts of pericellular HGF [106,107]. Therefore, the pro-HGF-activation system is significant in cancer progression. In urological cancers, several small molecule inhibitors targeting MET have been used in PC and RCC in clinical trials, and cavozantinib is used as a major agent targeting MET and VEGFR in patients with advanced RCC [90,91,108,109]. In addition, the efficacy of several agents for HGF and the activating TTSPs, including anti-HGF antibody, antibodies and small molecule inhibitors of hepsin and matriptase, has been reported [3,40,[110][111][112][113]. Unfortunately, however, no such specific inhibitors have been verified in clinical trials [3,10]. Both matriptase and hepsin have other independent functions besides HGF-activation [114]. Therefore, inhibition of these TTSPs may yield additional therapeutic benefits in cancer treatment. As a candidate, the inhibitor domain of HAIs (KD1) shows significant inhibition for matriptase, hepsin, and HGFA [3,11,12,69]. Therefore, we believe KD1 has favorable therapeutic potential in urological cancers.

Conflicts of Interest:
The authors declare no conflict of interest.