Research on cancer formation and treatment has finally started to reduce the mortality of cancer patients. In general, mortality rates decreased by 1.5% per year from 2001 through 2015, although these rates may differ depending on patient sex, ethnic group and type of malignancy. Nevertheless, cancer remains a significant global health problem, requiring constant search for novel treatments (1). The most common property of the malignant tumors is their ability to invade adjacent tissue and to metastasize to distant sites (2,3). This is due to increased expression or increased activity of numerous proteases associated with carcinogenesis (4-9). For example, cysteine proteases, such as cathepsin B and cathepsin D, can take part in dissolving of connective tissue and basement membrane needed for invasion and metastasis. However, these become activated at the low pH of lysosomes, requiring transporting into regions of tumor cell invasion (10-13). Aspartate proteases and other protease enzymes also serve a role in cancer development and metastasis (14-16). Nevertheless, among all these proteolytic events, the plasminogen and metalloproteinase family members have an exceptional significance due to their ability to cleave virtually any element of the extracellular matrix and basement membrane (17-20).

The components of the plasminogen activation system (PAS) and matrix metalloproteinase (MMP) families have been found to be overexpressed in malignant tumors, revealing that tumors are hijacking these systems and using them in the local invasion, metastasis and angiogenesis (21-23). One of the hallmarks of this cancer aggressiveness is the overexpression of proteolytic enzymes (24-27). For instance, prostate cancer is the most common non-cutaneous malignancy in men with >160,000 new cases in the United States each year, and the median age of diagnosis is 68 years. This cancer grows slowly, and is initially confined to the gland, requiring marginal or no treatment at all. However, certain types of prostate cancer are aggressive, can spread quickly and exhibit high proteolytic activity (28-31). This is also a case for other types of cancer, such as breast, colon and oral carcinomas among others (32-36).

In the present review, new findings on the role of plasminogen activation system (PAS) and metalloproteinase families in cancer formation and growth were summarized, and the study explored whether targeting these two protein families can serve as a possible treatment strategy.

The hallmark of metastatic disease is an altered equilibrium between the synthesis and degradation of the extracellular matrix proteins during pathological angiogenesis, invasion and metastasis (35,102).

It has previously been reported that receptor-bound uPA on the cancer cell surface is overexpressed in final step of malignant cell transformation that is responsible for invasion and metastasis (107). In addition, uPA has been implicated in the basement membrane and interstitial protein degradation during carcinogenesis, and is responsible for the disease progression (108-110). Therefore, inhibition of uPA may be beneficial in the treatment of cancer by limiting or preventing tumor growth and metastasis (26,111,112). Historically, one of the first uPA specific inhibitors identified was amiloride, which limits tumor growth and angiogenesis, as proven by in vitro and in vivo models (26,108,110,111,113). Amiloride has been approved by the Food and Drug Administration as a diuretic agent, however, it is not currently used in anticancer therapy.

The availability of the high resolution structure of uPA and sophisticated molecular modeling methods have aided the development of inhibitors binding to uPA. The specificity pocket of uPA is a part of this enzyme, which is responsible for the recognition of uPA substrates, therefore binding to the specificity pocket promises high specificity of inhibitors (35,67,105). Several 8-substituted 2-naphthamidine-based inhibitors with specific binding to uPA with high potency and selectivity were developed by Wendt et al (114). Among the multiple novel inhibitors developed by the Bruncko et al (115) and Katz et al (116) groups, only a limited number of inhibitors were translated into clinical practice. One of these is WX-UK1, also known as ethyl 4-[3-(3-carbamimidoylphenyl)- 2-[[2,4,6-tri(propan-2-yl)phenyl]sulfonylamino]propanoyl] piperazine-1-carboxylate or Mesupron^®, which is one of the most potent uPA inhibitors (0.41 µM), and is currently in Phase I clinical trial for pancreatic and breast cancer, with the possibility of application in ovarian and colon cancer as well (117-120).

Another possibility for inhibiting uPA is through the use of antibodies. For instance, a number of studies by Ossowski (121,122) have reported that the ability to invade the chick chorioallantoic membrane and metastasize from it to the embryo was evidently reduced for human carcinoma cells expressing a high amount of uPA during treatment with the antibody against the active site of uPA, in comparison to non-treated cells.

PAI-1 is also an attractive molecule for anticancer therapy, since it is a fast-acting, specific and high-affinity inhibitor of uPA (35,123). However, only active PAI-1 can be used to control angiogenesis, invasion and metastasis of malignant tumors. The half-life of wild-type PAI-1 is very short (t_1/2 = 1-2 h), after which it converts into its inactive latent form. This transformation is associated with the insertion of part of the reactive loop (P4-P10′) into the central β-sheet of the PAI-1 molecule, where P1-P1′ amino acids are not accessible for reaction with tPA or uPA (35,124). For that reason, wild-type PAI-1 cannot be used in anti-cancer therapy. Numerous engineered PAI-1 molecules with two or more mutations were developed, which exhibited a half-life extended from 2 h to >700 h, opening a possibility for their application in clinical settings (124-126). For instance, VLHL PAI-1 (t_1/2 = >700 h) has been demonstrated to inhibit angiogenesis and reduce tumor size in different ex vivo and in vivo models (26,71,110).

Notably, PAI-1-deficient mice exhibited lower tumor proliferation and higher apoptosis of implanted tumors when compared with their wild-type counterparts. Additionally, an elevated PAI-1 level is a predictive factor of poor prognosis in invasive breast cancer in humans (34,127,128). When PAI-1 was administrated in low concentrations, it was able to increase the metastatic potential and angiogenesis (34,127-129). These observations contradict our previous statements suggesting that PAI-1 is a potent inhibitor of angiogenesis and tumor growth (26,125,128,130). This paradox can be explained by the interaction of PAI-1 with vitronectin. This protein is a multifunctional glycoprotein present in the plasma, platelets and the extracellular matrix. PAI-1 binds to vitronectin and then binds with uPA/uPAR, forming a four protein complex (namely uPAR/uPA/PAI-1/vitronectin), which can be further combined with LDL receptor-related protein. This interaction weakens the PAI-1/vitronectin binding and triggers PAI-1/uPA/uPAR internalization. PAI-1 and uPA are degraded, whereas uPAR is recycled to the cell surface. Subsequently, uPAR can bind to uPA again and form a new PAI-1/uPA/uPAR/vitronectin complex. This process clears PAI-1 in the immediate vicinity of the cell, forcing it to migrate toward increased concentration of PAI-1 (131,132). Thus, PAI-1 can have a dual function as a pro-carcinogenic and anti-cancer agent, depending on its concentration. PAI-1 in physiological concentration acts by utilizing the vitro-nectin pathway. However, as it has been demonstrated in our previous studies and by other groups, when PAI-1 is present in much higher supraphysiologic concentrations, it suppresses the vitronectin pathway and acts as a potent inhibitor of angiogenesis by utilizing primarily its inhibitory properties to block proteinase activity (133-136).

uPAR (or CD87) is a multidomain glycoprotein attached to the cell membrane with a glycosylphosphatidylinositol (GPI) anchor. When binding to uPA, it creates high proteolytic activity in the proximity of the cell surface (137-139). Therefore, another approach to prevent invasion or metastasis is to block the binding of uPA to its receptor on the cancer cell surface. Similarly, limiting the binding of uPA to vascular cells would reduce cancer-associated angiogenesis. An octapeptide A6 (amino acids 136-143; nonreceptor-binding domain) of uPA inhibits the interaction of uPA with its receptor uPAR, and was reported to inhibit endothelial cell motility and tumor cell invasion (117,140,141). Furthermore, the use of this polypeptide has been reported in Phase I and II clinical studies of gynecological cancer (142). Mani et al (143) synthesized a methyl 3-aminobenzoate derivative that binds to uPAR with high affinity and demonstrated that this uPAR inhibitor lowers the invasion ability of breast MDA-MB-231 cells. In addition, female NSG mice inoculated with highly malignant TMD-MDA-MB-231 cells presented impaired metastasis to the lungs when treated with this uPAR inhibitor (143). In a subsequent study by Wang et al (144), the affinity of a large number of derivatives was measured using structure-based virtual screening. The synthesis of additional and cellular studies in cell lines (A549, H460 and H1299) revealed that these compounds blocked invasion, migration and adhesion, while the effects of these compounds on invasion were consistent with their inhibition of uPAR (144). Treatment of metastatic liver cells with 1 or 4 g/ml lectin from Bandeiraea simplicifolia seeds significantly inhibited the cell migration and invasion, and downregulated MMP2, MMP9 and uPA production (145). Furthermore, the AKT/GSK-3b/β-catenin pathway, which is upstream of MMP2, MMP9 and uPA, was found to be involved in the inhibition of cell migration and invasion by lectin (145). In a similar manner, quercetin functions on the uPA/uPAR system (146).

A total of 14 clinical studies can be found in the clinical-trials.gov registry on PAS and cancer. However, results have been posted for only one of these studies, and this study was abandoned due to limited activity of the agent (https://ccr.cancer.gov/news/clinical-trials). The diverse function of PAS is outlined in Fig. 1.

Inhibitors targeting MMP activity were initially intended to target the catalytic domain of these enzymes. The most clear approach was to synthetize polypeptides derived from the sequences of the amino acids of the MMP substrates, preventing hydrolysis of the scissile bond of proteins (147). Batimastat (INN/USAN, codenamed BB-94), a broad-spectrum, competitive, injectable drug was the first MMP inhibitor (MMPI) to go into clinical trials, acting as an angiogenesis inhibitor. While this agent reached Phase III trials, it was never marketed due to side effects (injection into the peritoneum caused peritonitis), poor solubility and very low oral bioavailability (147-149). Marimastat, another MMPI drug, exhibited some effects in delaying disease progression; however, it also had a dose-limiting toxicity. Patients experienced significant musculoskeletal pain and inflammation, and the clinical study was discontinued (147,150). The small molecular inhibitors of MMPs failed for a variety of reasons, despite preclinical data demonstrating their great potential for therapeutic use, leading to cancelled phase I trials on Prinomastat, and cancelled phase III trials on Batimastat, Marimastat, Tanomastat and CGS 27023A, which is a doxycycline tetracycline derivative (147).

Using hybridoma methods against the MMP-9 catalytic domain, Martens et al (151) generated the murine monoclonal antibody REGA-3G12. This antibody recognizes the W116 to K214 of the human MMP-9 catalytic domain, but not the Zn²⁺ binding site (151). The REGA-3G12 antibody inhibits MMP-9, but not MMP-2 (151,152). However, while MMPs have been recognized as a marker of poor prognosis and are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis, inhibitory monoclonal antibodies against MMPs have yet to find broad clinical applications.

The mainstream of the presently used chemotherapeutic agents attack all dividing cells, leading to the adverse effects that are typically associated with cancer treatment, such as hair loss or loss of appetite. For future therapeutics to be clinically successful, they need to be highly selective for a specific protein or proteins, and able to act on the cancerous tissues without causing adverse systemic effects. Despite unsuccessful early clinical studies on the inhibition of proteolysis in cancer (particularly MMPs), this approach still has great potential for therapeutic use. Inhibition of proteolysis in cancerous tissue has the ability to attenuate tumor invasion, angiogenesis and migration, which are the most fundamental properties of malignant tumors, thus deserving future development (147,153). This includes application of inhibitors at the time and stage of malignancy for which regulation of pathological proteolysis will be most beneficial. For instance, inhibiting cancer proteolytic enzymes during or after surgery can prevent cancer cell implantation and consequently cancer reemission (113). Inhibiting both uPA and MMPs is another beneficial approach, since tumors overexpress these groups of enzymes; therefore, employing this simple concept can be equally beneficial as cancer cell killing (Fig. 3). Epigallocatechin gallate, an inhibitor of uPA and MMPs, has previously been used, and this agent had a weak lethality, but prevented cancer cell implantation into the bladder wall following surgery and was slightly better in reducing cancer growth when compared with mitomycin C (Fig. 4) (113). Inhibition of PAS and MMPs simultaneously by a cocktail of inhibitors or a single inhibitor that is active against both proteolytic enzymes in all other types of cancer remains unexplored, but is a promising therapeutic approach deserving future development.