For the past few decades, the main focus of research in the field of proteases and their role in cancer progression has been on matrix metalloproteases and serine proteases. Cysteine cathepsins (CCs), a family of lysosomal proteases, and their endogenous inhibitors, cystatins, received little attention until the late 1990s. CCs are implicated in a number of tumorigenic processes, such as tumour growth, invasion and metastasis.1 With the failure of serine proteases and metalloprotease inhibitors in clinical trials, CCs have now emerged as targets for anticancer therapies.1
The human CCs comprise 11 members, namely CC B, C, F, H, K, L, O, S, V, W and X. The cystatins are grouped into five families: stefins, cystatins, latexins, fetuins and kininogens. These groups have physiological2 and pathological significance.3 In recent years, several reports have demonstrated the involvement of CCs in cancer progression.4 Up-regulation of CCs was reported in breast, brain, lung and gastrointestinal cancers, as well as colorectal cancer and melanoma.4 Several approaches have been developed to block CC activity. These include small-molecule inhibitors, antibodies and increased production of cystatins.1 Cystatins are reversible, competitive and endogenous inhibitors of CCs. Various reports have suggested that there exists an inverse correlation between the stage of tumour progression and the levels of cystatins.5 The tumour-suppressing properties of cystatin family members, such as stefin A, stefin B, fetuin B, cystatin M and cystatin C, have been demonstrated in several studies.6 Their antitumour activity is not attributed solely to the inhibition of CCs. For instance, cystatin C has recently been described as a tumour growth factor β-receptor antagonist.7
In a recent research article, Eliyahu et al.8 demonstrated a new mechanism of cystatin action that could be targeted for use in anticancer therapy. Using co-immunoprecipitation and co-localization techniques in two different cell lines – one (SK-MEL; human skin melanoma) with high and the other (HEK293T/17) with low endogenous acid ceramidase (AC; N-acylsphingosine deacylase; EC 126.96.36.199) expression – the authors elegantly demonstrated that cystatin SA (cysSA) is a potent physiological inhibitor of AC. This enzyme is very important in regulating the conversion of ceramide (Cer) to sphingosine-1-phosphate (S1P). Ceramide is a proapoptotic (tumour-suppressing) lipid involved in apoptosis, autophagy and growth inhibition. S1P is an antiapoptotic (tumour-promoting) lipid with roles in angiogenesis, cell survival and proliferation. The balance between Cer and S1P is known as the Cer–S1P rheostat, and is critical in determining whether a cell will die or proliferate. Owing to cysSA's ability to regulate the Cer-S1P rheostat, the inhibition of the AC enzyme has emerged as an attractive target for the treatment of cancer.
The involvement of AC in cancer development has been demonstrated in several studies, which report its overexpression in a wide variety of cancers including those of the prostate, head and neck, as well as melanoma and glioblastoma.9,10 Similarly, when AC was inhibited using N-oleoylethanolamine, an increase in ceramide formation and enhanced apoptosis was observed in glioma, fibrosarcoma and hepatoma cells.10 Another inhibitor of AC, B13, has also been shown to induce apoptosis in prostate and colon cancers and adenocarcinoma.11 Using recent advances in ribonucleic acid (RNA) interference technology, knockout of AC in L929 cells resulted in a significant accumulation of ceramide, and the induction of apoptosis.12 Furthermore, AC overexpression has been reported to confer resistance to common proapoptotic agents, such as tumour necrosis factors, doxorubicin, etoposide, cisplatin and gemcitabine.13,14 Thus, an inverse correlation appears to exist between AC activity and cell death.
These findings suggest that AC inhibition, alone or in combination with other anticancer treatments, may serve as a useful target for cancer therapy.14 This highlights the need for identification and synthesis of more specific potent inhibitors of AC. CysSA, identified by Eliyahu et al.8 as a novel endogenous inhibitor for AC, may be a candidate. These authors also predicted the mechanism of interaction between cysSA and AC using laboratory techniques and computer modelling. The authors demonstrated that the interaction between AC and cysSA occurs via a non-competitive inhibition mechanism, which may be facilitated by the AC-like domain in cysSA. They were also able to synthesize two small peptides based on the cysSA sequence, both of which were found to be effective inhibitors of AC in vitro.
In summary, cysSA is a possible therapeutic agent for the treatment of cancer, either alone or in combination with other drugs. However, longer-term studies are required to assess the full usefulness of this novel therapeutic weapon. In the future, we can also expect the use of cysSA-based peptides or inducers of cysSA for the treatment of various types of cancers.