Prognostic and predictive value of cathepsin X in serum from colorectal cancer patients

Discovery of molecules associated with colorectal cancer (CRC) whose levels may provide predictive and prognostic information, is important in order to improve treatment, optimize the quality of life and prolong survival of the patients. Several serum tumour markers are currently under evaluation, however, carcinoembryonic antigen (CEA) is the only one in regular clinical use for determining prognosis, for surveillance following curative resection and for monitoring therapy in advanced disease [1‐4].

Cysteine cathepsins, a group of lysosomal cysteine proteases, have been reported as being involved in the development and progression of cancer. They are used as targets for developing new antitumour therapies. Their expression, protein and activity levels [5‐7] have also been associated with prediction of prognosis in various cancer types, including CRC [8, 9]. Cathepsins B and L, in particular, are the most extensively studied cysteine cathepsins in colorectal diseases. In colorectal carcinoma they are generally overexpressed and their ability to degrade the components of extracellular matrix has been related to tumour invasion, migration and metastasis [8, 10]. Endogenous inhibitors of cysteine proteases, the cystatins, insufficiently impair the tumour associated activity of cathepsins; their lower levels in tumours, higher enzyme:inhibitor ratio and lower stability of the complex have been associated with cancer aggressiveness and patient survival [8, 11].

In recent studies the potential of other cathepsins, exhibiting more specific functions and localisation than cathepsins B and L, have been evaluated. Cathepsin X (Cat X), for example, is expressed specifically in various cells of the immune system [12] and regulates important processes such as proliferation, maturation, migration and adhesion of immune cells, phagocytosis and signal transduction [13‐15]. Elevated expression of Cat X has also been associated with the inflammatory processes in inflammatory related neurodegenerative disorders [16], Helicobacter pylori infection [17], multiple trauma [18] and tuberculosis [19]. However, there is increasing evidence that Cat X is also involved in malignant processes. The gene encoding Cat X is localized in chromosomal region 20q13, which is frequently amplified in several cancer types [20, 21]. In lung tumours, immunohistochemical analysis revealed strong staining of Cat X in infiltrated immune cells, but very weak staining in tumour cells [12]. On the other hand, increased expression of Cat X was found in both tumour and immune cells of prostate [22] and gastric cancer [17] and in the most aggressive phenotypes of malignant melanoma [23]. Further, breast cancer transgenic mice with Cat X deficiency had a prolonged tumour-free period for breast cancer, unlike wild type mice [24]. Moreover, Cat X deficiency leads to accelerated cell senescence, which is a powerful tumour suppressing mechanism [25]. Cat X may also promote tumour processes by mechanisms typical of immune cells, such as binding and activation of integrin receptors [15] and modulation of CXCL-12 chemokine [26] and gamma enolase [16].

Reports evaluating the clinical relevance of Cat X are less numerous. Wang et al. demonstrated that increased Cat X mRNA levels in hepatocellular carcinomas correlate with advanced tumour stage [27]. In a pilot study our group showed significantly lower levels of active Cat X in sera of patients with inflammatory breast cancer than with non-inflammatory breast cancer [28]. However, no significant correlation with any clinico-pathological parameter could be demonstrated in epithelial ovarian cancer cyst fluid [29]. On the other hand, higher levels of Cat X (both pro-form and active mature form) in sera from 77 patients with CRC correlated significantly to shorter overall survival [30].

In the present study, the aim was to confirm the results of the pilot study with an extended cohort of CRC patients and, further, to evaluate the use of serum levels of total Cat X for selecting patients who could benefit from chemotherapy.

Colorectal cancer is the second leading cause of cancer-related death in Europe and the US. About half of the patients develop recurrent disease within 3 to 5 years of resection of the primary tumour, and they are candidates for systemic chemotherapy. The availability of validated biological markers for selection of chemotherapy and prediction of treatment efficacy, together with monitoring, may increase patient survival. In this study we identified serum Cat X as a potential tumour marker in CRC, based on the demonstrated correlation of its high levels with shorter patient overall survival and the association with the adjuvant chemotherapy.

In our previous (pilot) study on 77 CRC patients [30] we found that levels of total Cat X in sera from patients with CRC were not significantly higher than those in groups of healthy persons, patients with adenomas or patients with non-neoplastic findings, all matched with CRC patients for age and gender. The mean level of total Cat X, determined in sera from the 264 CRC patients included in this confirmation study, also does not differ statistically from that in healthy persons (mean 23.4 ng/mL ± 6.4 SD vs. 18.8 ng/ml ± 11.4 SD, p > 0.05). As in the pilot study, there is no significant association with patient age or gender, tumour stage or location and CEA. In a univariate Cox regression model, the levels of total Cat X evaluated in this study did not confirm the significant relation to overall survival found in the pilot study [30], although the higher levels were associated with poor survival. When, as in the pilot study, the median was used as a cut off value, a similar result was obtained for the entire set of patients whereas, for patients with local resectable disease (stages I-III), the relation to overall survival was significant. In contrast, for patients with metastatic disease (stage IV), there was no relation between Cat X and overall survival. In multivariate analysis, including patients with stages I-III, a strong association (HR = 3.13) between high total Cat X levels and shorter overall survival was observed for the group of patients who did not receive adjuvant chemotherapy whereas, within the group of patients who received chemotherapy, there was no association between total Cat X and survival (HR = 0.51). Patient age, stage and CEA also contributed to the prognosis in multivariate analysis, whereas gender and location were not significant. The significant interaction between Cat X and adjuvant chemotherapy suggests its possible predictive value, since the data suggest that patients with high levels of Cat X benefit from the therapy.

Several mechanisms have been suggested that could link higher activity and/or concentration of Cat X with the progression of malignant disease. Cat X can be present in sera from healthy persons due to constitutive secretion by leukocytes during normal physiological immune cell activation and turnover [18]. Increased secretion of pro-Cat X could reflect the overexpression of Cat X in tumour and tumour associated immune cells, as observed in several cancer types. Secreted pro-Cat X most probably remains in the extracellular space, however, it can be activated by membrane bound cathepsin L [35] or surface heparan sulphate proteoglycans [13]. The activation by cathepsin L can also take place in endosomal/lysosomal vesicles during their translocation toward the plasma membrane [15, 35]. Pro-Cat X can modulate cell adhesion and motility through interaction of its pro-region with heparan sulphate proteoglycans [13] and integrin receptors [36‐38], while active Cat X can regulate cell functions through cleavage of C-terminal amino acids of chemokine CXCL-12 [26], beta-2 chain of integrin receptors [36], gamma-enolase (neuronal specific enolase - NSE) [16] and profilin [39, 40]. Therefore, both forms could promote tumour progression processes, resulting in enhanced adhesion and migration of tumour cells, induced epithelial-mesenchymal transition [27] and decreased cell senescence [25]. Results obtained on transgenic animals further confirm the active role of Cat X in tumour progression. It was suggested that Cat X, not being dependent on the degradation of extracellular matrix, a typical event for cathepsin B, may compensate the malignant effects of cathepsin B [24, 41] by changing the migration mode of tumour cells. Only mice with excluded expression of both cathepsin B and Cat X exhibited significantly lower tumour growth and metastasis formation. The relation between Cat X levels and response to therapy has not been reported, however, anticancer drugs may interfere with Cat X targets and processes, also regulating its expression and secretion.

From previous studies it is not clear whether Cat X is involved in early or late stages of cancer. The results by Sevenich et al. [24], using a PymT-induced metastatic breast cancer mouse model, showed that Cat X was related to the initial stages of the malignant process and less with tumour progression and metastasis. On the other hand, Hidaka et al. showed that Cat X could be involved in the later stages of cancer, since amplification of the region encoding for Cat X correlated with the metastatic potential and tumour progression in CRC [20]. A similar possibility was explored by Wang et al.[27], who showed that increased Cat X expression strongly correlated with advanced clinical stage, and induced metastatic potential and shorter overall survival of patients with hepatocellular carcinoma. Moreover, it was recently demonstrated that, in adenocarcinoma of the pancreatic duct, S100P-binding protein inversely regulates Cat X expression – lower levels of S100P-binding protein increased Cat X, cell adhesion and metastatic potential of pancreatic cancer cells [42]. Our findings are in accord with those of a recent study on lung cancer patients by Zhang et al., which also shows that higher Cat X serum levels are associated with shorter patient survival. However, lung cancer patients had significantly higher Cat X levels than healthy controls, which was not the case in our study [43]. Our combined results support the studies revealing the association of Cat X expression with the progression of CRC, since they provide the correlation of high levels of Cat X with shorter overall survival of CRC patients.

Our study confirms the previous results relating higher Cat X levels to progression of cancer and shorter overall survival of CRC patients. Although total Cat X serum levels did not differ between the entire group of patients and controls, a significant difference was found in the group of patients who did not receive adjuvant chemotherapy: patients with high total Cat X serum levels had significantly shorter overall survival than those with low levels. For patients who received adjuvant chemotherapy no difference in overall survival was observed between those groups with low and high total Cat X. Thus, total Cat X could be useful as a predictive, blood-based tumour marker that may allow selection of patients who could benefit from adjuvant chemotherapy. However, the results must be confirmed in a prospective clinical study.