Enzymes of the glycolytic pathway have been considered as multifunctional molecules that besides its glycolytic activity perform various additional regulatory functions. Gamma-enolase (neuron-specific enolase) is an enzyme of the glycolytic pathway that is overly-expressed in tumours of neurogenic and neuroendocrine origin and is a well-known tumour marker. As an enzyme of the glycolytic pathway it contributes to accelerated aerobic glycolysis, the main energy source of tumour cells. On the other hand, gamma-enolase might act as a pro-survival factor, as has been demonstrated for neuronal cells. This function is achieved through an additional active site localized at the C-terminal end of the molecule and cathepsin X, a cysteine protease, was shown to regulate this function by proteolytic cleavage of the C-terminal dipeptide. Cathepsin X has been confirmed as an important promoter of tumour progression, which participates in a variety of cancer related mechanisms. Both molecules are present in tumour cells; therefore their interaction might have a significant impact on the disease course. The purpose of the doctoral thesis was to contribute to the understanding of the role of gamma-enolase as a survival factor in cancer cells and the regulation of its effects by cathepsin X and to define the interplay, relation with clinical and pathological parameters and utility of both molecules in diagnosis and prognosis of cancer. In the first part we investigated the importance of gamma-enolase in adaptation and survival of tumour cells exposed to stressful conditions and its regulation by cathepsin X. Two adenocarcinoma (Caco-2, MCF7) and two glioblastoma (U-87 MG and U-251 MG) cell lines were exposed to serum starvation or media with hypoxia mimetic desferrioxamine. Using western blot we evaluated the changes in expression of uncleaved gamma-enolase (with intact C-terminus) in relation to cathepsin X proteolytic activity. Uncleaved gamma-enolase, rather than total (including uncleaved and cleaved forms) gamma-enolase was significantly up-regulated and related to cathepsin X proteolytic activity in all analysed cell lines. Consequently, gamma-enolase silencing led to significantly lower cell survival in stressful conditions. Cathepsin X silencing or specific inhibition with AMS36 significantly increased uncleaved gamma-enolase expression and consequently, promoted cell survival in stressful conditions. The results suggest that gamma-enolase function as a survival factor in cancer cells is related to its intact C-terminal end, promoting adaptation of tumour cells to stressful conditions; and cathepsin X regulates this function. Cathepsin X, which has been considered as a promoter of tumour processes, here suppresses the protective function of gamma-enolase, which suggests its multi-function role in cancer. In the second part, using Western blot and ELISA, we evaluated the expression of uncleaved and total gamma-enolase in different tumour cell lines (glioblastoma: U-87 MG, U-373 MG, U-251 MG; neuroblastoma: SH-SY5Y; colon cancer: Caco-2, human breast fibrocystic disease cells: MCF 10A; breast cancer cells: MCF7) and their relation to cathepsin X expression and activity. In addition, we investigated the clinical applicability of both forms in sera from patients with colorectal cancer. We have shown that uncleaved gamma-enolase is differentially expressed in the analysed cell lines with the highest levels in those cell lines, which were derived from metastatic sites (SH-SY5Y, MCF7) or highly invasive tumours (U-87 MG). Uncleaved gamma-enolase expression was inversely related to cathepsin X expression in cells, the latter being the lowest in more invasive cells. Total gamma-enolase expression did not differ among the analysed cell lines. Gamma-enolase is excreted into the cell supernatant predominantly as uncleaved form reflecting its expression in cells. Uncleaved and total gamma-enolase levels were evaluated in serum from colorectal cancer. A preliminary immunoprecipitation showed uncleaved gamma-enolase to be the predominant form in serum. Using ELISA, which specifically recognizes uncleaved gamma-enolase and ECLIA (Elecsys NSE assay, Roche Diagnostics), which recognizes total gamma-enolase we measured the values of both forms in sera from 264 patients with colorectal cancer. Results showed no association with clinical or pathological parameters. However, univariate survival analysis of the entire set showed that higher levels of both forms associated with shorter patient survival. Additionally, in multivariate analysis for the group of patients with metastatic colorectal cancer (stage IV) who did not receive palliative chemotherapy, higher levels of both forms significantly associated with shorter survival, while in patients who received palliative chemotherapy, no relation to survival could be observed. The latter suggests that gamma-enolase levels in serum can predict the disease course and response of patients with metastatic colorectal cancer to palliative chemotherapy, which may lead to significant improvements in patients’ treatment. Future studies should focus on examining the expression of uncleaved gamma-enolase in tumour samples, which might provide additional relations to various indicators of cancer progression allowing selection of patients with more aggressive tumour phenotype. In the last part of the thesis we evaluated diagnostic applicability of cathepsin X in serum from patients with colorectal cancer. We developed and validated an ELISA assay which detects total cathepsin X (procathepsin X and active cathepsin X) in serum. First, we conducted a pilot clinical study that included 77 patients with colorectal cancer, 77 patients with adenomas of the colon, 77 patients without neoplastic changes and 77 healthy controls. No statistically significant differences between the groups were observed, which means that total cathepsin X has no diagnostic value. Also, no significant association to any clinical or pathological parameter could be detected. Within the group of patients with colorectal cancer, higher levels of total cathepsin X significantly associated with shorter patients’ survival. These results were further evaluated by a confirmatory (including 264 patients) and validation (including 460 patients) clinical study. A larger cohort of patients provided additional evidence that higher total cathepsin X levels significantly associate with shorter survival of patients with non-metastatic cancer (stages I-III). For this group, multivariate Cox's regression analysis showed a strong association of higher cathepsin X levels to shorter survival of patients who did not receive adjuvant chemotherapy, while in patients who received chemotherapy, no difference could be detected. Values of cathepsin X in sera could therefore predict the disease course and response to chemotherapy in patients with non-metastatic colorectal cancer. The latter classifies cathepsin X as a new promising blood-based tumour marker in colorectal cancer. In the doctoral thesis we have identified an additional role of gamma-enolase, which supports the adaptation and survival of cancer cells exposed to stressful conditions. Understanding these processes along with inhibiting gamma-enolase glycolytic activity represents new possibilities in cancer therapy. The recognized clinical utility of gamma-enolase and cathepsin X opens up new possibilities in diagnosis and treatment of cancer patients.
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