Carboxypeptidase Cathepsin X Defines a Multifunctional Role of Gamma- Enolase in Cancer

Abstract

Janko Carboxypeptidase Cathepsin X Defines a Multifunctional Role of Gamma- Enolase In Cancer. Abstract Gamma-enolase enzymatic activity is involved in glycolysis, a prevalent process in cancer cell metabolism. Additionally, gamma-enolase has a pro-survival function, exhibited through the active site at the C-terminal end of the molecule. This activity is regulated by cysteine peptidase cathepsin X, which cleaves two amino acids at C-terminal end of gamma-enolase. In clinical practice, the determination of gamma-enolase as a tumour marker does not differ between total, uncleaved and C-terminally cleaved forms. However, levels of uncleaved gamma-enolase alone may provide additional clinical information. In this study we analysed cathepsin X, C- terminally uncleaved and total gamma-enolase in tumour cell lines and sera from 255 patients with colorectal cancer (CRC) by western blot, immunoprecipitation, enzymatic activity, ELISAs and ECLIA. Results show that uncleaved gamma-enolase, rather than total gamma- enolase, exhibits different levels in cells, being the highest in those, derived from metastatic sites or highly invasive tumours. Gamma-enolase is secreted into the extracellular space predominantly as an uncleaved form and levels were congruent to those within the cells. Furthermore, levels of uncleaved gamma-enolase in cells are inversely related to cathepsin X protein level and its enzymatic activity. Uncleaved gamma-enolase is also predominant form in sera of patients with CRC. Both forms exhibit similar stage dependent distribution, with slightly elevated levels in stage IV patients. Higher levels of total gamma-enolase are significantly related to shorter survival in patients with metastatic CRC. Results support evidence of additional pro-survival function of gamma-enolase in cancer. Future studies should focus on analysis of uncleaved gamma-enolase in tumour samples, which may provide additional relations to clinical indicators of disease progression. blot analysis (Figure 3A). Taken together, these data show that uncleaved gamma-enolase and total gamma-enolase have different expression and secretion profiles in different cancer cell lines and that the levels of uncleaved gamma-enolase, but not total gamma-enolase, inversely correlate to the levels of active Cat X in cell lysates. Higher levels of Cat X may therefore be related with more intensive gamma-enolase C-terminal end processing. Uncleaved gamma-enolase seems to be the predominant form to be secreted from cells and its levels reflect well those found in cell extracts. Extracellular forms are not dependent on Cat X, which is present in supernatants predominantly as inactive pro-enzyme. and lysates. While total gamma- enolase is uniformly in all analysed lines, uncleaved gamma-enolase has different expression levels. Cat X is expressed mainly as active enzyme and its expression is inversely related to uncleaved gamma-enolase expression: the highest expression of active Cat X can be detected in cell lines with the lowest uncleaved gamma-enolase expression. Graphs below western blot images indicate the relative protein amount of uncleaved gamma-enolase, and active Using ELISA, we measured the values of uncleaved in to confirm in accordance with western mg recombinant similar as the total gamma-enolase, Our study provides a new insight into the widely used tumour marker gamma-enolase. The C- terminally uncleaved gamma-enolase, which possesses an additional, pro-survival function, exhibits different expression levels in tumour cells, compared to total gamma-enolase, and is inversely related to Cat X expression. Uncleaved gamma-enolase is a predominant form in cell supernatants as well as in sera from patients with CRC. Only in a group of patients with metastatic CRC serum gamma-enolase correlated with survival. Further studies should focus on the analysis of uncleaved gamma-enolase in tumour samples, which may provide additional relations to clinical indicators of disease progression and enable the selection of patients with more aggressive tumor phenotype. multivariate Cox regression analysis was done in order to determine association of uncleaved and total gamma-enolase levels and other clinical and pathological parameters with overall survival of CRC patients. Both, uncleaved and total gamma-enolase, were scored as continuous log transformed covariates (using base 2), meaning that hazard ratios (HR) are for two fold differences in the markers levels. Model assessment was done using cumulated sums of martingale residuals. All results are presented with 95% confidence limits and p-values less than 5% are considered significant. Statistical analysis of the clinical study was done with SAS (v9.3, SAS Institute, Cary, N.C., USA) and R (R Core Team (2013). These results are reported in accordance with the REMARK guidelines native