Five-year survival in luminal breast cancer patients: relation with intratumoral activity of proteasomes.

Background: The purpose of the study was to analyze the relationship between the caspase-like (CL) and chymotrypsin-like (ChTL) activities of proteasomes and the 5-year overall and metastasis-free survival rates in patients with luminal breast cancer. Methods: The study included 117 patients with primary operable invasive breast cancer (T1-2N0-1M0). Tissue samples from breast cancer patients were obtained as a result of the radical mastectomy or breast conserving surgery, which was a first line of therapy. The ChTL and CL proteasomes activities in the tumor tissue and in the surrounding adjacent breast tissues were assessed using the fluorometric method. The coefficients of ChTL (cChTL) and CL (cCL) proteasomes activities were also determined. The coefficients were calculated as the ratio of the corresponding proteasomes activity in the tumor tissue to the surrounding adjacent breast tissues. Within 5 years of follow-up, hematogenous metastases occurred in 14% of patients with luminal A breast cancer, in 31% of patients with luminal B human epidermal growth factor receptor-2 (HER-2) negative and in 23% of patients with luminal B HER-2 positive breast cancers. The study protocol was approved by the Local Ethics Committee of the Cancer Research Institute of Tomsk National Research Medical Center. Written informed consent was obtained from all patients. Results: An increase in the ChTL and CL proteasomes activities was shown in all studied molecular subtypes of breast cancer compared to adjacent tissues. It was found that the cChTL of >35.9 U/mg protein and the cCL of >2.21 in breast cancer patients were associated with the development of distant metastases. In patients with luminal A breast cancer, the 5-year metastasis-free survival rates were associated only with the value of cCL of proteasomes (log-rank test: P=0.008). In patients with luminal B HER-2 negative breast cancer, the 5-year metastasis-free survival rates were associated with the levels of ChTL and cCL proteasomes activities (log-rank test: P=0.02 and P=0.04, respectively). Conclusions: The data obtained on the correlation of 5-year metastasis-free survival rates with the level of proteasomes activities indicate the possibility of their use as additional prognostic criteria for breast cancer.

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome.

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.

Exosomal Protease Cargo as Prognostic Biomarker in Colorectal Cancer.

OBJECTIVE: The aim of the study was to develop a model for predicting cancer risk in colorectal polyps' patients (CPPs), as well as to reveal additional prognosis factors for Stage III colorectal cancer based on differences in subpopulations of tetraspanins, tetraspanin-associated and tetraspanin-non-associated proteases in blood plasma exosomes of CPPs and colorectal cancer patients (CRCPs). METHODS: The subpopulations of CD151- and Tspan8-positive exosomes, the subpopulations of metalloproteinase at the surface of СD9-positive exosomes and the level of 20S proteasomes in plasma exosomes in 15 CPPs (tubulovillous adenomas) and 60 CRCPs were evaluated using flow cytometry and Western blotting. Logistic regression analysis was performed to predict cancer risk of CPPs. RESULTS: The levels of 20S proteasomes in exosomes, MMP9+, MMP9+/MMP2+/EMMPRIN+ in CD9-positive blood plasma exosomes are associated with the risk of malignant transformation of colorectal tubulovillous adenomas.  In patients with Stage III CRC, the levels of 20S proteasomes (less than 2 units) and MMP9+ subpopulations (more than 61%) in plasma exosomes are unfavorable prognostic factors for overall survival. The levels of 20S proteasomes and ADAM10+/ADAM17- subpopulations in CD9-positive blood plasma exosomes are the most significant values for predicting relapse-free survival. CONCLUSION: Protease cargo in CD9-positive blood plasma exosomes is prognostic biomarker for colorectal polyps and colorectal cancer.

Relation between Tetraspanin- Associated and Tetraspanin- Non- Associated Exosomal Proteases and Metabolic Syndrome in Colorectal Cancer Patients

Purpose: Exosomal proteases are important in regulation of molecular signaling from growth factor receptors and adhesion molecules and also the regulation of cell motility and protein folding. The aim of this study was to evaluate the level of ADAM10, ADAM17 and 20S proteasomes in exosomes isolated from colorectal cancer patients (CRCPs) in relation with clinical and histopathological parameters. Methods: Blood plasma exosomes of 60 CRCPs at stage T2-4N0-2M0-1 and 10 control subjects (CSs) with colorectal polyps were isolated using ultrafiltration in combination with ultracentrifugation. The level of tetraspanin-associated (ADAM20 and ADAM17) and tetraspanin-non-associated (20S proteasome) proteases were evaluated by flow cytometry and western blot analysis. Results: The ADAM10-/ ADAM17- population predominated in plasma exosomes of CRCPs and the level of ADAM10+ exosomes was significantly higher in exosomes of CSs compared with CRCPs. No difference was found between subpopulations of ADAM10/ADAM17 exosomes and level of exosomal 20S proteasomes in terms of sex, age and tumor grade. Simultaneous decrease of ADAM10+/ADAM17-subpopulation of exosomes and level of exosomal 20S proteasomes in patients with metastatic CRC was observed compared with patients with non-metastatic CRC. The level of ADAM17+ exosomes significantly reduced in exosomes of CRCPs with metabolic syndrome compared to CRCPs without metabolic syndrome( 3.97±0.71 (%) vs. 13.04±1.34 (%), respectively (p<0.05). A decrease in the 20S proteasomes level in plasma exosomes was revealed in CRCPs with metabolic syndrome compared with CRCPs without metabolic disorders ( 1.90±0.25 (r.u.) vs. 2.92±0.42 (r.u.) respectively( (p<0.05). Conclusion: According to findings of this study, it seems that exosomal proteases can be promising molecular predictors of hematogenous metastasis in patients with non-metastatic CRC. Further studies on subpopulation composition of exosomes CRCPs are need for elucidating the role of tetraspanin-associated and tetraspanin-non-associated exosomal proteases in CRC development and progression.

Association of IGFBP-6 Expression with Metabolic Syndrome and Adiponectin and IGF-IR Receptor Levels in Colorectal Cancer.

PURPOSE: To assess IGFBP-6 expression in relation with the presence of the metabolic syndrome, adiponectin receptors (AdipoR1 and AdipoR2) and IGF-IR levels in colorectal adenocarcinoma cases. MATERIALS AND METHODS: IGFBP-6 mRNA and protein levels were analyzed using real-time quantitative PCR and Western blotting in 46 patients. ELISA and ow cytometry were used for evaluation of AdipoR1, AdipoR2 and IGF-IR. RESULTS: The results showed that IGFBP-6 mRNA expression and the IGFBP-6 content were higher in tumor tissue samples of colorectal cancer patients with and without the metabolic syndrome. In addition, IGFBP-6 mRNA expression was associated with tumor invasion (tumor size) and the IGFBP-6 protein level was associated with nodal status. Positive correlations and positive nonlinear relations were found between the IGFBP-6 level and the AdipoR1 and AdipoR2 contents in colorectal cancer patients. CONCLUSIONS: The IGFBP-6 mRNA level and protein level were found to be associated with presence of the metabolic syndrome. Positive correlations indicated probable cross-talk between the IGF-IR-mediated and adiponectin-mediated signaling pathways in colorectal carcinomas. IGFBP-6 may be considered as a potential biomarker associated with lymphogenous metastasis and the metabolic syndrome in colorectal cancer.