Relationship between Telomere Length, TERT Genetic Variability and TERT, TP53, SP1, MYC Gene Co-Expression in the Clinicopathological Profile of Breast Cancer.

The molecular mechanisms of telomerase reverse transcriptase (TERT) upregulation in breast cancer (BC) are complex. We compared genetic variability within TERT and telomere length with the clinical data of patients with BC. Additionally, we assessed the expression of the TERT, MYC, TP53 and SP1 genes in BC patients and in BC organoids (3D cell cultures obtained from breast cancer tissues). We observed the same correlation in the blood of BC patients and in BC organoids between the expression of TERT and TP53. Only in BC patients was a correlation found between the expression of the TERT and MYC genes and between TP53 and MYC. We found associations between TERT genotypes (rs2735940 and rs10069690) and TP53 expression and telomere length. BC patients with the TT genotype rs2735940 have a shorter telomere length, but patients with A allele rs10069690 have a longer telomere length. BC patients with a short allele VNTR-MNS16A showed higher expression of the SP1 and had a longer telomere. Our results bring new insight into the regulation of TERT, MYC, TP53 and SP1 gene expression related to TERT genetic variability and telomere length. Our study also showed for the first time a similar relationship in the expression of the above genes in BC patients and in BC organoids. These findings suggest that TERT genetic variability, expression and telomere length might be useful biomarkers for BC, but their prognostic value may vary depending on the clinical parameters of BC patients and tumor aggressiveness.

Retinol-Binding Protein 4 Accelerates Metastatic Spread and Increases Impairment of Blood Flow in Mouse Mammary Gland Tumors.

Retinol-binding protein 4 (RBP4) is proposed as an adipokine that links obesity and cancer. We analyzed the role of RBP4 in metastasis of breast cancer in patients and in mice bearing metastatic 4T1 and nonmetastatic 67NR mammary gland cancer. We compared the metastatic and angiogenic potential of these cells transduced with Rbp4 (4T1/RBP4 and 67NR/RBP4 cell lines). Higher plasma levels of RBP4 were observed in breast cancer patients with metastatic tumors than in healthy donors and patients with nonmetastatic cancer. Increased levels of RBP4 were observed in plasma, tumor tissue, liver, and abdominal fat. Moreover, the blood vessel network was highly impaired in mice bearing 4T1 as compared to 67NR tumors. RBP4 transductants showed further impairment of blood flow and increased metastatic potential. Exogenous RBP4 increased lung settlement by 67NR and 4T1 cells. In vitro studies showed increased invasive and clonogenic potential of cancer cells treated with or overexpressing RBP4. This effect is not dependent on STAT3 phosphorylation. RBP4 enhances the metastatic potential of breast cancer tumors through a direct effect on cancer cells and through increased endothelial dysfunction and impairment of blood vessels within the tumor.

Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy.

Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.

Combination Therapy with DETA/NO and Clopidogrel Inhibits Metastasis in Murine Mammary Gland Cancer Models via Improved Vasoprotection.

Vascular endothelial dysfunction and platelet activation play a key role in tumor metastasis, and therefore, both of these processes are considered important therapeutic targets in cancer. The aim of our studies was to analyze antimetastatic activity of combination therapy using nitric oxide donor DETA/NO and antiplatelet drug clopidogrel. Nitric oxide acts as a vasoprotective mediator, while clopidogrel inhibits ADP-mediated platelet aggregation. 4T1-luc2-tdTomato cell line transplanted intravenously (i.v.) and 4T1 cell line transplanted orthotopically were used as metastatic mammary gland cancer models. Moreover, antiaggregation action of compounds was tested ex vivo on the blood samples taken from breast cancer patients. We have shown that in selected dosage regimes, DETA/NO combined with clopidogrel significantly reduced lung metastatic foci formation in an i.v. model, and such inhibition was transiently observed also in an orthotopic model. The antimetastatic effect was correlated with a significant increase of prostacyclin (PGI2) metabolite and reduction of endothelin-1, sE-selectin, sI-CAM, and TGF-ß plasma levels as well as decreased V-CAM expression on the endothelium. Combination therapy decreased fibrinogen binding to the resting platelets at the early stage of tumor progression (day 14). However, at the later stages (days 21 and 28), the markers of platelet activation were detected (increased JON/A antibody bound, P-selectin level, binding of fibrinogen, and vWf). Decreased aggregation as well as a lower release of TGF-ß were detected in platelets incubated ex vivo with compounds tested from metastatic breast cancer patients. Although combination therapy increases E-cadherin, the increase of N-cadherin and α-SMA in tumor tissue was also observed. The results showed that at the early stages of tumor progression, combined therapy with DETA/NO and clopidogrel improves vasoprotective and antiplatelet activity. However, in advanced tumors, some adverse effects toward platelet activation can be observed.

Elevated nuclear S100P expression is associated with poor survival in early breast cancer patients.

S100P - low molecular weight acidic protein has been shown to be involved in processes of proliferation, survival, angiogenesis, multidrug resistance and metastasis in various human malignancies. In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumour behaviour, indicating that this protein may have adverse prognostic value. We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer patients with a median follow up of 17 years. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. We also studied prognostic value of S100P mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. Moreover, the relationship was examined between expression of S100P in cells of four breast cancer cell lines and their sensitivity to the 11 most frequently applied cytotoxic drugs. Univariate and multivariate analyses showed that higher expression of nuclear S100P (S100Pn) was typical for cases of a shorter overall survival and disease-free time. KM plotter analysis showed that elevated S100P expression was specific for cases of a relapse-free survival and distant metastases-free survival. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. We demonstrated that a high S100Pn expression level was associated with poor survival in early stage breast cancer patients. Since preliminary data indicated that expression of S100P was up-regulated by activation of glucocorticoid receptor and several agents manifested potential to activate or inhibit S100P promoter activity, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value.

Elevated nuclear YB1 expression is associated with poor survival of patients with early breast cancer.

BACKGROUND: Y-Box-Binding (YB1) protein represents a multifunctional protein, which plays a significant role in processes of proliferation, apoptosis and control of tumour cell response to toxic agents, including chemotherapy. The present study aimed at evaluating the prognostic significance of YB1 expression in breast cancer. MATERIALS AND METHODS: We analyzed nuclear and cytoplasmic expression of YB1 in 101 patients with stage II breast cancer, with 17 years of follow-up. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against YB1. Results were tested for their correlation with clinical and pathological data. RESULTS: Patients with a pronounced expression of the nuclear form the YB1 protein demonstrated a highly significant shortening of disease-free survival, disease-specific survival and overall survival. The prognostic value of YB1 was also corroborated by multivariate analysis. CONCLUSION: We demonstrated that high nuclear expression of YB1 is associated with poor survival of patients with early-stage breast cancer.

[Analysis of BCRP expression in breast cancer patients]. / Analiza ekspresji BCRP u pacjentek z rakiem piersi.

UNLABELLED: Breast cancer resistance protein (BCRP, ABCG2) is a xenobiotic half-transporter protein. It is a member of the ATP-binding cassette protein family and functions as an energy-dependent efflux pump. BCRP is involved in multidrug resistance. The study aimed at examining BCRP expression in breast cancers and at defining a relationship between activity of this protein and clinical course of the cancer. MATERIALS AND METHODS: We analyzed the expression of BCRP in 101 stage II breast cancer patients. All the patients were diagnosed and treated at the Lower Silesia Oncology Centre (LSOC) between January 1993 and June 1994. After the treatment the patients remained under constant control at LSOC. Mean duration of the observation was 14.2 years (ranging between 9.1 and 16.5 years). Data related to relapse of the disease and deaths were obtained from medical documentation stored in LSOC. The immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against BCRP. The intensity of immunohistochemical reactions with BCRP antibody was evaluated using the semi-quantitative IRS (ImmunoReactive Score) scale, which took into account the intensity of the colour reaction and percentage of positive cells. Results of the immunohistochemical reactions, pathological and of clinical observations were subjected to statistical analysis. Correlations between these factors and BCRP were analyzed using Spearman and Chi2 tests. In order to estimate the survival rate, we used Kaplan Meier statistics, log-rank tests and Cox proportional hazard regression. RESULTS: In our analysis we observed a positive correlation between the expression of the BCRP protein and grade of tumour advancement (r = 0.2 p = 0.03). We found also a negative correlation between the expression of BCRP and the estrogen (r = 0.24 p = 0.02) and progesteron (r = 0.28 p = 0.02) receptors. In a univariate analysis a significantly shorter disease free survival (DFS) and disease specific survival (DSS) was noted in patients with metastases to the lymph nodes (p = 0.003 and p = 0.0006), over the age of 50 years old ((p = 0.02 and p = 0.04) and clearly statistically significant in patients with a high expression of BCRP (p = 0.00044 and p = 0.00005). Overall survival (OS) was shorter in patients over the age of 50 (p = 0.01), with higher stage of the disease - IIB (p = 0.025), with metastases to the lymph nodes (p = 0.003) and also clearly statistically significant in patients with a high expression of BCRP (p = 0.00004). A multivariate analysis allowed to reveal that only higher expression of BCRP and metastases to lymph nodes were typical for cases of DFS (p = 0.,028 and p = 0.00015), DSS (p = 0.00052 and 0.000017) and OS (p = 0.0018 and p = 0.000007) time. CONCLUSIONS: We demonstrated that high BCRP expression level is associated with poor survival in early stage breast cancer patients.