Elżbieta Płuciennik

Prognostic Relevance of Basal Cytokeratin Expression in Operable Breast Cancer

Objective: We investigated whether basal cytokeratin (CK5/6 or CK17) expression had an impact on survival in patients with operable breast cancer. Methods: Expression of CK5/6 or CK17 was analyzed by immunohistochemistry in 195 women with breast cancer. Results: In total, 72 (37%) tumor samples were regarded as being positive for CK5/6 or CK17. The basal-like phenotype as defined by basal cytokeratin expression, lack of estrogen receptor (ER) and absence of HER2 overexpression was found in 48 (25%) cases. Positive staining for CK5/6 or CK17 was associated with worse prognosis when compared with patients negative for basal cytokeratins in all cases (5-year cancer-specific survival rate 59.4 vs. 77.5%, p = 0.0273) and in the node-negative group (70.5 vs. 90.8%, p = 0.0208) but not in the node-positive group (43.9 vs. 65.4%, p = 0.1182). To determine the real prognostic value of basal cytokeratins, survival in a group of ER-negative patients was analyzed depending on CK5/6 or CK 17 expression. No influence on survival was observed. The outcome of patients whose cancers were positive for cyclin E regardless of ER status was not changed by CK5/6 or CK17 expression. In multivariate analysis, independent prognostic factors affecting survival in the whole group included: nodal involvement, HER2 status and cyclin E expression. Neither ER status nor basal cytokeratin expression retained statistical significance. Conclusion: We demonstrated that the poor prognosis associated with the basal-like phenotype of breast cancer was determined by ER absence and cyclin E expression and not by CK5/6 or CK17 expression.

Molecular analysis of WWOX expression correlation with proliferation and apoptosis in glioblastoma multiforme

Glioblastoma multiforme is the most common type of primary brain tumor in adults. WWOX is a tumor suppressor gene involved in carcinogenesis and cancer progression in many different neoplasms. Reduced WWOX expression is associated with more aggressive phenotype and poor patient outcome in several cancers. We investigated alternations of WWOX expression and its correlation with proliferation, apoptosis and signal trafficking in 67 glioblastoma multiforme specimens. Moreover, we examined the level of WWOX LOH and methylation status in WWOX promoter region. Our results suggest that loss of heterozygosity (relatively frequent in glioblastoma multiforme) along with promoter methylation may decrease the expression of this tumor suppressor gene. Our experiment revealed positive correlations between WWOX and Bcl2 and between WWOX and Ki67. We also confirmed that WWOX is positively correlated with ErbB4 signaling pathway in glioblastoma multiforme.

WWOX expression in colorectal cancer—a real-time quantitative RT-PCR study

The WWOX gene is a tumour suppressor gene affected in various types of malignancies. Numerous studies showed either loss or reduction of the WWOX expression in variety of tumours, including breast, ovary, liver, stomach and pancreas. Recent study demonstrated that breast cancer patients exhibiting higher WWOX expression showed significantly longer disease-free survival in contrast to the group with lower relative WWOX level. This work was undertaken to show whether similar phenomena take place in colon tumours and cell lines. To assess the correlation of WWOX gene expression with prognosis and cancer recurrence in 99 colorectal cancer patients, we performed qRT-PCR analysis. We also performed analysis of WWOX promoter methylation status using MethylScreen method and analysis of loss of heterozygosity (LOH) status at two WWOX-related loci, previously shown to be frequently deleted in various types of tumours. A significantly better disease-free survival was observed among patients with tumours exhibiting high level of WWOX (hazard ratio = 0.39; p = 0.0452; Mantel–Cox log-rank test), but in multivariate analysis it was not an independent prognostic factor. We also found that although in colorectal cancer WWOX expression varies among patients and correlates with DFS, the exact mode of decrease in this type of tumour was not found. We failed to find the evidence of LOH in WWOX region, or hypermethylation in promoter regions of this gene. Although we provide the evidence for tumour-suppressive role of WWOX gene expression in colon, we were unable to identify the molecular mechanism responsible for this.

Analysis of the expression of angiotensin II type 1 receptor and VEGF in endometrial adenocarcinoma with different clinicopathological characteristics.

In Poland, endometrial carcinoma takes second place after breast cancer among all cancers in women and is considered the most common genital cancer. It has been repeatedly reported that angiotensin is involved in the development and invasion of some cancers including breast, ovarian, and pancreatic ones. It is suggested that angiotensin two and its receptors are actively involved in tumour biology in endometrial adenocarcinoma. In the present study, we identify a possible relationship between the expression of AT1-R, AT2-R, ERα, and VEGF and clinicopathological characteristics of primary endometrial adenocarcinoma. We determined the above components both at the mRNA (real-time RT-PCR) and protein levels (Western Blot assay). Our results indicate that in patients with grade G3 adenocarcinoma, the expression of AT1-R significantly decreased in comparison with G1 patients (p = 0.034), but the level of ERα was the highest in G2 and the lowest in G3. Moreover, the level of VEGF mRNA significantly increased between G2 and G3 (p = 0.034). We also noted a significant correlation between the expression of AT1-R and AT2-R in FIGO stage 1 (Rs = 0.9636; p = 0.0001) and that of AT2-R and VEGF (Rs = 0.5377; p = 0.005). In grade G1 and G2 carcinoma, a significant correlation was also found between the expression of AT1-R and AT2-R (Rs = 0.9924; p = 0.0001; Rs = 0.8717, p = 0.0005, respectively), but in grade G1, a negative correlation was observed between AT1-R and VEGF (Rs = −0.8945, p = 0.0005). Further studies are required to clarify the biological function of the angiotensin receptor in regulating VEGF expression in endometrial carcinoma.

Basal keratin expression in breast cancer by quantification of mRNA and by immunohistochemistry

Definitions of basal-like breast cancer phenotype vary, and microarray-based expression profiling analysis remains the gold standard for the identification of these tumors. Immunohistochemical identification of basal-like carcinomas is hindered with a fact, that on microarray level not all of them express basal-type cytokeratin 5/6, 14 and 17. We compared expression of cytokeratin 5, 14 and 17 in 115 patients with operable breast cancer estimated by real-time RT-PCR and immunohistochemistry.Despite the method of dichotomization and statistical analysis, there were cases with discordant results comparing immunohistochemistry and RT-PCR analysis. For dichotomisation based on quartiles and ROC, 14% of cases were negative on immunohistochemical examination for CK5/6, but presented high CK5 mRNA levels. There were also 48-55% cases, which were CK5/6-immunopositive, but were negative by mRNA examination. Similar discordances were observed for CK14 and CK17.Basal keratin mRNAs did not correlate with ER mRNA levels, while immunohistochemistry produced significant relationship with ER status.Our observation suggest that both method may produce different results in a small proportion of cases. Discordance between immunohistochemistry and RT-PCR may confound attempts to establish a simple methods for identification of basal-like tumors.

Evaluation of oestrogen receptor expression in breast cancer by quantification of mRNA.

Aims:  cDNA microarrays have subclassified breast carcinomas into molecular subtypes with oestrogen receptor‐α (ER) gene expression as a main marker. The aim was to compare ER expression in 97 patients with operable breast cancer estimated by real‐time reverse transcriptase‐polymerase chain reaction (real‐time RT‐PCR) and by routine immunohistochemistry, and to determine which method was reliable for molecular subtyping in relation to basal‐type keratins and HER2 gene expression.

A comparison of the effects of Angiotensin IV on androgen-dependent and androgen-independent prostate cancer cell lines

Introduction: Angiotensin IV is one of the biologically active peptides of the renin–angiotensin system. Limited data suggests that this hexapeptide could contribute to cancer development and/or progression. Materials and methods: Using the MTT reduction assay as an indicator of cell viability, and the bromodeoxyuridine incorporation assay as an indicator of cell proliferation, the influence of Angiotensin IV was evaluated on two human prostate cancer lines: androgen-dependent (LNCaP) and androgen-independent (DU-145). The potential effect of Angiotensin IV classic angiotensin receptors was examined by using the selective antagonists losartan and PD123319. Finally, the changes in expression levels of AT1 and AT2 receptors were compared, before and after angiotensin treatment. Results: Angiotensin IV caused significant changes in cell viability and proliferation in LNCaP cells but not in DU-145. It was found that AT2 receptor blocker (PD123319) was able to diminish the suppressor effect of Angiotensin IV on bromodeoxyuridine incorporation into the DNA of androgen-dependent prostate cancer cells. Simultaneously, it was reported that Angiotensin IV is the factor that modulates the density of AT1 and AT2 receptors in prostate cancer cells. Conclusions: These findings suggested that Angiotensin IV can modulate tumour cell proliferation in the early stage of androgen-dependent prostate cancer. The effect might be promoted by the change of the angiotensin receptor level.

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer.

PURPOSE Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. METHODS The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. RESULTS We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R(s)=0.50; p=0.002, R(s)=0.69; p=0.0001, R(s)=0.52; p=0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R(s)=0.70, p=0.0001, R(s)=0.67; p=0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R(s)=0.54, p=0.0001, R(s)=0.68; p=0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). CONCLUSIONS Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.

Proteins in cancer multidrug resistance

Multidrug Resistance (MDR) is defined as insensitivity to administered medicines that are structurally unrelated and have different molecular targets. Cancers possess numerous mechanisms of drug resistance, involving various aspects of cell biology. A pivotal role in this phenomenon is played by proteins--enzymatic or structural parts of the cell. Membrane transporters, including the main members of ABC protein family--P-gp, MRP1 and BCRP, as well as LRP, which builds structure of vaults, determine the multidrug-resistant phenotype by decreasing drug concentration within the cell or modifying its distribution to intracellular compartments. The π isoform of protein enzyme--glutathione S-transferase (GSTP-1), is responsible for excessive intensity of detoxification of cytostatics. A common example of altered drug target site that does not respond to chemotherapy is topoisomerase II α (TopoIIa). Alterations of programmed cell death result from expression of metallothionein (MT)--inhibitor of the process, and cytokeratin 18 (CK18), which, if in high concentration, also prevents apoptosis of cells. Several methods of decreasing activity of these proteins have been developed, aiming to overcome MDR in cancer cells. However, for a variety of reasons, their clinical suitability is still very low, leading to continuous increase in death rate among patients. This paper presents current state of knowledge on the most important examples of proteins responsible for MDR of cancer cells and molecular mechanisms of their action.

The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer

This study defines the role of WWOX in the regulation of epithelial to mesenchymal transition. A group of 164 endometrial adenocarcinoma patients was studied as well as an ECC1 well-differentiated steroid-responsive endometrial cell line, which was transducted with WWOX cDNA by a retroviral system. The relationship between WWOX gene and EMT marker (CDH1, VIM, ZEB1, SNAI1) expression on mRNA (RT-qPCR) and protein levels (western blotting) was evaluated. The EMT processes were also analysed in vitro by adhesion of cells to extracellular matrix proteins, migration through a basement membrane, anchorage-independent growth and MMP activity assay. DNA microarrays (HumanOneArray™) were used to determine WWOX-dependent pathways in an ECC1 cell line. A positive correlation was observed between WWOX and ZEB1, and a negative correlation between CDH1 and VIM. WWOX expression was found to inversely correlate with the risk of recurrence of tumors in patients. However, in the WWOX-expressing ECC1 cell line, WWOX expression was found to be inversely related with VIM and positively with CDH1. The ECC1/WWOX cell line variant demonstrated increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. However, these cells were not able to form colonies in suspension and revealed decreased adhesion to fibronectin and fibrinogen. Microarray analysis demonstrated that WWOX has an impact on the variety of cellular pathways including the cadherin and integrin signalling pathways. Our results suggest that the WWOX gene plays a role in the regulation of EMT processes in endometrial cancer by controlling the expression of proteins associated with cell motility, thus influencing tissue remodeling, with the suppression of mesenchymal markers.