High mRNA Expression Levels of Heat Shock Protein Family B Member 2 (HSPB2) Are Associated with Breast Cancer Patients' Relapse and Poor Survival.

Small heat shock proteins (sHSPs) are ubiquitous ATP-independent chaperones that contribute to the maintenance of proteome integrity and functionality. Recent evidence suggests that sHSPs are ubiquitously expressed in numerous types of tumors and have been proposed to be implicated in oncogenesis and malignant progression. Heat shock protein family B member 2 (HSPB2) is a member of the sHSPs, which is found to be expressed, among others, in human breast cancer cell lines and constitutes an inhibitor of apical caspase activation in the extrinsic apoptotic pathway. In this study, we investigated the potential prognostic significance of HSPB2 mRNA expression levels in breast cancer, which represents the most frequent malignancy in females and one of the three most common cancer types worldwide. To this end, malignant breast tumors along with paired non-cancerous breast tissue specimens were used. HSPB2 expression levels were quantified in these two cohorts using a sensitive and accurate SYBR green-based quantitative real-time polymerase chain reaction (q-RT-PCR). Extensive biostatistical analyses were performed including Kaplan-Meier and Cox regression survival analyses for the assessment of the results. The significant downregulation of HSPB2 gene expression was revealed in breast tumors compared to their adjacent non-cancerous breast tissues. Notably, high HSPB2 mRNA expression predicts poor disease-free survival and overall survival of breast cancer patients. Multivariate Cox regression analysis revealed that HSPB2 mRNA overexpression is a significant predictor of poor prognosis in breast cancer, independent of other clinicopathological factors. In conclusion, high HSPB2 mRNA expression levels are associated with breast cancer patients' relapse and poor survival.

Uncovering the clinical impact of kallikrein-related peptidase 5 (KLK5) mRNA expression in the colorectal adenoma-carcinoma sequence.

Background Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases located on chromosome 19q13.3. Most KLKs have been extensively studied as potential biomarkers for several carcinomas and other diseases. KLK5 was originally identified from a keratinocyte library, and its enzyme was purified from the stratum corneum of human skin. KLK5 was shown to be differentially expressed in a variety of endocrine tumors, although it is not as yet examined widely in colorectal cancer (CRC). Methods In this study, we quantitatively assessed the mRNA expression status of KLK5 in 197 colorectal tissues from 133 patients (70 cancerous and their paired normal colonic mucosa for 64 of them, as well as 63 colorectal adenomas) by quantitative real-time PCR (qPCR) using TaqMan probes. Statistical analysis evaluated the results. Results It was shown that KLK5 expression is reduced following the histologically non-cancerous-adenoma sequence (p<0.001), whereas it is increased during the sequence adenoma-carcinoma (p<0.001). Furthermore, KLK5 positive expression is associated with positive nodal status (p=0.022), advanced tumor stage (p=0.038) and high histological grade (p=0.033). Cox univariate analysis revealed that KLK5 positive expression is associated with disease-free survival (DFS) (p=0.028) and overall survival (OS) of patients (p=0.048). Kaplan-Meyer survival models showed that patients with positive KLK5 expression have lower DFS (p=0.009) and OS (p=0.019). Receiver operating characteristic (ROC) analysis demonstrated for first time that KLK5 expression had significant discriminatory values between cancer and adenoma tissues (area under the curve [AUC] 0.77; 95% confidence interval [CI]=0.69-0.85, p=0.03). Conclusions KLK5 mRNA expression may be useful for the differentiation of CRC from colorectal adenoma and represents a potential unfavorable prognostic biomarker for CRC.

A comprehensive clinicopathological evaluation of the differential expression of microRNA-331 in breast tumors and its diagnostic significance.

OBJECTIVE: MicroRNA-331 (miR-331) has shown regulatory activity against several genes whose expression has been claimed to be deregulated in breast tumors, including that of epidermal growth factor receptor 2 (HER2). Herein, the clinical value of miR-331 expression was investigated by analyzing its levels in breast benign and malignant tumors. METHODS: The expression levels of miR-331 were quantified via real-time PCR in 130 malignant and 66 benign breast tissue specimens collected after surgical resection of primary tumors. The generated data were analyzed by applying several statistical tests in order to examine the relationship of miR-331 expression with various established clinicopathological features and survival data of patients. RESULTS: Our data showed that miR-331 was overexpressed in malignant breast tumors compared to their benign counterparts both overall (P = 0.026) and individually when the subgroups of fibroadenoma and invasive ductal carcinoma were analyzed with each other (P = 0.001). ROC curve analysis confirmed the diagnostic value of these variations, providing an AUC value equal to 0.597 (P = 0.026) and 0.663 (P = 0.001), respectively. Furthermore, miR-331 levels were elevated (P = 0.026) in ductal cancerous specimens compared to the lobular ones but failed to correlate with other clinicopathological features or survival data of the breast cancer patients. CONCLUSIONS: Our results provide evidence that miR-331 levels might provide valuable information regarding the differential diagnosis of benign and malignant breast tumors but present no prognostic value for breast cancer.

Human kallikrein-related peptidase 12 (KLK12) splice variants discriminate benign from cancerous breast tumors.

OBJECTIVES: As kallikrein-related peptidase 12 (KLK12) has been implicated in the cancer progression and alternative splicing plays significant role in this disease, the aim of this study was to examine the expression profile and the clinical impact of the KLK12 splice variants in breast cancer. DESIGN AND METHODS: Total RNA was isolated and reverse transcripted from 141 tissues. Afterwards, quantitative real-time PCR were conducted, followed by the performance of the comparative CT (2-ΔΔCT) method for relative quantification, whilst their correlation with the clinicopathological features of breast malignancies were assessed by statistical analysis. RESULTS: Both KLK12sv1/2 and KLK12sv3 showed higher expression in non-cancerous than in cancerous samples. KLKsv1/2 (P = 0.001) upregulated and KLK12sv3 (P < 0.001) downregulated in the malignant compared to the benign tumors and their discriminative ability was verified by ROC curve analysis. Moreover, KLK12sv3 was associated with grade (P = 0.012) and hormonal receptor status (P = 0.001). Furthermore, Kaplan-Meier and Cox regression analyses showed that patients with positive KLK12sv1/2 and KLK12sv3 levels presented a significantly longer disease-free survival (P = 0.014 and P = 0.013, respectively) and overall survival (P = 0.062 and P = 0.004, respectively). CONCLUSIONS: Our results demonstrate the discriminative value of KLK12sv1/2 and KLK12sv3 between benign and malignant breast tumors as well as their potential favorable prognostic significance in breast adenocarcinoma.

Expression Analysis of miR-29b in Malignant and Benign Breast Tumors: A Promising Prognostic Biomarker for Invasive Ductal Carcinoma With a Possible Histotype-Related Expression Status.

BACKGROUND: Aberrations in microRNA levels seem to provide valuable information regarding breast cancer prognosis and therapy. In this study, we sought to analyze miR-29b expression in breast tumors and thus explore its clinical value. MATERIALS AND METHODS: One hundred twenty-one malignant and 56 benign breast tissue specimens were collected and subjected to extraction of total RNA, which was polyadenylated and reverse transcribed to cDNA. Subsequently, a highly sensitive quantitative real-time polymerase chain reaction protocol was developed and miR-29b levels, estimated via the comparative CT method, were finally subjected to comprehensive statistical analysis. RESULTS: MiR-29b levels did not differ between the analyzed benign and malignant breast tissue specimens, but were found to be significantly (P = .010) decreased in invasive ductal adenocarcinomas compared with their lobular counterparts, albeit receiver operating characteristics curve analysis did not verify the latter correlation. Additionally, miR-29b expression was elevated in samples with positive estrogen receptor status (P = .021) in the overall population, whereas it was negatively correlated (P = .035) with primary tumor staging in the ductal subset and increased in poorly-differentiated tumors of lobular origin (P = .041). Furthermore, Kaplan-Meier and Cox regression analyses showed that patients with ductal carcinoma and elevated miR-29b levels had a significantly longer disease-free survival (P = .010) and a lower risk to relapse (hazard ratio = 0.35, 95% confidence interval, 0.15-0.81; P = .014). CONCLUSION: Our results provide evidence that miR-29b levels constitute a promising biomarker of favorable prognosis for patients with invasive ductal breast carcinoma and imply that its expression status might be affected by the histological origin of breast malignancy.

Unravelling a p73-regulated network: The role of a novel p73-dependent target, MIR3158, in cancer cell migration and invasiveness.

The transcription factor p73 is homologous to the well-known tumor-suppressor p53. The p73-regulated networks are of significant clinical interest, because they may substitute for impaired p53-regulated networks which are commonly perturbed in cancer. Herein, we aimed to characterize a p73-regulated network that mediates cell migration and restores anti-oncogenic responses in p53-mutant cancer cells. In this study, we demonstrate that p73 regulates a network underlying cell migration, which consists of MIR34A/MIR3158/vimentin/ß-catenin/lef1. The p73 isoforms transactivate the miRNA components (MIR34A/MIR3158) of this network, which in turn, downregulate their EMT-related mRNA co-targets (vimentin/ß-catenin/lef1) to decrease cell-migration. Modulation of this network, by increasing the level of the novel p73-dependent target MIR3158, was found to induce anti-oncogenic/anti-invasive responses in p53-mutant cancer cells. Taken together, a p73-regulated, MIR3158-containing, network restores anti-invasive phenotypes in p53-mutant cancer cells; this property could be exploited towards the development of anticancer therapeutics.

Kallikrein-related peptidase 6 (KLK6) expression in the progression of colon adenoma to carcinoma.

KLK6 is a secreted trypsin-like serine protease. KLK6 mRNA expression and its association with colon cancer (CC) progression was studied using quantitative real-time PCR. We examined the expression of KLK6 in 232 colon tissues (cancerous, non-cancerous, and adenomatous). We proved that KLK6 expression in CC behaves as a continuous variable, as its expression correlates significantly with increasing tumor stage (p=0.004) and histological grade (p=0.007). Interestingly, the expression of KLK6 in adenomas was significantly higher than that in the cancerous or non-cancerous tissues examined (p<0.001). Cox proportional hazard regression model using univariate analysis revealed that positive KLK6 expression is a significant factor for disease-free survival (DFS) (p=0.017) and overall survival (OS) (p=0.002) of patients. Kaplan-Meier survival curves demonstrated that KLK6-negative expression is significantly associated with longer DFS (p=0.009) and OS (p=0.001). ROC analysis showed that KLK6 expression has significant discriminatory power in distinguishing cancerous from non-cancerous colon tissues (p<0.001), or cancerous from adenoma tissues (p=0.001), or adenoma from non-cancerous colon tissues (p<0.001). Additionally, strong KLK6 immunostaining was seen in the cancer cells of selected CC sections, as well as in glandular cells and inflammatory cells of adenomas. In conclusion, KLK6 may represent a potential unfavorable prognostic biomarker for CC.

Identification of a STAT5 target gene, Dpf3, provides novel insights in chronic lymphocytic leukemia.

STAT5 controls essential cellular functions and is encoded by two genes, Stat5a and Stat5b. To provide insight to the mechanisms linking hematologic malignancy to STAT5 activation/regulation of target genes, we identified STAT5 target genes and focused on Dpf3 gene, which encodes for an epigenetic factor. Dpf3 expression was induced upon IL-3 stimulation in Ba/F3 cells, while strong binding of both STAT5a and STAT5b was detected in its promoter. Reduced expression of Dpf3 was detected in Ba/F3 cells with Stat5a and Stat5b knock-down, suggesting that this gene is positively regulated by STAT5, upon IL-3 stimulation. Furthermore, this gene was significantly up-regulated in CLL patients, where DPF3 gene/protein up-regulation and strong STAT5 binding to the DPF3 promoter, correlated with increased STAT5 activation, mainly in non-malignant myeloid cells (granulocytes). Our findings provide insights in the STAT5 dependent transcriptional regulation of Dpf3, and demonstrate for the first time increased STAT5 activation in granulocytes of CLL patients. Novel routes of investigation are opened to facilitate the understanding of the role of STAT5 activation in the communication between non-malignant myeloid and malignant B-cells, and the functions of STAT5 target genes networks in CLL biology.

Significant alterations in the expression pattern of kallikrein-related peptidase genes KLK4, KLK5 and KLK14 after treatment of breast cancer cells with the chemotherapeutic agents epirubicin, docetaxel and methotrexate.

Given that 1.3 million new cases of breast cancer are universally registered among women and approximately 36 % of the patients die annually, the revelation of new predictive markers for treatment efficiency is of vital importance. Recently, our group has depicted that KLK4, KLK5, and KLK14 are differentially expressed in breast carcinoma. The objective of this study was to determine and investigate the expression pattern of the KLK4, KLK5, and KLK14 genes in breast cancer cells after treatment with established chemotherapeutic agents. We evaluated these genes' expression after treatment of the BT-20 cells with epirubicin, docetaxel and methotrexate, determining their cytotoxic effect by MTT and trypan blue assays. The relative quantification of genes' mRNA levels was performed by using the SYBR Green® chemistry, and the HPRT1 served as an endogenous control gene. The drugs triggered apoptosis in treated cells and induced deregulations in the expression of the above KLKs. The most significant alterations were a 12-fold and tenfold increase of KLK5 in docetaxel and methotrexate-treated cells, respectively, while the KLK4 levels decreased by ten-fold in epirubicin, five-fold in docetaxel and twenty-fold in methotrexate treated-cells, compared to the untreated ones. In the case of KLK14 levels, a twofold increase in epirubicin and threefold decrease in methotrexate-treated cells were observed. Present significant alterations in the expression pattern of KLK4, KLK5, and KLK14 could comprise an initial stage for predicting chemotherapy response in breast cancer and should be further investigated as predictive markers in the future.

Down-regulation of kallikrein-related peptidase 5 (KLK5) expression in breast cancer patients: a biomarker for the differential diagnosis of breast lesions.

BACKGROUND: Kallikrein-related peptidase 5 (KLK5) is a secreted trypsin-like protease of the KLK family, encoded by the KLK5 gene. KLK5 has been found to cleave various extracellular matrix components, as well as to activate several other KLK proteases, triggering the stimulation of tissue microenvironment proteolytic cascades. MATERIAL AND METHODS: KLK5 expression levels were quantified in 102 cancerous and benign breast tissue specimens, obtained by randomly chosen patients, using RT-qPCR assay. Subsequently, advanced biostatistics were applied in order to analyze the KLK5 expression profile in the two patients' cohorts and also to evaluate its clinical significance for the discrimination of breast tumors. RESULTS: A statistically significant (p < 0.001) down-regulation of the KLK5 expression levels were observed in the malignant specimens compared to the benign ones. Logistic regression and ROC curve analysis revealed the significant (p < 0.001) and the independent (p < 0.001) value of the KLK5 expression quantification, for the discrimination of the malignant from the benign mammary gland biopsies. Moreover, KLK5 expression levels correlate with the pre-menopausal status (p < 0.005) as well as the ER-negative staining (p = 0.028) of women with breast cancer. CONCLUSIONS: The quantification of KLK5 expression in breast tissue biopsies may be considered as a novel and independent biomarker for the differential diagnosis between malignant and benign tumors of the mammary gland.

Quantitative expression analysis and study of the novel human kallikrein-related peptidase 14 gene (KLK14) in malignant and benign breast tissues.

Human kallikrein-related peptidase 14 gene (KLK14) is regulated by androgens and progestins. This gene is expressed in the central nervous system and endocrine tissues such as the breast, prostate and ovary. The differential KLK14 mRNA expression levels are related to several human neoplasias, among them breast cancer. The aim of this study was to analyse the KLK14 expression in breast tissues and to investigate its differential diagnostic and prognostic value in the mammary carcinomas. For this purpose, we isolated total RNA from 70 malignant and 33 benign specimens. After testing RNA quality, we synthesised cDNA by reverse transcription and applied a highly sensitive quantitative real-time PCR (qRT-PCR) method for KLK14 mRNA quantification using the SYBR Green® chemistry. HPRT1 was used as a reference gene and the BT20 breast cancer cell line as a calibrator. Relative quantification analysis was performed using the comparative CT method 2-ΔΔCT. KLK14 expression was detected in both types of breast tumours. However, a statistically significant increase of the KLK14 mRNA level was observed in the malignant, compared to the benign tumour samples (p<0.001), highlighting its value in discriminating these breast lesions. Elevated KLK14 expression profiles were associated with higher tumour grade (p=0.043) and size (p=0.007) in cancerous samples. Furthermore, KLK14 mRNA expression showed negative correlation in a statistically significant manner with estrogen receptor status (p=0.024). In accordance with logistic regression models (p=0.012) and receiver-operating-characteristics analysis (p<0.001), KLK14 gene expression could be evaluated as a putative independent diagnostic biomarker in breast tumour biopsies.

Expression analysis and study of KLK4 in benign and malignant breast tumours.

The steroid hormone-regulated gene KLK4 (kallikrein 4) is a new member of the human kallikrein-related peptidase gene family. Up to date, studies report that KLK4 is differentially expressed in many tumours. The purpose of this study was the expression analysis and study of KLK4 in benign and malignant breast tumours. Total RNA was isolated from 16 benign and 45 malignant breast tissue specimens. After testing RNA quality, cDNA was prepared by reverse transcription. Highly sensitive quantitative real-time PCR method for KLK4 mRNA quantification was developed using the SYBR Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative C(T) method 2(-DeltaDeltaC)(T) KLK4 expression was found to vary in both patients' cohorts; however, a statistically significant elevation of the KLK4 mRNA levels was observed in malignant compared to benign tumour patients. Low KLK4 expression levels were found in well-differentiated tumours (p = 0.011) as well as in stage I (p = 0.024) patients. Moreover, a statistically significant (r(s) = -0.318, p = 0.035) negative correlation between the KLK4 expression and progesterone receptor staining was observed. ROC and logistic regression analysis recommended that KLK4 gene expression may be used as a new potential biomarker in breast cancer.