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.

Mesenchymal stem cells in preclinical cancer cytotherapy: a systematic review.

Mesenchymal stem cells (MSC) comprise a heterogeneous population of rapidly proliferating cells that can be isolated from adult (e.g., bone marrow, adipose tissue) as well as fetal (e.g., umbilical cord) tissues (termed bone marrow (BM)-, adipose tissue (AT)-, and umbilical cord (UC)-MSC, respectively) and are capable of differentiation into a wide range of non-hematopoietic cell types. An additional, unique attribute of MSC is their ability to home to tumor sites and to interact with the local supportive microenvironment which rapidly conceptualized into MSC-based experimental cancer cytotherapy at the turn of the century. Towards this purpose, both naïve (unmodified) and genetically modified MSC (GM-MSC; used as delivery vehicles for the controlled expression and release of antitumorigenic molecules) have been employed using well-established in vitro and in vivo cancer models, albeit with variable success. The first approach is hampered by contradictory findings regarding the effects of naïve MSC of different origins on tumor growth and metastasis, largely attributed to inherent biological heterogeneity of MSC as well as experimental discrepancies. In the second case, although the anti-cancer effect of GM-MSC is markedly improved over that of naïve cells, it is yet apparent that some protocols are more efficient against some types of cancer than others. Regardless, in order to maximize therapeutic consistency and efficacy, a deeper understanding of the complex interaction between MSC and the tumor microenvironment is required, as well as examination of the role of key experimental parameters in shaping the final cytotherapy outcome. This systematic review represents, to the best of our knowledge, the first thorough evaluation of the impact of experimental anti-cancer therapies based on MSC of human origin (with special focus on human BM-/AT-/UC-MSC). Importantly, we dissect the commonalities and differences as well as address the shortcomings of work accumulated over the last two decades and discuss how this information can serve as a guide map for optimal experimental design implementation ultimately aiding the effective transition into clinical trials.

Genetically­modified stem cells in treatment of human diseases: Tissue kallikrein (KLK1)­based targeted therapy (Review).

The tissue kallikrein­kinin system (KKS) is an endogenous multiprotein metabolic cascade which is implicated in the homeostasis of the cardiovascular, renal and central nervous system. Human tissue kallikrein (KLK1) is a serine protease, component of the KKS that has been demonstrated to exert pleiotropic beneficial effects in protection from tissue injury through its anti­inflammatory, anti­apoptotic, anti­fibrotic and anti­oxidative actions. Mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs) constitute populations of well­characterized, readily obtainable multipotent cells with special immunomodulatory, migratory and paracrine properties rendering them appealing potential therapeutics in experimental animal models of various diseases. Genetic modification enhances their inherent properties. MSCs or EPCs are competent cellular vehicles for drug and/or gene delivery in the targeted treatment of diseases. KLK1 gene delivery using adenoviral vectors or KLK1 protein infusion into injured tissues of animal models has provided particularly encouraging results in attenuating or reversing myocardial, renal and cerebrovascular ischemic phenotype and tissue damage, thus paving the way for the administration of genetically modified MSCs or EPCs with the human tissue KLK1 gene. Engraftment of KLK1­modified MSCs and/or KLK1­modified EPCs resulted in advanced beneficial outcome regarding heart and kidney protection and recovery from ischemic insults. Collectively, findings from pre­clinical studies raise the possibility that tissue KLK1 may be a novel future therapeutic target in the treatment of a wide range of cardiovascular, cerebrovascular and renal disorders.

Death receptor 5 (DR5) and a 5-gene apoptotic biomarker panel with significant differential diagnostic potential in colorectal cancer.

High expression of Inhibitor of apoptosis proteins (IAPs) has been related to colorectal cancer (CRC) progression, resistance to treatment and poor prognosis. TRAIL (TNF-related apoptosis-inducing ligand) through its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) can selectively induce cancer cell apoptosis. The mRNA expression of DR4, DR5, c-IAP1, c-IAP2, XIAP and BIRC5/Survivin genes was examined in 100 paired (cancerous-normal) colorectal tissue specimens by real-time PCR, 50 of which were KRAS wild-type and 50 KRAS-mutant. DR5, XIAP and BIRC5/Survivin genes are significantly up-regulated (p < 0.0001, p = 0.012 and p = 0.0003, respectively), whereas c-IAP1 and c-IAP2 genes are significantly down-regulated at mRNA and protein levels in CRC (p < 0.0001 for both). ROC analyses showed that DR5, cIAP1 and cIAP2 expression has discriminatory value between CRC and normal tissue (AUC = 0.700, p < 0.0001 for DR5; AUC = 0.628, p = 0.011 for cIAP1; AUC = 0.673, p < 0.0001 for cIAP2). Combinatorial ROC analysis revealed the marginally fair discriminatory value of 5 genes as a panel (AUC = 0.685, p < 0.0001). Kaplan-Meier survival curves revealed significant association of cIAP2 down-regulation in CRC with lower overall survival probability of CRC patients (p = 0.0098). DR5, BIRC5/Survivin, XIAP, c-IAP1 and c-IAP2 mRNA expression are significantly deregulated in CRC and could provide a panel of markers with significant discriminatory value between CRC and normal colorectal tissue.

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.

Parallel overexpression and clinical significance of kallikrein-related peptidases 7 and 14 (KLK7KLK14) in colon cancer.

Currently available colon cancer (CC) markers lack sensitivity and specificity. Kallikrein-related peptidases (KLKs) present a new class of biomarkers under investigation for diverse diseases, including cancer. KLKs are co-expressed in various tissues participating in proteolytic cascades. KLK7 in human tumours facilitates metastasis by degrading components of the extracellular matrix. KLK14 promotes tumourigenesis by activating proteinase-activated receptors. In the present study we examined the concomitant expression of KLK7 and KLK14 in245 colonic tissue specimens from 175 patients; 70 were pairs of cancerous-normal tissues, 31 were cancerous tissues and 74 were colonic adenomas. We used quantitative real-time PCR and proved that both genes are up-regulated in CC at the mRNA level. Receiver-operating characteristic (ROC) analysis of our results showed that both genes have discriminatory value between CC and adenoma tissues, with KLK14 obtaining greater distinguishing power (area under the curve [AUC]=0.708 for KLK14; AUC=0.669 for KLK7). Current work showed that the two genes are fairly co-expressed in all three types of colon tissues examined (normal rs=0.667, p<0.001, adenomas rs=0.373, p=0.001, carcinomas rs=0.478, p<0.001). KLK14 is associated with shorter disease-free survival (DFS) and overall survival (OS) of patients (p=0.003, p=0.016 respectively), whereas KLK7only with shorter DFS (p=0.004). KLK7 and KLK14 gene expression can be regarded as markers of poor prognosis for CC patients with discriminating power between CC and adenoma patients.

Kallikrein-related peptidase 6 (KLK6)gene expression in intracranial tumors.

Kallikrein-related peptidases (KLKs) are emerging novel new biomarkers for prognosis, diagnosis and therapeutic intervention of cancer. Kallikrein-related peptidase 6 (KLK6) has the highest expression in normal brain among other tissues. Although its expression has been extensively studied in many types of cancer and in neurodegenerative diseases, very little is known for its expression in intracranial tumors. In the present study, 73 intracranial tumor samples were examined for KLK6 messenger ribunucleic acid (mRNA) gene expression using quantitative real-time polymerase chain reaction. Statistical analysis revealed the significant association of KLK6 expression with clinical and pathological parameters. Follow-up information was available for a median time of 20 months (range 1-59 months). KLK6 is expressed more frequently in tumors of high malignancy like the glioblastomas (70.6 %) and less in tumors of low malignancy like the meningiomas (12.5 %). KLK6 positive expression is associated with tumor grade (p < 0.001), malignancy status (p < 0.001), and tumor histologic type (p = 0.001). Cox proportional hazard regression model using univariate analysis revealed for the first time that positive KLK6 expression is a significant factor for disease-free survival (DFS; p = 0.041) of patients suffering from intracranial tumors. Kaplan-Meier survival curves demonstrated that negative KLK6 expression is significantly associated with longer DFS (p = 0.032). KLK6 gene expression may have clinical utility as a marker of unfavorable prognosis for intracranial tumors, and consequently, it could be used as target for therapeutic intervention.

Human kallikrein-related peptidase 12 (KLK12) splice variants expression in breast cancer and their clinical impact.

Kallikrein-related peptidases (KLKs) are a group of 15 serine proteases, hormonally regulated, and localized on chromosome 19q13.4. Alternative splicing is a process that plays significant role in the development, physiology, and different diseases, like cancer. Kallikrein family numbers more than 82 alternative transcripts. Understanding the role that those gene transcripts play in various cancer types, could lead to the discovery of diagnostic markers or drug targets. The present study was designed to analyze the expression profile of the splice variants of kallikrein-related peptidase 12 (KLK12) in breast cancer patients and to evaluate their clinical significance. KLK12 splice variants (KLK12sv3 and KLK12sv1/KLK12sv2) were examined in 69 tissue samples of breast cancer using quantitative real-time PCR as well as semi-quantitative PCR. Relative quantitative expression of KLK12 was statistically associated to clinicopathological parameters. From the splice variants examined, statistical associations with clinicopathological parameters were obtained only from KLK12sv3 variant. KLK12sv3 is more frequently expressed in tumors of lower grade (p = 0.040), early patient TNM stage (p = 0.024), and smaller tumor size (p = 0.023). Positive KLK12sv3 expression is associated with longer patient disease-free survival (DFS) (p = 0.042) and higher progesterone receptor concentration (p = 0.008). KLK12sv1/KLK12sv2 expression is statistically associated with KLK12sv3 expression (p = 0.001). KLK12sv3 can be regarded as a marker of good prognosis in breast cancer.

Evaluation of kallikrein-related peptidase 5 expression and its significance for breast cancer patients: association with kallikrein-related peptidase 7 expression.

BACKGROUND: Kallikrein-related peptidases (KLKs) have been proposed as potential cancer biomarkers. Contradictions in literature led us to clarify the role of KLK5 as a breast cancer predictor, as well as its association with KLK7 expression. PATIENTS AND METHODS: Semi-quantitative RT-PCR detected KLKs 5 and 7 in 80 breast tissues, 74 neoplastic and 6 normal. Steroid hormone receptors were quantified in all samples. Associations between KLK5 status and clinicopathological variables, as well as disease-free survival (DFS) and overall survival (OS) of patients were analyzed. RESULTS: Forty tumor tissues showed high KLK5 expression, which was significantly associated with estrogen receptor status. Significant co-expression of KLKs 5 and 7 was observed in the same cancer samples (p=0.02). Increased KLK5 expression was a statistically significant independent prognostic factor for DFS (p=0.009 univariate analysis and p=0.028 multivariate analysis) and OS of patients (p=0.014, univariate analysis). CONCLUSION: Increased KLK5 expression can contribute to the prognosis of DFS and OS of breast cancer patients. KLKs 5 and 7 are co-expressed in breast cancer.

Expression analysis and clinical evaluation of kallikrein-related peptidase 10 (KLK10) in colorectal cancer.

Kallikrein-related peptidases (KLKs) represent a serine protease family having 15 members. KLK10 is a secreted protease with a trypsin-like activity. The function of KLK10 is poorly understood, although it has been suggested that KLK10 may function as a tumor suppressor gene. In human cancer, KLK10 gene shows organ-specific up- or down-regulation. Since KLKs are promising tumor biomarkers, the examination of KLK10 mRNA expression and its association with colorectal cancer (CRC) progression was studied using semi-quantitative PCR. One hundred and nineteen primary CRC specimens were examined for which follow-up information was available for a median period of 29 months (range, 1-104 months). KLK10 expression was found to be significantly associated with TNM stage (p=0.028). Cox proportional hazard regression model using univariate analysis revealed for the first time that high status KLK10 expression is a significant factor for disease-free survival (DFS; p=0.002) and overall survival (OS; p=0.026) of patients. Kaplan-Meier survival curves demonstrated that KLK10 expression of low status is significantly associated with longer DFS (p=0.001) as well as OS (p=0.021), suggesting that KLK10 gene expression may be used as a marker of unfavorable prognosis for CRC. As the epigenetics of cancer are unraveled, KLK10 may represent not only a novel biomarker, but also a promising future therapeutic target for the disease.

Clinical evaluation of PRMT1 gene expression in breast cancer.

Methylation of arginine residues has been implicated in many cellular activities like mRNA splicing, transcription regulation, signal transduction and protein-protein interactions. Protein arginine methyltransferases are the enzymes responsible for this modification in living cells. The most commonly used methyltransferase in man is protein arginine methyltransferase 1 (PRMT1). Since methylation processes appear to interfere in the emergence of several diseases, including cancer, we investigated the localisation of the protein in cancer tissue and, for the first time, the relation that possibly exists between the expression of PRMT1 gene and breast cancer progression. We used tumour specimens from 62 breast cancer patients and semi-quantitative RT-PCR to determine the expression of PRMT1 gene and was found to be associated with patient's age (p = 0.002), menopausal status (p = 0.006), tumour grade (p = 0.03), and progesterone receptor status (p = 0.001). Survival curves revealed that PRMT1-v1 status-low expression relates to longer disease-free survival (DFS; p = 0.036). To the contrary, PRMT1-v2 status is not associated neither with the clinical or pathological parameters nor with DFS (p = 0.31). PRMT1-v3 was not statistically significantly expressed in breast cancer tissue. Selected cancer and normal breast samples were stained for PRMT1. In both normal and cancerous breast tissues, staining was in the cytoplasm and only in rare cases the cell nucleus appeared stained. Present results show a potential use for this gene as a marker of unfavourable prognosis for breast cancer patients.

Cathepsin B protein levels in endometrial cancer: Potential value as a tumour biomarker.

OBJECTIVE: Lysosomal cysteine protease Cathepsin-B has been implicated in the progression of various human tumours. We examined Cathepsin-B protein levels in endometrial carcinoma patients-mainly post-menopausal-and investigated their possible association with clinical and pathological parameters in order to assess Cathepsin-B's significance as a potential tumour biomarker. METHODS: The indirect immunoperoxidase method was used for Cathepsin-B immunohistochemical staining of 64 paraffin-embedded endometrial tumour tissues, having follow-up period of 18-240 months. Steroid hormone receptors were measured as well. Tissue samples were staged following the FIGO criteria. RESULTS: Positive Cathepsin-B immunostaining was observed in 27 patients (42.2%) and was significantly associated with the FIGO stage of the disease (p=0.006), as well as cervical and stromal invasion (p=0.001 and p=0.037, respectively) and progesterone receptor status (p=0.027). Positive Cathepsin-B expression was also inversely related to Disease-free Survival (p=0.034) and Overall Survival (p=0.035) in univariate analysis, as well as in multivariate analysis (p=0.022 and p=0.035, respectively). CONCLUSION: Increased Cathepsin-B expression was found to be predictive of more aggressive tumour behaviour over time and can be regarded as an unfavourable and independent tumour marker for endometrial cancer patients with a long follow-up.