Chris Planque

Alternative Splicing Variant of Kallikrein-Related Peptidase 8 as an Independent Predictor of Unfavorable Prognosis in Lung Cancer

BACKGROUND A relatively unexplored area for biomarker identification is alternative splice variants. We undertook this study to evaluate the usefulness of mRNA isoforms encoded by the KLK8 (kallikrein-related peptidase 8) gene as prognostic markers for lung cancer. METHODS Real-time reverse-transcription PCR was used to analyze the mRNAs encoded by KLK8 (particularly 2 mRNA splice variants, KLK8-T3 and KLK8-T4) in 60 non-small-cell lung cancer (NSCLC) tumors and in paired unaffected tissues. The ratios of these mRNAs to those encoded by the KLK5, KLK6, KLK7, KLK10, KLK11, KLK13, and KLK14 genes were also determined and analyzed for correlations with various clinicopathologic variables. RESULTS KLK8-T3 and KLK8-T4 were the most abundant of the 6 mRNA isoforms identified in lung tissues. The overall expression of the KLK8 gene and the amounts of the KLK8-T3 and KLK8-T4 mRNAs were significantly increased in lung tumor tissue (P < 0.0001). Univariate survival analysis revealed significant relationships of the relative concentrations of mRNA splice variants KLK8 (P = 0.043), KLK8-T3 (P = 0.037), and KLK8-T4 (P = 0.009) with overall survival (OS). Cox multivariate analysis indicated that the amount of KLK8-T4 mRNA was an independent prognostic factor for OS (relative risk = 3.90; P = 0.016) and that high KLK8-T4/KLK7, KLK8-T4/KLK10, and KLK8-T4/KLK11 mRNA ratios in NSCLC indicated increased risk of death. The increase was approximately 5-fold for the KLK8-T4/KLK7 and KLK8-T4/KLK10 ratios (P = 0.006, and P = 0.011, respectively) and 8-fold for the KLK8-T4/KLK11 ratio (P = 0.001). CONCLUSIONS The KLK8-T4 alternative splice variant, alone or in combination, may be a new independent marker of unfavorable prognosis in lung cancer.

Expression of the human kallikrein genes 10 (KLK10) and 11 (KLK11) in cancerous and non-cancerous lung tissues

Abstract Only one transcript for KLK10 was identified by RT-PCR in lung tissue, whereas KLK11 expressed at least four alternative transcripts. Quantitative analysis of KLK10 and KLK11 expression levels was assessed by real-time PCR, in a cohort of 47 patients with non-small-cell lung cancer (NSCLC). Expression levels of these genes were widely distributed in the population studied. Multivariate analysis revealed a correlation between KLK10 over-expression and the squamous cell carcinoma histotype (p=0.034). There was no correlation between gene expression and patient survival. Overall, both genes behaved similarly (p<0.001). These results suggest a co-regulation of KLK10 and KLK11 expression in lung and a lack of KLK10 suppressor role in NSCLC. Finally, our findings indicate that these genes are likely involved in normal physiology processes in bronchus.

Recombinant kallikrein expression: site-specific integration for hK6 production in human cells.

Abstract Kallikreins have been implicated in carcinogenesis and are promising biomarkers for the diagnosis and follow-up of various cancers. To evaluate the functions and clinical interest of kallikreins, it is important to be able to produce them as recombinant proteins. Here we summarize the various strategies used to produce kallikreins, emphasizing their advantages and limitations. We also describe an approach to achieve high-level production of kallikreins, such as hK6, with correct folding and activity, combining an expression system with targeted transgene integration and an efficient cultivation device to increase yield, the CELLine bioreactor. This novel method for recombinant kallikrein production will be useful to study their bio-pathological functions and to develop anti-bodies.

Kallikrein-Related Peptidase 6 Overexpression Promotes Non-Small Cell Lung Cancer Cell Proliferation and is associated with poor patient outcome

Introduction The human kallikrein-related peptidases (KLK) are a family of serine proteases that are often aberrantly expressed in common human malignancies and contribute to neoplastic progression through multifaceted roles. Methods We evaluated KLK6 expression in the tumoral and normal adjacent lung tissue of 56 patients with Non-Small Cell Lung Cancer (NSCLC) by real-time RT-PCR and immunohistochemistry. To determine the impact of KLK6 overexpression on the growth of lung cancer cells, we integrated the cDNA encoding the complete sequence of KLK6, through homolog recombination, in a NSCLC line (A549 Flp-In) and determined the growth rate of two independent clones. Progression of the KLK6- and parental cells inside the cell cycle was assessed by flow cytometry following synchronization of cells at the end of the G1 phase with starvation and hydroxyurea treatments. Key regulator proteins of the cell cycle were analyzed by Western blot in synchronized and unsynchronized cells. Results We found KLK6 transcript up-regulation in tumor tissues from patients with NSCLC and association of KLK6 status with low patient survival. KLK6 immunoreactivity was restricted to epithelial cells of normal bronchi and detected in most of cancer samples, in which KLK6 signal intensity correlated with well differentiated tumors. Ectopic expression of KLK6 dramatically enhanced NSCLC cell growth. Analysis of cell cycle progression revealed that promotion of cell growth caused by KLK6 results from an acceleration of cell cycle progression through G1/S transition, which was accompanied with a marked increase of cyclin E and repression of p21. In addition, expression of KLK6 in NSCLC cells was associated with an increase of c-Myc that is well-know to promote cell-cycle progression via regulation of cyclin D/E activation and down-regulation of p21. Conclusion ectopic expression of KLK6 facilitates cell cycle progression, certainly through alteration of c-Myc and downstream key regulators, and thus promotes cell proliferation. Moreover, KLK6 is overexpressed in NSCLC and associated with poor prognosis. Altogether, those findings suggest that KLK6 might play a central role in NSCLC development and progression.

059 Différentiel expression of the kallikrein 5 and kallikrein 7 gènes in non-small cell lung cancers subtypes

Introduction Human tissue kallikreins (hKs) are secreted serine proteases with diverse expression patterns and physiological roles. Several members of the kallikrein gene (KLK) family, which now includes 15 genes, are differentially expressed in various cancers and some emerge as useful diagnostic/ prognostic markers for hormone dependent cancers. Although clinically effective non-invasive meth-ods for early detection and screening of lung cancer are lacking, analysis of kallikrein gene expression has never been performed in lung. The aim of this study was to investigate KLK5 and KLK7 gene expression in non small-cell lung cancers (NSCLC). Methods We used both RT-PCR and Western blotting to analyse KLK5 and KLK7 gene expression and their respective products (hK5 and hK7) in lung. Gene expression was then quantified by real-time PCR in matched malignant and nonmalignant lung tissue samples obtained from 56 patients with NSCLC and results were compared to clinico-pathological parameters. Results We detected for the first time the expression of both KLK5 and KLK7 genes and their protein products (hK5 and hK7) in tumoral and nontumoral lung tissues. A significant increase in KLK5 expression was observed in squamous cell carcinoma (P = 0.02) compared to matched nontumoral tissue, whereas KLK7 expression was decreased in adenocarcinoma (P = 0.003). In non-cancerous tissues, multivariate analysis revealed a negative correlation between KLK5 expression and the metastatic status of the paired tumors (P = 0.05) and, a positive correlation between KLK7 expression and tumor differentiation (P = 0.02). In cancerous tissues, KLK5 and KLK7 expressions were mainly associated with squamous cell carcinoma (P = 0.04 and P = 0.01, respectively). Fur-thermore, KLK7 expression correlated positively with differentiation (P = 0.03) and negatively with adenocarcinoma histotype (P = 0.003). Conclusions Our results demonstrate that KLK5 and KLK7 are differentially expressed in NSCLC subtypes and indicate that the variability in KLK5 and KLK7 expressions hâve to be taken into account not only to better understand the lung tumorogenesis but also for clinical purposes.