EXPRESSION AND EPIGENETIC REGULATION OF KALLIKREINS AND KININ RECEPTORS IN LUNG CARCINOMA CELLS Kanti Bhoola, Yee Yen Sia, Odette Shaw, Neil Misso & Philip Thompson Lung Institute of Western Australia, Centre for Asthma, Allergy & Respiratory Research, The University of Western Australia, Perth, Australia Results – expression of kallikreins, kininogen and kinin receptor proteins Results – epigenetic regulation of kallikrein-kinin genes Introduction Lung cancer is the leading cause of cancer-related death in men and there is a strong causal relationship between cigarette smoking and deaths from lung cancer. The pathology of lung cancer is a multi-step process, associated with multifocal metaplasia or dysplasia in the airways and there are a number of histological types of lung cancer: Squamous cell carcinoma (epidermoid and spindle) Adenocarcinoma (acinar, papillary, bronchoalveolar, solid muco-ca) Large cell carcinoma (giant cell, clear cell) Small cell carcinoma (oat cell, intermediate, combined oat-int.) Carcinoid tumours Tissue kallikrein (KLK1) and plasma kallikrein (KLKB1) are serine proteases that act on kininogens to produce biologically active kinin peptides. The cellular actions of the kinin peptides are mediated through kinin B1 and B2 receptors. The kallikrein-kinin cascade is implicated in tumorigenesis, angiogenesis, cell proliferation and metastasis. In cancers, expression of genes is repressed by promoter methylation and abnormal DNA patterns (epimutations) promote tumorigenesis. Epigenetic regulation involves the covalent methylation of CpG sites by DNA methyltranferases, a family of enzymes that methylate DNA at C-5 of cytosine. The methylated DNA is bound by methyl-binding proteins (MBP), which link methlyated DNA to the histone modifying enzymes. Expression of KKC genes following treatment with 25 and 6.25 µM of 5-Aza. Cell lines KLK1 KLKB1 BDKRB1 BDKRB2 KNG1 Cells incubated with 6.25 uM 5-Aza for 24 hours BEAS-2B 3.0 ↓ 1.4 ↑ 7.1 ↓ 7.8 ↓ Not detected H2126 1.3 ↑ 2.4 ↑ 2.0 ↓ 1.7 ↑ H520 3.4 ↑ 1.9 ↓ 1.8 ↑ 4.8 ↑ 8.0 ↑ Cells incubated with 6.25 uM 5-Aza for 24 hours and then cultured in normal medium for further 24 hours 1.1 ↑ 2.0↓ 2.9 ↓ 2.1 ↓ 1.5 ↓ 1.6 ↓ 7.5 ↓ 2 ↓ 9.5 ↓ 19.0 ↑ 11.0 ↓ 3.7 ↑ 3.0 ↑ Cell lines KLK1 KLKB1 BDKRB1 BDKRB2 KNG1 Cells incubated with 25 uM 5-Aza for 24 hours BEAS-2B 4.0 ↓ 1.1 ↑ 6.3 ↓ 2.6 ↓ Not detected H2126 1.3 ↓ 4.0 ↑ 2.8 ↓ 2.2 ↓ 1.2 ↑ H520 1.6 ↑ 5.3 ↑ 7.1 ↑ 45 ↑ 3.9 ↑ Cells incubated with 25 uM 5-Aza for 24 hours and then cultured in normal medium for further 24 hours 1.4 ↓ 1.1 ↓ 5.9 ↓ 4.4 ↓ 1.7 ↓ 4.6 ↓ 14.3 ↓ 16.0 ↑ 5.4 ↑ 11.1 ↑ 44 ↑ 8.3 ↑ DAB staining for tissue kallikiren (A, F, K), plasma kallikrien (B, G, L), B1 receptor (C, H, M), B2 receptor (D, I, N) and kininogen (E, I, O) in Beas 2B (A-E), H2126 (F-J) and H520 (K-O) cells. Scale bar is 25 µm Values are from one set of experiments performed in triplicate and indicate fold changes. Arrows signify increase or decrease in gene expression compared to control (acetic acid treated) cells 1 1 2 M CpG islands in the kallikrein-kinin cascade genes 2 KLK1 gene Human Chromosome 19q13.3 (4640 bp)(56014216 – 56018855) KLKB1 gene Human Chromosome 4q34-35 (30954 bp)(187385666 – 187416619) BDKRB1 gene Human Chromosome 14q32.1-q32.2 (8542 bp)(95792312 – 95800853) BDKRB2 gene Human Chromosome 14q32.1-q32.2 (39593 bp)(95740950 – 95780542) Immunofluorescence labelling for plasma kallikrein (1) and tissue kallikrein (2) in Beas 2B (A-E), H2126 (F-J) and H520 (K-O). Cells were grown in normal media (A, F, K), media containing acetic acid (B, G, L), media containing 25 µM 5-aza (C, H, M), media containing 12.5 µM 5-aza (D, I, N) or media containing 6.25 µM 5-aza (E, I, O) for 24 h prior to staining. Scale bar is 50 µm 3 1 2 Change in KKC gene expression for Beas 2B (1), H2126 (2) and H520 (3) cells. Graphs show one set of experiments performed in triplicate. Bars indicate fold increase or decrease compared to control (acetic acid treated) cells. Cells were treated with 25 µM or 6.25 µM Aza for 24 h, then either harvested or cultured in normal media for a further 24 h before RNA extraction and real-time PCR. KNG1 gene Human Chromosome 3q27 (26624 bp)(187917814 – 187944437) Immunofluorescence labelling for B1 receptor (1) and B2 receptor (2) in Beas 2B (A-E), H2126 (F-J) and H520 (K-O). Cells were grown in normal media (A, F, K), media containing acetic acid (B, G, L), media containing 25 µM 5-aza (C, H, M), media containing 12.5 µM 5-aza (D, I, N) or media containing 6.25 µM 5-aza (E, I, O) for 24 h prior to staining. Scale bar is 50 µm Conclusions Aims The KKC proteins are differentially expressed in normal lung and lung carcinoma cells. Expression of the KKC proteins in normal lung epithelial cells support their presence in tumours of the lung. Higher concentraions of 5-Aza caused marked down-regulation of KLK1, BDKRB1 and BDKRB2 mRNA. This was reversed in a dose-dependent manner, and with 6.25 µM 5-Aza the decrease in mRNA expression was minimal. Explanations: 1) suppression of transcription factors; 2) remethylation by RNAi machinery Suppresion of the KLK1, BDKRB1 and BDKRB2 genes by 5-Aza in lung cancer cells suggests that demethylating drugs may be beneficial in the treatment of solid tumours. KLK1 is probably an oncogene that transforms normal cells and increases tumour invasiveness and metastasis. Increased expression of KLKB1 in 5-Aza treated lung cancer cells suggests a critical role for this gene in the formation of new blood vessels in human lung cancers. Detection of methylated DNA in plasma of cancer patients may provide a non-invasive means of diagnosis or monitoring response to treatment. 1 1. To assess the expression of tissue kallikrein (KLK1, TK, TproK), plasma kallikrein (KLKB1, PPK, PK11) and kinin receptor (B1R, B2R) proteins in normal lung epithelial cells (Beas 2B) and lung adenocarcinoma (H2126) and squamous carcinoma (H520) cells 2. To investigate the expression of tissue kallikrein (KLK1), plasma kallikrein (KLKB1), kininogen (KNG1) and kinin receptor (BDKRB1, BDKRB2) mRNA and protein, following inhibition of DNA methylation