1. EXPRESSION AND DEMETHYLATION OF KALLIKREIN AND KININ RECEPTOR KALLIKREIN AND KININ RECEPTOR GENES IN LUNG CARCINOMA CELLS GENES IN LUNG CARCINOMA CELLS Kanti Bhoola, Yeeyen Sia, Odette Shaw, Neil Misso & Philip Thompson. Neil Misso & Philip Thompson. Lung Institute of Western Australia, Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, Perth, Australia

2. Kallikreins Tissue (hK1) and plasma (hKB1) kallikreins are serine proteases that release bradykinin and lys-bradykinin from H- and L-kininogens. TK (hK1) is implicated in carcinogenesis through the induction of gene expression. Kinins Kinins act through B1 and B2 receptors, which are seven transmembrane receptors coupled to G proteins. Kinins are mitogenic and enhance the proliferation of tumour cells. Dimeric kinin receptor antagonists have been shown to cause apoptosis of cancer cells through a novel mechanism. Kallikrein- Kinin Cascade INTRODUCTION

3. Epidemiology of lung carcinoma: ♦ Leading cause of cancer death in men ♦ Strong causal relationship between cigarette smoking and deaths from lung cancer (Doll et.al. 2004) ♦ A multi-step process, associated with multifocal metaplasia or dysplasia in the airways – field carcinogenesis Pathology: ♦ The kallikrein-kinin cascade is implicated in tumorigenesis, angiogenesis, cell proliferation and metastasis. Biology

4. Histological Classification of lung cancers 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

5. proKLK1 in lung carcinomas- Confocal microscopy images: (A) Adenocarcinoma; (B) Squamous cell carcinoma; (C)Large cell carcinoma; (D) Small cell carcinoma; (E) Lung carcinoid; (F) Salivary gland (Positive control); (G) (primary antibody omitted, greyscale image).

6. preKLKB1 in lung carcinomas- Confocal microscopy images: ( A) Adenocarcinoma; (B) Squamous cell carcinoma; (C); Large cell carcinoma; (D) Small cell carcinoma; (E) Lung carcinoid; (F) Salivary gland (Positive control); (G) Primary antibody omitted, greyscale image).

7. B1R of in lung carcinomas- Confocal microscopy images: (A) Adenocarcinoma; (B) Squamous cell carcinoma; (C) Large cell carcinoma; (D) Small cell carcinoma; (E) Lung carcinoid; (F) Salivary gland (Positive control); (G) Primary antibody omitted, greyscale image).

8. B2R of in lung carcinomas- Confocal microscopy images: (A) Adenocarcinoma; (B) Squamous cell carcinoma; (C) Large cell carcinoma; (D) Small cell carcinoma; (E) Lung carcinoid; (F) Salivary gland (Positive control); (G) Primary antibody omitted, greyscale image.

9. What is? Epigenomics and Epigenetics Epigenomicsgenome-wide approach to ■- Epigenomics: genome-wide approach to study epigenetics. Central goal is to define study epigenetics. Central goal is to define the DNA sequence features that direct the DNA sequence features that direct epigenetic processes epigenetic processes Epigenetics:Inheritable changes in gene ■- Epigenetics: Inheritable changes in gene expression that cannot be attributed to expression that cannot be attributed to changes in DNA sequence changes in DNA sequence Significance: Epigenetic and genetic attributes- ■- Significance: Epigenetic and genetic attributes- Functionally: These changes dominate the Functionally: These changes dominate the growth of cancer and embryonic stem cells growth of cancer and embryonic stem cells

10. Epigenetic regulation involves the covalent methylation of CpG sites by mehyltransferases and deacetylation of of CpG sites by mehyltransferases and deacetylation of by histone modifying enzymes. by histone modifying enzymes. DNA methyltranferases are a family of enzymes that methylate DNA at C-5 of cytosines. methylate DNA at C-5 of cytosines. The methylated DNA is then bound by methyl-binding The methylated DNA is then bound by methyl-binding proteins (MBP), which links methly DNA to the histone proteins (MBP), which links methly DNA to the histone modifying enzymes, histone deacetylases (HDAC)& modifying enzymes, histone deacetylases (HDAC)& histonemethyltranferases (HMT). The HDAC and HMT histonemethyltranferases (HMT). The HDAC and HMT covalently modify H4K16 and H3K9 respectively. covalently modify H4K16 and H3K9 respectively. In cancers, expression of genes is repressed by promoter methylation and abnormal DNA patterns (epimutations) methylation and abnormal DNA patterns (epimutations) promote tumourignesis. promote tumourignesis.

11. Diagrams showing CpG islands in the kallikrein-kinin cascade genes :

12. EXPRESSION AND DEMETHYLATION OF KALLIKREIN AND KININ RECEPTOR GENES IN LUNG CARCINOMA CELLS Kanti Bhoola, Yeeyen Sia, Odette Shaw, Neil Misso & Philip Thompson. Lung Institute of Western Australia, Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, Perth, Australia KLK1 gene Human Chromosome 19q13.3 (4640 bp) (56014216 – 56018855) KLKB1 gene Human Chromosome 4q34-35 (30954 bp) (187385666 – 187416619)

13. BDKRB1 gene Human Chromosome 14q32.1-q32.2 (8542 bp) (95792312 – 95800853) BDKRB2 gene Human Chromosome 14q32.1-q32.2 (39593 bp) (95740950 – 95780542)

14. KNG1 gene Human Chromosome 3q27 (26624 bp) (187917814 – 187944437)

15. Results – expression of kallikreins, kininogen and kinin receptor proteins

16. To assess the expression of tissue kallikrein (KLK1, TK), ♦ To assess the expression of tissue kallikrein (KLK1, TK), plasma kallikrein (KLKB1) and kinin receptor (B1R, B2R) plasma kallikrein (KLKB1) and kinin receptor (B1R, B2R) proteins in normal lung epithelial cells (Beas-2b) and proteins in normal lung epithelial cells (Beas-2b) and in lung cancer cell lines ( H2126- adenoocarcinoma and in lung cancer cell lines ( H2126- adenoocarcinoma and H520- Sqamous cell carcinoma) H520- Sqamous cell carcinoma) To investigate the expression of tissue kallikrein (KLK1), ♦ To investigate the expression of tissue kallikrein (KLK1), plasma kallikrein (KLKB1), kininogen (KNG1) and kinin plasma kallikrein (KLKB1), kininogen (KNG1) and kinin receptor (BDKRB1, BDKRB2) mRNA and protein, receptor (BDKRB1, BDKRB2) mRNA and protein, following inhibition of DNA methylation following inhibition of DNA methylation Aims

17. Expression of KKC genes with a different incubation period design using 25 and 6.25 uM of 5-Aza. One set of separate experiments performed in triplicate and are represented as fold change. Arrows signify increase or decrease in the gene expression when compared to control (corresponding acetic acid treated cells)

18. Cell linesKLK1KLKB1BDKRB1BDKRB2KNG1 25 uM 5-Aza treated cells, incubated for 24 hours BEAS-2B4.0 ↓1.1 ↑6.3 ↓2.6 ↓Not detected H21261.3 ↓4.0 ↑2.8 ↓2.2 ↓1.2 ↑ H5201.6 ↑5.3 ↑7.1 ↑45 ↑3.9 ↑ 25 uM 5-Aza treated cells, only normal medium replaced for a further 24 hours BEAS-2B1.4 ↓1.1 ↓1.2 ↑5.9 ↓4.4 ↓ H21262.2 ↓1.3 ↓1.7 ↓4.6 ↓14.3 ↓ H52016.0 ↑5.4 ↑11.1 ↑44 ↑8.3 ↑ 6.25 uM 5-Aza treated cells, incubated for 24 hours BEAS-2B3.0 ↓1.4 ↑7.1 ↓7.8 ↓Not Detected H21261.3 ↑2.4 ↑2.0 ↓1.3 ↑1.7 ↑ H5203.4 ↑1.9 ↓1.8 ↑4.8 ↑8.0 ↑ 6.25 uM 5-Aza treated cells, with 5-Aza incubated for a further 24 hours BEAS-2B1.1 ↑2.0↓2.9 ↓2.1 ↓Not detected H21261.5 ↓1.6 ↓7.5 ↓2 ↓9.5 ↓ H520 19.0 ↑11.0 ↓1.7 ↑3.7 ↑3.0 ↑

19. Expression of KLK1, KLKB1, BDKRB1, BDKRB2, KNG1 genes in lung normal and cancer cells treated with two different concentrations of 5- azacytidine. Bees-2b

20. H2126 Lung Adenocarcinoma

21. H520 Squamous lung cell carcinoma

22. Cytotoxicity effect on normal lung epithelial cells assessed by cell proliferation assay after treatment with 5-AZA by cell proliferation assay after treatment with 5-AZA Bees-2b

23. Cytotoxicity effect onlung cancer cells assessed by cell proliferation assay after treatment with 5-AZA H2126

24. Cytotoxicity effect on lung cancer cells assessed by cell proliferation assay after treatment with 5-AZA by cell proliferation assay after treatment with 5-AZA H520

25. B1 B2 PK TKBees2A

26. PK TK B1 B2 H2126

27. BEAS-2B

28. B(H2126)

29. C) H520

30. The KKC proteins are differentially expressed in normal epithelial (Bees- 2A) and adeno-(H2126) and squamous-(H520) carcinoma cells This study adds further credence to the proposed tumourigenic role of the KKC proteins in human lung cancers. Higher concentraions of 5-AZA caused marked reduction in the expression of mRNA for KLK1, BDKRB1 and BDKRB2. This was reversed in a dose-dependent manner, and with 6.25 µM 5-AZA the reduction in mRNA expression was minimal.Exp: 1- suppression of transcription factors 2- remethylation by RNAi machinery Higher concentraions of 5-AZA caused marked reduction in the expression of mRNA for KLK1, BDKRB1 and BDKRB2. This was reversed in a dose-dependent manner, and with 6.25 µM 5-AZA the reduction in mRNA expression was minimal. Exp: 1- suppression of transcription factors 2- remethylation by RNAi machinery KLK1 is most probably an oncogene. Suppresion of the KLK1, BDKRB1 and BDKRB2 genes by 5-AZA in lung cancer cells suggests that trials with demethylating KLK1 is most probably an oncogene. Suppresion of the KLK1, BDKRB1 and BDKRB2 genes by 5-AZA in lung cancer cells suggests that trials with demethylating drugs may be beneficial in the treatment of solid tumours DNA methylation patterns appear to be increasingly important in the therapeutic management of cancer patients. The detection of methylated DNA in plasma provides a non-invasive means of diagnosis or monitoring response to treatment Increased expression of KLKB1 in 5-AZA treated mesothelioma cells suggests a critical role for this gene in the formation of new blood vessels in human lung cancer.