High expression of apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3)

1. High expression of apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) in human cholangiocarcinoma Ph.D. student: Daraporn Chua-on Faculty of Associated Medical Sciences KhonKaen University, Thailand 25 May 2017

2. Worldwide incidence of CCA Worldwide incidence (cases per 100,000) of cholangiocarcinoma (CCA) The temporal trend of incidence (↑increasing trend; ↔ stable trend; ↓decreasing trend) Banales et al,. 2016

3. Diagnosis and therapy Surgical procedures : It is a potentially curative approach, which unfortunately have no improvement with long term survival. Chemotherapy : It is ineffective. Therefore, searching for the effective drugs with less adverse effect and high sensitivity to cholangiocarcinoma are needed. Biomarkers : No specific serum, urine, billiary or histological biomarkers are currently available for the diagnosis of CCA. Specific Biomarkers ?

4. Mitochondria http://www.dreamstime.com/royalty-free-stock-images-mitochondria-image14218389 • Mitochondrial defects contribute to the development of numerous human diseases including cancer (Taylor et al., Nat Rev Genet. 2005) • Mitochondrial deregulation plays a key role to trigger tumorigenesis and metastasis (Gogvadze et al., Trends Cell Biol. 2008)

5. In cholangiocarcinoma, Muisuk and colleagues (2015) explained that mitochondrial DNA (mtDNA) mutation may not significantly associate with carcinogenesis and tumor progression of CCA in the Northeast Thai population

6. Mitochondrial proteins • The subset of the 20,000 distinct mammalian proteins • Approximately 2,000 to 2,500 mitochondrial proteins • Approximately 1,100 of these • proteins have been identified to date http://www.plantmethods.com/content/2/1/8/figure/F4?highres=y

7. Hypothesis : CCA tissues have mitochondrial protein patterns that are different from non-cancerous tissues Purpose :To identify potential candidate proteins for CCA diagnosis using mitochondrial proteome analysis in CCA patients

8. Proteomic studies Mitochondrial isolation Methodology Data analysis and candidate protein selection Protein validation

9. Characteristic of papillary CCA and non-papillary CCA There is now increasing evidence that the genetic alterations and phenotypic changes are different between papillary-CCA and non-papillary CCA. Zen et al ,2006; Yoon, 2013

10. Mitochondria isolation and mitochondrial proteins separation Canc_nonpap Adj_nonpap Canc_pap Adj_pap a b

11. Heatmap of expression protein profile pattern of total 105 proteins 11

12. Identification of AIFM3 as a candidate protein a b 12

13. AIFM3 expression patterns in papillary CCA Adjacent Adjacent Papillary CCA Papillary CCA Expression patterns of AIFM3 in adjacent non-cancerous and cancerous tissues from papillary CCA, (magnification, x400). Scale bar is 50 µm. The AIFM3 expression pattern in papillary CCA was different when compared to its pair of adjacent non-cancerous tissues. 13

14. AIFM3 expression patterns in non-papillary CCA Non-papillary CCA Non-papillary CCA Adjacent Adjacent Expression patterns of AIFM3 in adjacent non-cancerous and cancerous tissues from non-papillary CCA, (magnification, x400). Scale bar is 50 µm. The AIFM3 expression pattern in non-papillary was different when compared to its pair of adjacent non-cancerous tissues. 14

15. Adjacent tissue of papillary CCA b a Papillary CCA c d Non-papillary CCA Adjacent tissue of non-papillary CCA 15

16. Interaction between AIFM3 and mitochondrial carcinogenesis-related proteins Interaction of AIFM3 and mitochondrial carcinogenesis-related proteins including Superoxide dismutase 2 (SOD2), NADPH oxidase 1 (NOX1), Serine hydroxymethyltransferase 2 (SHMT2), Complement component 1 (C1QBP), Ornithine aminotransferase (OAT), 3-oxoacid CoA transferase 1 (OXCT1), Anamorsin (CIAPIN1), Heat shock 70kDa protein 9 (mortalin or HSPA9) and Protein kinase C, alpha (PRKCA) 16

17. Apoptosis-inducing factor, mitochondrion- associated 3 (AIFM3) • - It is also called AIF-like (AIFL). • Mitochondrial flavoenzyme • 598 amino acids • The predictive structures of human AIFM3 revealed two regions: a Rieske domain induced apoptosis. Another domain is pyridine nucleotide-disulfide oxidoreductase domain (Pyr_redox) did not contribute to the pro-apoptotic function. Domain structure of AIFM3 protein Apoptotic activity Oxidoreductase activity (Non-apoptotic) Xie Q et al., J Biol Chem, 2005 17

18. AIF • Apoptotic activity • Regulation of the permeability of the mitochondrial membrane upon apoptosis • Caspase-independent cell death • Translocation to cytosol and nucleus for trigger DNA • Oxidoretuctase activity (play role in respiratory chain complex I for energy production) • Free radical scavenger • ROS production AIFM3 • Apoptotic activity • Caspase-dependent cell death • There is no translocation to cytosol and nucleus for trigger DNA • Oxidoretuctase activity Thus, AIF may be regulated the life-death balances may be rather specific for the cell type and death inducer. 18

19. Investigation of AIFM3 in serum by western blot a b • - 100 ug of serum sample • Dilution of antibody (AIFM3) 1:500 • Detection time Auto 2 min AIFM3 expressions were increased in CCA groups. 19

20. CONCLUSION • Expression patterns of mitochondrial proteome were different in two types of CCA when compared to their adjacent non-cancerous tissues. • Expression patterns of mitochondrial proteome were different between papillary CCA and non-papillary CCA. • Overexpression of AIFM3 wasfound in human CCA tissues and serum of CCA patients. AIFM3 could be a potential biomarker for CCA diagnosis. 20

21. Acknowledgement Advisor : Assoc. Prof. Dr. Siriporn Proungvitaya Co-advisors : Asst. Prof. Dr. Tanakorn Proungvitaya Dr. Anchalee Techasen Committee Prof. Dr. Temduang Limpaiboon Dr. Sittirak Roytrakul and Suthathip Kittisenachai for technical support on LC-MS/MS and mass data analysis (BIOTEC) Manuscript editing Asst.Prof.Dr.Tanakorn Proungvitaya, Dr.Anchalee Techasen, Prof.Dr.Temduang Limpaiboon, Dr.Sittiruk Roytrakul, Prof.Dr.Sopit Wongkham, Assoc.Prof.Dr.Chaisiri Wongkham, Ongart Somintara, Sakkarn Sungkhamanon, Asst.Prof.Dr.Siriporn Proungvitayaand Prof. Dr. Yukifumi Nawa

22. Thank you

23. -AIF is necessary for organizing and maintaining mitochondrial respiratory chain complex I. - Respiratory chain complex I are the primary site and the major site for ROS production of eukaryotic cells in disease state. Vahsen N et al., EMBO J, 2004

24. Conceptual framework Long time survival Chronic inflammation Cholangiocyte Cholangiocarcinoma Alterations of mitochondrial proteins -OV infection -Nitrosocompound -Carcinogens Mitochondrial protein patterns Comparison of proteome between CCA and non-cancerous tissues Important mitochondrial protein Targeted molecular therapy

25. Hypothesis : CCA tissues have mitochondrial protein patterns that are different from non-cancerous tissues Purpose :To identify potential candidate proteins for chemotherapy using mitochondrial proteome analysis for CCA tissues