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

2. Worldwide incidence of CCA
The risk factors of cholangiocarcinoma in this region have been reported to be associated with infection of liver fluke, Opisthorchis viverrini from eating uncooked food and poor sanitation practice. At present, opisthorchiasis-associated CCA is still serious public health problem in this region.
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 Specific Biomarkers ?
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.
Although, Surgery to completely remove the cancer is the only possible curative treatment for CCA, it will be effectively based on tumor type and tumor stage. In addition, the chemotherapy is ineffective and there are no specific tumor markers that can indicate the early stages and status of CCA. Diagnostic biomarker of cholangiocarcinoma (CCA) patients is absolutely poor in spite of extensive efforts for the improvment of diagnosis.
Biomarkers : No specific serum, urine, billiary or histological biomarkers are currently available for the diagnosis of CCA.
Specific Biomarkers ?

4. Mitochondria
Mitochondria are considered as targeted organelle for cancer therapy. Mitochondria contain five distinct parts including the outer mitochondrial membrane, the intermembrane space, the inner mitochondrial membrane, the cristae space, and the matrix. Moreover, they posses their own circular genome with mitochondria specific transcription ,translation and protein assembly systems. Mitochondria are the important organelles that involved in many cellular process such as oxidative phosphorylation, apoptosis, redox, Ca2+ regulation. Several studies have been reported that mitochondrial defects contribute to the development of numerous human diseases including tumorigenesis and metastasis . Therefore, it implies that the alteration of mitochondrial-related proteins a is significantly contributes to cancer development. In cancer, mitochondria disorder as a result of several factors both genetic and protein level was observed.
Mitochondria are considered as the target organelles for discovering biomarkers of several cancers including CCA.
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
Recently,…………………………………. Since, mtDNA is not final gene product that expressed many functions in biological system.
Thus, study of protein level may be other ways lead to understanding role of carcinogenesis in CCA.

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

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
..ต่อกรอบแรก in both papillary and non-papillary CCA.

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

9. Characteristic of papillary CCA and non-papillary CCA
Papillary proliferation in the bile duct lumen (Intraductal growth type)
Other types were not intraductal papillary growth such as mass-like, periductal infiltrating, hilar
Less invasive
High invasive
Well-differentiated
Poorly-differentiated
Less common (Rare cancer)
Most common
Good prognosis
Poor prognosis
Good indicator for surgical resection
Poor indicator for surgical resection
Long-term survival of patients
Short-term survival of patients
Other histologic types of CCA included 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
Canc_pap
Adj_nonpap
Adj_pap a b
Successful separation of both fractions was validated by western blot analysis using antibody against mitochondria-specific marker protein. VDAC (Fig. 1a) shows strongly positive reaction in mitochondrial fractions but negative in cytosolic fractions. After dividing into 12 ranges according to the molecular size, each gel plug was digested in-gel. The extracted tryptic peptides were subjected to LC-MS/MS analysis. The differentially expressed peptides data obtained from DecyderMS software were searched against the NCBI human mitochondria database for protein identification.

11. 11 Heatmap of expression protein profile pattern of total 105 proteins
In total, 281 mitochondrial proteins were identified. Using the MultiExperiment Viewer (MeV, version 4.6.1) software, 105 proteins were selected based on statistically significant (P < 0.05) and protein expression levels as shown in the heatmap (Fig. 2). 11

12. Identification of AIFM3 as a candidate protein
show that 5 and 7 proteins are over-expressed in papillary and non-papillary CCA, respectively. However, only one protein was commonly found in both papillary and non-papillary type CCA tissues, but not in the adjacent tissues (Fig. 3a). This protein was identified as AIFM3. Western blot analysis of the mitochondrial solutions of the 3 cases each of papillary and non-papillary CCA cases revealed that AIFM3 protein was uniquely expressed in all 3 CCA tissues, but not in the corresponding adjacent non-cancerous tissues b 12

13. AIFM3 expression patterns in papillary CCA
Adjacent
Papillary CCA
Adjacent
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
Adjacent
Non-papillary CCA
Adjacent
Non-papillary CCA
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 Adjacent tissue of non-papillary CCAb c d
To investigate the expression of AIFM3 in clinical specimens, apart from 3 cases each of papillary and non-papillary CCA used for mitochondrial extraction, 25 more human CCA and their corresponding adjacent non-cancerous tissues were examined by immunohistochemistry. The results show that H-scores of the AIFM3 staining were significantly higher in CCA than in the corresponding adjacent non-cancerous tissues. 15

16. Interaction between AIFM3 and mitochondrial
Interaction between AIFM3 and mitochondrial carcinogenesis-related proteins
To speculate the potential role of AIFM3 in CCA-genesis, possible interaction of AIFM3 and mitochondrial carcinogenesis-related proteins were predicted by STITCH version 4.0 (Fig. 5). The results show that 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) were identified as proteins interacting with AIFM3
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 AIFM3 18 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
However, the structure and the role of AIFM3 has not been fully elucidated. These should be further investigated.
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
- 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 was found 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 Proungvitaya and Prof. Dr. Yukifumi Nawa
Before I finish my talk, I would like to thanks my colleague Asst.Prof.Dr. Siriporn Proungvitaya, Asst.Prof.Dr. Tanakorn Proungvitaya, Dr.อัญชลี เตชะเสน and Prof. Dr. Temduang Limpaiboon , And a few consultant to help me in my laboratory.

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
Chronic inflammation
Cholangiocarcinoma
Alterations of mitochondrial proteins
-OV infection
-Nitrosocompound
-Carcinogens
Mitochondrial protein patterns
Comparison of proteome between CCA and non-cancerous tissues
The conceptual framework of this research. After Ov infection in combination with other risk factors causing chronic inflammation and finally cholangiocarcinoma developed which may lead to the alteration of mitochondrial proteins. In this research, I focus only investigation of mitochondrial protein patterns and compared different patterns between CCA and non-cancerous tissues.
if I could find the important mitochondrial protein that may be overexressed or exclusively expressed in CCA, I may use it as a molecular target for CCA therapy leading to long time survival of patients.
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