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Title Page 1

ABSTRACT 3

Contents 7

Abbreviations 13

1. Introduction 14

1.1. Pancreatic cancer 14

1.1.1. Pancreatic cancer 14

1.1.2. Mutant Kras in pancreatic cancer 16

1.2. Mitophagy 18

1.2.1. Mitophagy 18

1.2.2. mt-Keima system 22

1.2.3. The role of mutant Kras 25

1.2.4. The role of mutant p53 26

1.2.5. The role of mitophagy in cancer 27

2. Purpose of this study 29

3. Materials and Methods 30

3.1. Cell lines and culture conditions 30

3.2. Antibodies and reagents 30

3.3. DNA Construct and siRNA Transfections 31

3.4. Generation of Pancreatic cancer model mouse 32

3.5. Hematoxylin and Eosin (H&E) staining and Immunohistochemistry (IHC) 33

3.6. Western blotting 34

3.7. Tissue collection and Cryosection 34

3.8. Quantification of mitophagy activity using mt-Keima 35

3.9. Statistical analysis 36

4. Results 37

4.1. Generation of Kras mutant cell lines and experimental scheme 37

4.2. Mitochondria mass decreases in inducible Kras mutant cell 41

4.3. Mitochondria mass decreases in inducible Kras mutant cell compared to when p53 is additionally knocked down 44

4.4. Mitochondrial length is more fragmented in Kras mutant cells compared to when p53 is additionally knocked down 46

4.5. Mitochondrial length is more fragmented in inducible Kras mutant cells compared to when p53 is additionally knocked down 51

4.6. Generation of stable mt-Keima cell lines 54

4.7. Mitophagy activity using mt-Keima system increases in Kras mutant cells but restores in additional knock down p53 57

4.8. In vivo mitophagy activity increases in pancreas from KC mice 59

4.9. Mitochondrial mass increases in pancreas from KPC mice compared to KC mice 64

5. Discussion 67

BIBLIOGRAPHY 70

List of Tables 10

Table 1. Genetically engineered mouse (GEM) models of pancreatic cancer to be used in this study 17

List of Figures 11

Figure 1. Pancreatic cancer progression model. Infiltrating cancer develops from normal duct epithelium (left to right) via a sequence of histologically identified... 15

Figure 2. An overview of the mitophagy process. Mitochondria dysfunctionality occur due to ROS, aging, and nutrient deficiency. Damaged mitochondria are then... 20

Figure 3. The molecular mechanism of mitophagy. Two primary pathways－PINK1-Parkin-dependent and PINK1-Parkin-independent mitophagy－make up... 21

Figure 4. The scheme of assessing mitophagy activity using mt-Keima reporter. A coral-derived fluorescent protein called Keima changes its color based on the... 24

Figure 5. Generation of stable Kras mutant cell lines (A, B) and experimental scheme (C). To generate a cell line expressing Kras G12D, lentivirus was created... 40

Figure 6. Mitochondria mass decreases in inducible Kras mutant cell but retores in additional knock down of p53 cell. (A) WT MEF expressing Tet-on Kras G12D,... 42

Figure 7. Mitochondiral length is reduced in Kras mutant cells, compare to WT cells. (A) HPNE cells stably expressing Kras G12D and additional p53 knock out... 50

Figure 8. Mitochondrial length is more fragmented in inducible Kras mutant cells compared to when p53 is additionally knocked down. (A) MEF expressing Tet-on... 52

Figure 9. Establishment of cell lines with stable expression of mt-Keima. (A) The fluorescent protein Keima has an excitation spectrum that changes according to... 56

Figure 10. Mitophagy activity increases in Kras mutant cell, but restores in additional p53 knock-down onto Kras mutation. (A) HPNE cells stably expressing... 58

Figure 11. Mitophagy activity in pancreas from KC mice increases but restores in KPC mice. (A) Breeding scheme to generate the genetically engineered mice for... 62

Figure 12. Mitochondria mass decreases in pancreas of KC mice but restores in the pancreas of KPC mice. (A) Representative images of tumor duct, CK19, and... 66

초록보기

 Mitophagy is a selective autophagy to remove the damaged mitochondria, which is critical for maintaining proper cellular functions such as regulating cellular energy homeostasis and cell death. In cancer progression mitophagy plays dual roles. Mitophagy acts as a tumor-suppressive role by eliminating dysfunctional mitochondria, leading to the prevention of genomic instability, oxidative stress, and apoptosis resistance. On the other hand, mitophagy tend to promote tumor growth and survival by fulfilling cellular energy requirements through effective elimination of dysfunctional mitochondria. While mitochondria play a critical role in cellular energy production and metabolic regulation, current understanding of mitophagy regulation at different stages of pancreatic cancer development remains limited.

To evaluate mitophagy activity effectively, a mitochondrial-targeted variant of Keima is employed, utilizing its pH-dependent fluorescence characteristics of Keima protein. Although previous reports reported relatively higher levels of autophagy activity in pancreatic cancers, it is still largely unknown whether a type of selective autophagy, mitophagy is changed on the distinct stages of pancreatic cancer development.

In this study, I investigated the alteration of mitochondria dynamics and mitophagy activities during cancer progression by using Kras mutant cells. Furthermore, a pancreatic cancer genetically engineered mouse model (GEM) harboring mt-Keima, a mitophagy reporter system, is generated to utilize for detecting mitophagy events within lysosomes during pancreatic cancer development in vivo and the levels of mitochondrial marker proteins in mutant Kras expressed cells is investigated.

Firstly, observations of mitochondrial mass in Kras activation demonstrated the levels of mitochondria marker decreased significantly in both Kras inducible and stable Kras expressing human pancreatic epithelial cells (HPNE). Next, to examine the effect of Kras on the mitochondrial degradation, the Kras mutant cell lines were established to harbor mt-Keima and utilized for measuring mitophagy activity in confocal microscope. The relative levels of mt-Keima red signal increases in mutant Kras expressed cells compared to the levels of normal cells, suggesting that mitophagy activity is also enhanced in the Kras mutation.

Moreover, the measurement of mitochondrial morphology in mutant Kras expressed cells showed increase of mitochondrial fragmentation rather than the length of mitochondria in normal cells. Interestingly, Kras-mediated mitochondria fragmentation restored in additional p53 knock down in Kras mutants.

Consistent of cellular results, mitophagy activity in vivo system utilizing mt-Keima significantly increased in pancreas from LSL- Krasᴳ¹²ᴰ., Pdx1-Cre (KC) mice, whereas it decreases in the pancreas of LSL- Krasᴳ¹²ᴰ, LSL-Trp53ᴿ¹⁷²ᴴ., Pdx1-Cre (KPC) mice, suggesting that mitophagy activity in pancreatic mouse model substantially enhanced in response to Kras mutation showing the early stage of pancreatic cancer progression.

Taken together, mitochondria morphology and degradation activity are dependent on the Kras mutant and p53 status. These results provide critical insights to the molecular roles of mitophagy in tumor development mediated with certain oncogenes or tumor suppressors. Furthermore, identifying novel target molecules involved in mitophagy regulation would be important for developing potential anticancer therapeutics in the future.

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