在癌症患者中具有高侵襲性的腫瘤之惡性程度與病理組織分化具有關聯。然而，結腸直腸癌病理組織分化與癌細胞幹性之間的聯繫仍然不清楚。DNA結合與分化抑制因子(Inhibitors of DNA binding and cell differentiation, ID)家族蛋白已知能夠調節細胞分化和腫瘤進程。在本研究中，我們發現 ID4 的表達與結腸癌幹細胞中幹性標誌物 LGR5 的表達呈正相關，但其他 ID 的表達並非如此。ID4的高表達與晚期結腸直腸癌腫瘤的病理分化有關，並能夠預測結腸直腸癌患者的生存預後較差。我們發現腦源性神經營養因子 (Brain Derived Neurotrophic Factor, BDNF)為結腸直腸癌中 ID4 的下游靶標。 ID4 調節 BDNF 表達以維持結腸癌細胞的存活和傳播。 ID4 或 BDNF的抑制降低癌細胞生長能力，同時減少轉移能力並伴隨著細胞凋亡增加。 BDNF的過度表達增強了結腸癌細胞的失巢凋亡之抵抗性和轉移能力。此外，我們觀察到 ID4-BDNF 信號調節結腸癌細胞的上皮間質細胞轉化 (Epithelial Mesenchymal Transition, EMT) 和化療抗性。間質細胞標記物Vimentin、CDH2 和 SNAI1 在 ID4 或 BDNF 抑制的細胞中表現量降低。 ID4 的抑制則增強了結腸癌細胞的化療敏感性，BDNF 過度表達使細胞具有高化療抗性。我們的研究結果提供新的ID4-BDNF 信號路徑對於腫瘤進程的致癌能力，因此可作為結腸直腸癌的預後標誌物和治療的靶點。

The poor pathological differentiation has been linked to tumor malignancy with a high potential for aggressiveness and invasiveness in cancer patients. However, the connection between pathological differentiation and cancer stemness in colorectal cancer is still elusive. Inhibitors of DNA binding and cell differentiation (ID) proteins regulate cellular differentiation and tumor progression. In this study, we found ID4 expression, but not that of other ID members, was positively correlated with the expression of stemness marker LGR5 in colon cancer stem cells. Its high expression was associated with advanced pathological differentiation of colorectal tumors and predicted poor survival in colorectal cancer patients. We identified brain-derived neurotrophic factor (BDNF) as a downstream target of ID4 expression in colorectal cancer. ID4 regulated BDNF expression to maintain the survival and dissemination of colon cancer cells. Knockdown of either ID4 or BDNF attenuated the colony-forming ability and decreased migration accompanied by increased apoptosis. BDNF expression enhanced anoikis resistance and migration of colon cancer cells. In addition, we observed that ID4-BDNF signaling regulated epithelial-to-mesenchymal transition (EMT) pattern and chemoresistance in colon cancer cells. Mesenchymal markers Vimentin, CDH2, and SNAI1 were suppressed in ID4- or BDNF-silenced cells. ID4 knockdown enhanced chemosensitivity of colon cancer cells, and BDNF overexpression endowed cells with high chemoresistant ability. Our findings provided novel insights into the oncogenic role of ID4-BDNF signaling in colorectal cancer, and suggested molecules of ID4-BDNF signaling could serve as prognostic markers and therapeutic target in colorectal cancer.

ABSTRACT 2
ACKNOWLEDGEMENTS 4
TABLE OF CONTENTS 6
LIST OF FIGURES 8
LIST OF TABLES 10
LIST OF ABBREVIATIONS 11
CHAPTER 1. INTRODUCTION 12
1.1. The overview of colorectal cancer 12
1.2. The overview of the inhibitors of DNA binding and cell differentiation 15
1.3. The overview of brain-derived neurotrophic factor 17
1.4. The overview of leucine-rich repeat-containing G-protein-coupled receptor 5 in colorectal cancer 18
1.5. The overview of epithelial-mesenchymal transition in colorectal cancer 21
1.6. Aims of study 22
CHAPTER 2. MATERIALS AND METHODS 23
2.1. Cell lines 23
2.2. Plasmids 23
2.3. Quantitative real-time polymerase chain reaction 24
2.4. Clonogenic assay 25
2.5. Spheroid formation assay 26
2.6. Cell tracking assay 26
2.7. Immunoblotting 26
2.8. Immunohistochemistry staining 27
2.9. Enzyme-linked immunosorbent assay 28
2.10. Cell apoptosis assay 29
2.11. Cell cycle assay 29
2.12. Statistical analysis 29
CHAPTER 3. RESULTS 31
3.1. ID4 expression correlates with advanced grade and poor survival of colorectal cancer 31
3.2. ID4 is essential for the maintenance of growth and dissemination in colon cancer cells 38
3.3. ID4 induces BDNF to promote the growth and migration of colon cancer cells 44
3.4. ID4-BDNF signaling regulates the EMT, dissemination and chemoresistance of colon cancer cells 49
CHAPTER 4. DISCUSSION 58
4.1. Conclusion 58
4.2. Discussion 59
4.3. Novelties of study 62
4.4. Limitations and future works 62
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