自2016年以來，結直腸癌在癌症死亡率居高不下，排名第三。此外，有轉移的患者比例超過80％，這增加了復發的可能性和預後不良。近年來，結直腸癌的研究從未停止過，但仍有許多未知之處，包括腫瘤微環境及糖代謝與結直腸癌轉移之間的相互作用。
我們透過結直腸癌臨床樣本的微陣列與臨床數據庫進行比較選擇了兩種與糖代謝相關的蛋白，SLC16A4（Solute carrier family 16 member 4）和ALDOB（Aldolase B），並過度表達它們作為我們的實驗模型。 在腫瘤增長過程中，還會伴隨著結締組織異常增生，並改變細胞表現。研究中，用低氧環境和聚丙烯酰胺凝膠（硬度~8.64 kPa）模擬結直腸癌微環境，從葡萄糖攝取、細胞內訊號調控、糖代謝及細胞骨架分布進而到細胞牽引力和集結能力的現象研究代謝與機械轉導之間的機制。HCT-116細胞中，葡萄糖攝取能力隨基質硬度提升細胞骨架排列穩定而下降，並由糖解及有氧呼吸維持細胞能量水平 其與胞內FAK/PI3K/Rho訊傳調控葡萄糖之攝取有關；當ALDOB的過度表達，可維持高度的糖解作用能力，並隨著基質硬度上升而下調粒線體功能，並同時調節葡萄糖攝取來使細胞內維持能量水平，以應付不同環境的差異。而SLC1616A4和ALDOB均促進糖解作用的發生，而有相似的趨勢。由本實驗證實，當ALDOB與SLC16A4上調時，改變糖解與有氧呼吸之平衡，且提升對缺氧及基質硬度較的敏感性，且在缺氧及大腸腫瘤微環境下，隨時間所觀察到其細胞牽引力下降，可能與細胞轉移能力的提升有關。提升微環境的硬度伴隨細胞骨架的重組誘導腫瘤細胞轉移，並重塑糖解及有氧呼吸的平衡。
研究中，我們從細胞力學的角度探討了結直腸癌代謝機制與機械傳導的關係，藉以增加對大腸直腸癌之了解，最終希望以減少結直腸癌的復發和死亡率，改善預後。

Colorectal cancer (CRC) is one of the most common cancer worldwide, and various studies have been explored the underlying mechanism of CRC progression. However, the interaction between microenvironment, metabolism and colorectal cancer metastasis is still largely unknown. CRC progression is highly associated with desmoplasia, which composed of a hypoxia and stiff ECM microenvironment. In this study, we selected two metabolic proteins, SLC16A4 (Solute carrier family 16 member 4) and ALDOB (Aldolase B), as our experiment model, and to investigate the CRC metabolism reprogramming in vitro.
In the engineered desmoplastic microenvironment, using hypoxia environment and polyacrylamide gel (1.4~8.64 kPa), we observed that glucose uptake of HCT-116 decreased when stiffness increased, and SLC16A4- and ALDOB- overexpressed cells showed significantly increasing glucose uptake under tumor-like stiffness (8.64 kPa). In ALDOB up-regulate cells, glycolysis ability stayed in high level, but mitochondrial respiraiton ability decreased. Our data showed both FAK/PI3K/HIF and FAK/Rho signal pathways involved in the metabolic shift, and the actin dynamic was associated as well. Moreover, SLC16A4 and ALDOB can even change the response of CRC traction force, which related to tumour metastasis, to hypoxia/normoxia.

In summary, our data implied a counter influence between hypoxia and substrate stiffness on CRC glucose uptake, and the SLC16A4 and ALDOB upregulation can interfere this balance, which drive aerobatic glycolysis synergistically. Our results not only highlight the potential metabolic reprogramming leads by physical alterations in desmoplastic microenvironment, but also extend our understand about the essential role of SLC16A4 and ALDOB in CRC progression from biophysical perspective.

摘要 1
Abstract 2
Table of contents 3
List of Table and Figure 5
Chapter 1 Introduction 6
1.1 Colorectal cancer 6
1.2 The interactions between tumor microenvironment stiffness and the colorectal cancer cells 6
1.3 The relation between physical microenvironment and metastasis of the colorectal cancer cells 8
1.4 Cytoskeleton assembly related to microenvironment alteration and ATP regulation of the colorectal cancer cells 8
1.5 Glucose metabolism in colorectal cancer cells 9
1.6 The role of SLC16A4 and ALDOB in ATP production of colorectal cancer 10
1.7 Motivation and Purpose 11
Chapter 2 Materials and Methods 12
2.1 Cell culture 12
2.2 Preparation of hydrogel substrate 12
2.3 Hypoxia environment preparation 13
2.4 Quantitative real-time polymerase chain reaction (qPCR) 13
2.5 Glucose uptake staining assay 14
2.6 Seahorse assay 14
2.7 Actin staining assay 15
2.8 Traction force microscopy assay 15
2.9 Cluster formation 16
2.10 Data statistics 16
Chapter 3 Results and Discussion 17
3.1 SLC16A4 and ALDOB in Clinical dataset analysis 17
3.2 Glucose uptake of colorectal cancer cells 19
3.4 Metabolism of colorectal cancer 24
3.4.1 Glycolysis ability of HCT-116/SLC16A4/ALDOB 24
3.4.2 Mito stress of HCT-116/SLC16A4/ALDOB 27
3.3 Signal pathway in colorectal cancer cells 31
3.3.1 FAK/PI3K signal pathway by qPCR 31
3.3.2 FAK/Rho signal pathway by qPCR 33
3.5 Colorectal cancer cells mechanobiological behavior 35
3.5.1 Cytoskeleton distribution of colorectal cancer cells 35
3.5.3 Cluster and spheroid formation of colorectal cancer cells in hypoxia environment 41
3.5.2 Traction force of colorectal cancer cells 44
Chapter 4 Conclusion 47
References 49

1. Cunningham, D., et al., Colorectal cancer. The Lancet, 2010. 375(9719): p. 1030-1047.
2. Fedirko, V., et al., Alcohol drinking and colorectal cancer risk: an overall and dose–response meta-analysis of published studies. Annals of Oncology, 2011. 22(9): p. 1958-1972.
3. Itzkowitz, S.H. and X. Yio, Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2004. 287(1): p. G7-G17.
4. Lozano, R., et al., Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The Lancet, 2012. 380(9859): p. 2095-2128.
5. Merika, E., et al., Colon cancer vaccines: an update. In vivo, 2010. 24(5): p. 607-628.
6. Triantafillidis, J.K., G. Nasioulas, and P.A. Kosmidis, Colorectal cancer and inflammatory bowel disease: epidemiology, risk factors, mechanisms of carcinogenesis and prevention strategies. Anticancer research, 2009. 29(7): p. 2727-2737.
7. Valtin, H., “Drink at least eight glasses of water a day.” Really? Is there scientific evidence for “8 × 8”? American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2002. 283(5): p. R993-R1004.
8. Watson, A.J. and P.D. Collins, Colon cancer: a civilization disorder. Digestive diseases, 2011. 29(2): p. 222-228.
9. Johnson, L.A., et al., Matrix stiffness corresponding to strictured bowel induces a fibrogenic response in human colonic fibroblasts. Inflammatory bowel diseases, 2013. 19(5): p. 891-903.
10. 田中昌彦, et al., ヒト大腸癌の進展に伴う細胞外基質の相互作用の変化 とくに脈管侵襲および壁深達度との関連について. 日本大腸肛門病学会雑誌, 1994. 47(1): p. 48-58.
11. Kawano, S., et al., Assessment of elasticity of colorectal cancer tissue, clinical utility, pathological and phenotypical relevance. Cancer Science, 2015. 106(9): p. 1232-1239.
12. Marin-Hernandez, A., et al., Hypoglycemia Enhances Epithelial-Mesenchymal Transition and Invasiveness, and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and Microspheroids. J Cell Physiol, 2017. 232(6): p. 1346-1359.
13. Gómez‐Lechón, M.J., L. Tolosa, and M.T. Donato, Metabolic activation and drug‐induced liver injury: in vitro approaches for the safety risk assessment of new drugs. Journal of Applied Toxicology, 2016. 36(6): p. 752-768.
14. Bernstein, B.W. and J.R. Bamburg, Actin-ATP hydrolysis is a major energy drain for neurons. Journal of Neuroscience, 2003. 23(1): p. 1-6.
15. Christofori, G., New signals from the invasive front. Nature, 2006. 441(7092): p. 444.
16. Ganz, A., et al., Traction forces exerted through N‐cadherin contacts. Biology of the Cell, 2006. 98(12): p. 721-730.
17. Lu, P., V.M. Weaver, and Z. Werb, The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol, 2012. 196(4): p. 395-406.
18. Plikus, M.V., et al., Regeneration of fat cells from myofibroblasts during wound healing. Science, 2017. 355(6326): p. 748-752.
19. Polackwich, R.J., et al., Traction force and tension fluctuations in growing axons. Frontiers in cellular neuroscience, 2015. 9: p. 417.
20. Spano, D., et al. Molecular networks that regulate cancer metastasis. in Seminars in cancer biology. 2012. Elsevier.
21. Stetler-Stevenson, W.G., S. Aznavoorian, and L.A. Liotta, Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annual review of cell biology, 1993. 9(1): p. 541-573.
22. Stivarou, T. and E. Patsavoudi, Extracellular molecules involved in cancer cell invasion. Cancers, 2015. 7(1): p. 238-265.
23. Yu, H., et al., Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations. PLoS computational biology, 2007. 3(2): p. e21.
24. Zaman, M.H., et al., Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proceedings of the National Academy of Sciences, 2006. 103(29): p. 10889-10894.
25. Wirtz, D., K. Konstantopoulos, and P.C. Searson, The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nature Reviews Cancer, 2011. 11(7): p. 512.
26. Ghosh, D., et al., iTRAQ based quantitative proteomics approach validated the role of calcyclin binding protein (CacyBP) in promoting colorectal cancer metastasis. Molecular & cellular proteomics : MCP, 2013. 12(7): p. 1865-1880.
27. Droppelmann, C.A., et al., A new level of regulation in gluconeogenesis: metabolic state modulates the intracellular localization of aldolase B and its interaction with liver fructose-1, 6-bisphosphatase. Biochemical Journal, 2015. 472(2): p. 225-237.
28. Marín‐Hernández, Á., et al., Hypoglycemia Enhances Epithelial‐Mesenchymal Transition and Invasiveness, and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and Microspheroids. Journal of cellular physiology, 2017. 232(6): p. 1346-1359.
29. Lehmann, S., et al., Hypoxia Induces a HIF-1-Dependent Transition from Collective-to-Amoeboid Dissemination in Epithelial Cancer Cells. Current Biology, 2017. 27(3): p. 392-400.
30. Hirakawa, M., et al., Sequential activation of RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium. The Journal of physiology, 2004. 558(Pt 2): p. 479-488.
31. Bae, Y.-S. and S.H. Ryu, ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells. Experimental & Molecular Medicine, 2001. 33(3): p. 150-155.
32. Sulzmaier, F.J., C. Jean, and D.D. Schlaepfer, FAK in cancer: mechanistic findings and clinical applications. Nature reviews. Cancer, 2014. 14(9): p. 598-610.
33. Bernstein, B.W. and J.R. Bamburg, Actin-ATP Hydrolysis Is a Major Energy Drain for Neurons. The Journal of Neuroscience, 2003. 23(1): p. 1-6.
34. Fang, S. and X. Fang, Advances in glucose metabolism research in colorectal cancer. Biomedical reports, 2016. 5(3): p. 289-295.
35. Hsu, P.P. and D.M. Sabatini, Cancer Cell Metabolism: Warburg and Beyond. Cell, 2008. 134(5): p. 703-707.
36. Droppelmann, C.A., et al., A new level of regulation in gluconeogenesis: metabolic state modulates the intracellular localization of aldolase B and its interaction with liver fructose-1,6-bisphosphatase. Biochem J, 2015. 472(2): p. 225-37.
37. Li, Q., et al., Aldolase B Overexpression is Associated with Poor Prognosis and Promotes Tumor Progression by Epithelial-Mesenchymal Transition in Colorectal Adenocarcinoma. Cell Physiol Biochem, 2017. 42(1): p. 397-406.
38. Kishnani, P.S. and Y.-T. Chen, Disorders of Carbohydrate Metabolism, in Emery and Rimoin's Principles and Practice of Medical Genetics. 2013. p. 1-36.
39. Campbell, E., et al., Fructose-Induced Hypertriglyceridemia: A Review, in Nutrition in the Prevention and Treatment of Abdominal Obesity. 2014. p. 197-205.
40. Chang, Y.C., et al., Roles of Aldolase Family Genes in Human Cancers and Diseases. Trends Endocrinol Metab, 2018. 29(8): p. 549-559.
41. Wang, J., et al., The Molecular Nature of the F-actin Binding Activity of Aldolase Revealed with Site-directed Mutants. Journal of Biological Chemistry, 1996. 271(12): p. 6861-6865.
42. Halestrap, A.P. and D. Meredith, The SLC16 gene family-from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond. Pflugers Arch, 2004. 447(5): p. 619-28.
43. Dembo, M. and Y.-L. Wang, Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophysical journal, 1999. 76(4): p. 2307-2316.
44. Fischer, R.S., et al., Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior. Nature protocols, 2012. 7(11): p. 2056.
45. Wu, D. and P. Yotnda, Induction and testing of hypoxia in cell culture. JoVE (Journal of Visualized Experiments), 2011(54): p. e2899.
46. Said, H.M., et al., Response of the plasma hypoxia marker osteopontin to in vitro hypoxia in human tumor cells. Radiotherapy and oncology, 2005. 76(2): p. 200-205.
47. Chen, C., et al., Inositol hexaphosphate hydrolysate competitively binds to AKT to inhibit the proliferation of colon carcinoma. Oncol Rep, 2017. 38(5): p. 2901-2910.
48. Ding, Z., et al., Expression and significance of hypoxia-inducible factor-1 alpha and MDR1/P-glycoprotein in human colon carcinoma tissue and cells. J Cancer Res Clin Oncol, 2010. 136(11): p. 1697-707.
49. Song, L., Y. Guo, and B. Xu, Expressions of Ras Homolog Gene Family, Member A (RhoA) and Cyclooxygenase-2 (COX-2) Proteins in Early Gastric Cancer and Their Role in the Development of Gastric Cancer. Med Sci Monit, 2017. 23: p. 2979-2984.
50. Hariharan, R., et al., Fundamental limitations of [18F] 2-deoxy-2-fluoro-D-glucose for assessing myocardial glucose uptake. Circulation, 1995. 91(9): p. 2435-2444.
51. TeSlaa, T. and M.A. Teitell, Techniques to monitor glycolysis, in Methods in enzymology. 2014, Elsevier. p. 91-114.
52. Zou, C., Y. Wang, and Z. Shen, 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. Journal of biochemical and biophysical methods, 2005. 64(3): p. 207-215.
53. Hu, Y., et al., Colorectal cancer susceptibility loci as predictive markers of rectal cancer prognosis after surgery. Genes, chromosomes & cancer, 2018. 57(3): p. 140-149.
54. Chekulayev, V., et al., Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes. Biochemistry and Biophysics Reports, 2015. 4: p. 111-125.
55. Park, H.S., et al., Hypoxia induces glucose uptake and metabolism of adipose‑derived stem cells. Molecular medicine reports, 2016. 14(5): p. 4706-4714.
56. Zhang, J.-Z., A. Behrooz, and F. Ismail-Beigi, Regulation of glucose transport by hypoxia. American Journal of Kidney Diseases, 1999. 34(1): p. 189-202.
57. Ida-Yonemochi, H., et al., Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars. Developmental biology, 2012. 363(1): p. 52-61.
58. Head, B.P., H.H. Patel, and P.A. Insel, Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling. Biochimica et biophysica acta, 2014. 1838(2): p. 532-545.
59. Bryan, T.O., et al., Follow the ATP: Tumor Energy Production: A Perspective. Anti-Cancer Agents in Medicinal Chemistry, 2014. 14(9): p. 1187-1198.
60. Chaube, B. and M.K. Bhat, AMPK, a key regulator of metabolic/energy homeostasis and mitochondrial biogenesis in cancer cells. Cell Death &Amp; Disease, 2016. 7: p. e2044.
61. Fu, Y., et al., The reverse Warburg effect is likely to be an Achilles' heel of cancer that can be exploited for cancer therapy. Oncotarget, 2017. 8(34): p. 57813-57825.
62. Tung, J.C., et al., Tumor mechanics and metabolic dysfunction. Free radical biology & medicine, 2015. 79: p. 269-280.
63. Zong, S., et al., Identification of hypoxia-regulated angiogenic genes in colorectal cancer. Biochemical and biophysical research communications, 2017. 493(1): p. 461-467.
64. Gan, L., et al., Extracellular matrix protein 1 promotes cell metastasis and glucose metabolism by inducing integrin β4/FAK/SOX2/HIF-1α signaling pathway in gastric cancer. Oncogene, 2018. 37(6): p. 744.
65. Xu, F., et al., Hypoxia and TGF-β1 induced PLOD2 expression improve the migration and invasion of cervical cancer cells by promoting epithelial-to-mesenchymal transition (EMT) and focal adhesion formation. Cancer cell international, 2017. 17(1): p. 54.
66. Hussain, A., et al., A novel PI3K axis selective molecule exhibits potent tumor inhibition in colorectal carcinogenesis. Molecular carcinogenesis, 2016. 55(12): p. 2135-2155.
67. Hu, H., et al., Phosphoinositide 3-kinase regulates glycolysis through mobilization of aldolase from the actin cytoskeleton. Cell, 2016. 164(3): p. 433-446.
68. Sahai, E. and C.J. Marshall, RHO-GTPases and cancer. Nat Rev Cancer, 2002. 2(2): p. 133-42.
69. Hu, H., et al., Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin Cytoskeleton. Cell, 2016. 164(3): p. 433-46.
70. Ochsner, M., et al., Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape. PLoS One, 2010. 5(3): p. e9445.
71. Peng, J.M., et al., Actin cytoskeleton remodeling drives epithelial-mesenchymal transition for hepatoma invasion and metastasis in mice. Hepatology, 2018. 67(6): p. 2226-2243.
72. Chen, Y.-K., A.H.-C. Huang, and L.-M. Lin, Sphere-forming-like cells (squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma. International Journal Of Oral Science, 2014. 6: p. 212.
73. Mah, E.J., et al., Collagen Stiffness Modulates MDA-MB231 Cell Metabolism Through Adhesion-Mediated Contractility. SSRN Electronic Journal, 2018.
74. Han, X.-Y., et al., Epithelial-Mesenchymal Transition Associates with Maintenance of Stemness in Spheroid-Derived Stem-Like Colon Cancer Cells. PLOS ONE, 2013. 8(9): p. e73341.
75. Peschetola, V., Determination of traction force exerted by tumor cells during migration on a deformable substrate. 2011, Université de Grenoble.
76. Gkretsi, V. and T. Stylianopoulos, Cell Adhesion and Matrix Stiffness: Coordinating Cancer Cell Invasion and Metastasis. Frontiers in oncology, 2018. 8: p. 145-145.
77. Tang, X., et al., A mechanically-induced colon cancer cell population shows increased metastatic potential. Molecular Cancer, 2014. 13(1): p. 131.