||Investigating the molecular roles and mechanisms of Galectin-3 on drug resistance in colon cancer
||Institute of Basic Medical Sciences
大腸直腸癌是全球十大癌症死亡原因之一，也是近十年來台灣腫瘤發生率與發生人數最多的一種癌症。由於手術後的高復發率以及化療無效反應(抗藥性)的結果，每年的死亡率和死亡人數也是居高不下。許多研究發現，癌症的高復發率以及高轉移率與兩個因素有高度相關性，其一是多重要抗藥性相關蛋白的表現量上升，另一則是腫瘤中存在的癌症起始細胞(cancer initiating cells, CICs or cancer stem cells, CSCs)。腫瘤幹細胞除了具有分化成癌細胞以及自我修復等特性之外，也具備較高的抵抗藥物能力。因此，研究大腸癌細胞抗藥性的分子機制，進而探討細胞抗藥性在癌症起始細胞中的角色，為本論文主要的目標。我們首先發現半乳糖凝集素3 (galectin-3) 同時參與在大腸癌抗藥能力的調控，以及癌症起始細胞特性的維持。大腸癌細胞會透過大量表現galectin-3，經由β-catenin/GSK-3β路徑會促進細胞內多重抗藥蛋白的表現上升。我們接著發現大腸癌細胞腫瘤懸浮球體(spheres)中的 galectin-3表現量有明顯增加；透過RNAi系統抑制大腸癌細胞中的galectin-3表現，則會減少腫瘤懸浮球體的大小、數量、以及相關蛋白的表現，而外加galectin-3抑制劑也可獲得類似的效果。根據以上的研究結果顯示，大腸癌細胞在抗癌藥物或是塑化劑的刺激之下，可以透過不同的分子機制促進多重抗藥蛋白的表現，以及表現出癌症起始細胞的相關基因與能力；而galectin-3在大腸癌細胞的多重抗藥性與癌症起始細胞特性上，也扮演相當關鍵的角色。基於這些結果，我們建議針對galectin-3可能是改善結腸癌治療的有效方法。
Colorectal cancer (CRC) is among the top ten causes of cancer-related death worldwide, and it also accounts for the highest incidence of cancer in Taiwan over the past decade. The prognosis of CRC remains poor because of the high recurrence rates and low response to chemotherapy (drug resistance). Many studies have demonstrated two factors, namely the upregulation of multidrug resistance (MDR)-associated proteins and presence of tumor-initiating cells (cancer-initiating cells, CICs, or cancer stem cells, CSCs), that are correlated with a high rate of recurrence and high metastatic ability. CSCs are capable of self-renewal and differentiation into various types of cancer. Moreover, CSCs are proposed to be responsible for resistance to chemotherapy. Therefore, this study investigated the roles and molecular mechanisms of drug resistance in CRC, in addition to elucidating the drug resistance mechanisms in colon CSCs. We determined that galectin-3 (Gal-3) participated in drug resistance modulation in CRC and in maintaining stemness in CSCs. Increased Gal-3 expression in colon cancer cells involves the upregulation of MDR-related proteins through the induction of β-catenin/GSK-3β signaling. Furthermore, the expression of Gal-3 increased significantly in colon tumor spheres. The suppression of Gal-3 through RNA interference in colon cancer cells decreased Gal-3 expression and reduced the size, number, and stemness-related proteins in tumor spheres. In addition, these phenomena could be rescued by Gal-3 inhibitor treatment. Thus, the exposure of colon cancer cells to anticancer drug stimulation or plasticizing agents may promote the expression of multidrug-resistant proteins through different molecular mechanisms and exhibit CIC-related gene expression and activity. Taken together, Gal-3 plays key roles in MDR and tumor-initiating cell properties. Accordingly, we suggest that targeting galectin-3 may be a potent approach for improving colon cancer therapy.
Abstract in Chinese I
Table of contents V
List of Abbreviations VIII
Colorectal cancer and multidrug resistance 1
Galectin-3 in cancer 3
Gal-3 and MDR 4
Colon cancer-initiating cells 6
Colon CICs and MDR 7
Specific aims 9
Combined epirubicin and shGal-3 treatment significantly intensified epirubicin cytotoxicity 11
Combined epirubicin and shGal-3 treatment induced apoptosis-mediated morphological changes in Caco-2 cells and increased sub-G1 accumulation in the Caco-2 cell cycle 11
shGal-3 significantly increased intracellular epirubicin retention in Caco-2 cells 12
Combined shGal-3 and epirubicin treatment significantly inhibited the mRNA expression of Gal-3 and β-catenin but increased the expression of GSK-3β 12
Combined shGal-3 and epirubicin treatment inhibited the mRNA expression levels of the cyclin D1 and c-myc that were induced by the epirubicin treatment 12
Combined shGal-3 and epirubicin treatment significantly inhibited the mRNA expression levels of MDR1, MRP1, and MRP2 12
Confirmation of the protein expression level of the Wnt/β-catenin signaling pathway and apoptosis-associated pathway components after epirubicin treatment in wild-type or Gal-3 knockdown Caco-2 cells 13
Knockdown of Gal-3 in Caco-2 cells inhibited P-gp and cyclin-D1 protein expression after epirubicin treatment 13
Identification of a new monoclonal antibody recognizing CD133 14
Gal-3 attenuation reduced sphere formation and stemness-related gene expression 15
Cancer sphere formation indeed requires Gal-3 15
Lactose suppressed colon cancer sphere formation with Oct4 downregulation in the cancer sphere formation process 16
Gal-3 induces the formation of Gal-3/Oct4 complexes for cancer sphere formation 16
Nuclear interaction of Gal-3 with Oct4 in cancer spheres 17
TD-139 inhibited colon cancer sphere formation in a dose-dependent manner 17
Use of efflux pump inhibitors significantly reduces cell viability in DEHP/MEHP-treated HCT116 colon cancer cells 17
DEHP/MEHP-induced drug resistance and antiapoptosis but without Gal-3 increase in HCT116 colon cancer cells receiving anticancer drug treatment 18
Long-term DEHP/MEHP exposure can induce sphere formation, which was inhibited by Gal-3 knockdown in HCT116 colon cancer cells 19
Materials and Methods 25
Consumables and devices 27
Cell culture and experimental conditions 28
Construction of galectin-3 shRNA 28
Cytotoxicity assay 28
Apoptosis detection assay 29
Observation of chromatin condensation using a fluorescence microscope 29
Cell cycle analysis 30
Intracellular epirubicin accumulation 30
Construction and overexpression of galectin-3 30
Real-time PCR of P-gp, MRP1, MRP2, Bax, and Bcl-2 31
Western blotting 32
Statistical analysis 32
CD133 antigen design and generation 33
Immunization and monoclonal antibody production 33
Cell culture and experimental conditions 33
FACS staining 34
Lentivirus-based RNAi knockdown 35
Construction and overexpression of galectin-3 35
Western blotting 35
Total RNA preparation and RT-PCR 36
Test of Lactose (Lac) effect on spheres formation 36
Co-mmunoprecipitation Assays 37
Test of TD-139 effect on spheres formation 37
Preparation of cell nuclear and cytosol fractions 38
Statistical analysis 38
Cell culture and experimental conditions 39
Western blotting 39
P-glycoprotein activity assay 40
Cell proliferation analysis 40
Sphere formation analysis 41
Statistical analysis 41
Table 1. The sequences of shRNA and primer sequences used for knock-down and real-time PCR. 42
1. Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA Cancer J Clin, 2013. 63(1): p. 11-30.
2. Chang, H.C., et al., Evaluation of the appropriate age range of colorectal cancer screening based on the changing epidemiology in the past 20 years in taiwan. ISRN Gastroenterol, 2012. 2012: p. 960867.
3. Changchien, C.-R., Epidemiology of Colorectal Cancer in Taiwan. J. Chinese Oncol. Soc, 2008. 24: p. 143-147.
4. Primrose, J.N., Surgery for colorectal liver metastases. Br J Cancer, 2010. 102(9): p. 1313-8.
5. Aghili, M., et al., Clinical and pathological evaluation of patients with early and late recurrence of colorectal cancer. Asia Pac J Clin Oncol, 2010. 6(1): p. 35-41.
6. Anderson, E.C., et al., The role of colorectal cancer stem cells in metastatic disease and therapeutic response. Cancers (Basel), 2011. 3(1): p. 319-39.
7. Goldstein, L.J., et al., Expression of a multidrug resistance gene in human cancers. J Natl Cancer Inst, 1989. 81(2): p. 116-24.
8. Kimura, Y., et al., Mechanism of multidrug recognition by MDR1/ABCB1. Cancer Sci, 2007. 98(9): p. 1303-10.
9. Leslie, E.M., R.G. Deeley, and S.P. Cole, Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol Appl Pharmacol, 2005. 204(3): p. 216-37.
10. Riordan, J.R., et al., Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. Nature, 1985. 316(6031): p. 817-9.
11. Loo, T.W. and D.M. Clarke, Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux. J Membr Biol, 2005. 206(3): p. 173-85.
12. Kajiji, S., et al., Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site. Biochemistry, 1993. 32(16): p. 4185-94.
13. Tsuruo, T., et al., Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal. Cancer Sci, 2003. 94(1): p. 15-21.
14. Zhao, B.X., et al., Grape seed procyanidin reversal of p-glycoprotein associated multi-drug resistance via down-regulation of NF-kappaB and MAPK/ERK mediated YB-1 activity in A2780/T cells. PLoS One, 2013. 8(8): p. e71071.
15. Tomiyasu, H., et al., Regulation of expression of ABCB1 and LRP genes by mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and its role in generation of side population cells in canine lymphoma cell lines. Leuk Lymphoma, 2013. 54(6): p. 1309-15.
16. Song, S., et al., Galectin-3 mediates nuclear beta-catenin accumulation and Wnt signaling in human colon cancer cells by regulation of glycogen synthase kinase-3beta activity. Cancer Res, 2009. 69(4): p. 1343-9.
17. Ma, S., et al., CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene, 2008. 27(12): p. 1749-58.
18. Bansal, T., et al., Novel formulation approaches for optimising delivery of anticancer drugs based on P-glycoprotein modulation. Drug Discov Today, 2009. 14(21-22): p. 1067-74.
19. Martin, C., et al., The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein. Br J Pharmacol, 1999. 128(2): p. 403-11.
20. Houzelstein, D., et al., Phylogenetic analysis of the vertebrate galectin family. Mol Biol Evol, 2004. 21(7): p. 1177-87.
21. Califice, S., et al., Dual activities of galectin-3 in human prostate cancer: tumor suppression of nuclear galectin-3 vs tumor promotion of cytoplasmic galectin-3. Oncogene, 2004. 23(45): p. 7527-36.
22. Inohara, H. and A. Raz, Effects of natural complex carbohydrate (citrus pectin) on murine melanoma cell properties related to galectin-3 functions. Glycoconj J, 1994. 11(6): p. 527-32.
23. Liang, Y., et al., [The expression of galectin-3 and osteopontin in occult metastasis of non-small cell lung cancer]. Zhonghua Wai Ke Za Zhi, 2009. 47(14): p. 1061-3.
24. Fukumori, T., et al., Galectin-3 regulates mitochondrial stability and antiapoptotic function in response to anticancer drug in prostate cancer. Cancer Res, 2006. 66(6): p. 3114-9.
25. Song, S., et al., Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding Ras and activating Ras signaling. PLoS One, 2012. 7(8): p. e42699.
26. Kobayashi, T., et al., Transient silencing of galectin-3 expression promotes both in vitro and in vivo drug-induced apoptosis of human pancreatic carcinoma cells. Clin Exp Metastasis, 2011. 28(4): p. 367-76.
27. Kobayashi, T., et al., Transient gene silencing of galectin-3 suppresses pancreatic cancer cell migration and invasion through degradation of beta-catenin. Int J Cancer, 2011. 129(12): p. 2775-86.
28. Acikalin, M.F., et al., Prognostic significance of galectin-3 and cyclin D1 expression in undifferentiated nasopharyngeal carcinoma. Med Oncol, 2012. 29(2): p. 742-9.
29. Shi, Y., et al., Inhibition of Wnt-2 and galectin-3 synergistically destabilizes beta-catenin and induces apoptosis in human colorectal cancer cells. Int J Cancer, 2007. 121(6): p. 1175-81.
30. Nakamura, M., et al., Involvement of galectin-3 expression in colorectal cancer progression and metastasis. Int J Oncol, 1999. 15(1): p. 143-8.
31. Endo, K., et al., Galectin-3 expression is a potent prognostic marker in colorectal cancer. Anticancer Res, 2005. 25(4): p. 3117-21.
32. Tsuboi, K., et al., Galectin-3 expression in colorectal cancer: relation to invasion and metastasis. Anticancer Res, 2007. 27(4B): p. 2289-96.
33. Takenaka, Y., et al., Nuclear export of phosphorylated galectin-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs. Mol Cell Biol, 2004. 24(10): p. 4395-406.
34. Akahani, S., et al., Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res, 1997. 57(23): p. 5272-6.
35. Mirandola, L., et al., Galectin-3 inhibition suppresses drug resistance, motility, invasion and angiogenic potential in ovarian cancer. Gynecol Oncol, 2014. 135(3): p. 573-9.
36. Lin, C.I., et al., Galectin-3 regulates apoptosis and doxorubicin chemoresistance in papillary thyroid cancer cells. Biochem Biophys Res Commun, 2009. 379(2): p. 626-31.
37. Nakahara, S., et al., Characterization of the nuclear import pathways of galectin-3. Cancer Res, 2006. 66(20): p. 9995-10006.
38. Oka, N., et al., Galectin-3 inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by activating Akt in human bladder carcinoma cells. Cancer Res, 2005. 65(17): p. 7546-53.
39. Lapidot, T., et al., A Cell Initiating Human Acute Myeloid-Leukemia after Transplantation into Scid Mice. Nature, 1994. 367(6464): p. 645-648.
40. Du, L., et al., CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res, 2008. 14(21): p. 6751-60.
41. Huang, E.H., et al., Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res, 2009. 69(8): p. 3382-9.
42. Ricci-Vitiani, L., et al., Identification and expansion of human colon-cancer-initiating cells. Nature, 2007. 445(7123): p. 111-5.
43. Singh, S.K., et al., Cancer stem cells in nervous system tumors. Oncogene, 2004. 23(43): p. 7267-73.
44. Collins, A.T., et al., Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res, 2005. 65(23): p. 10946-51.
45. Vermeulen, L., et al., Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U S A, 2008. 105(36): p. 13427-32.
46. Al-Hajj, M., et al., Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A, 2003. 100(7): p. 3983-8.
47. Li, C., et al., Identification of pancreatic cancer stem cells. Cancer Res, 2007. 67(3): p. 1030-7.
48. Dalerba, P., et al., Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A, 2007. 104(24): p. 10158-63.
49. O'Brien, C.A., et al., A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature, 2007. 445(7123): p. 106-10.
50. Puglisi, M.A., et al., Isolation and characterization of CD133+ cell population within human primary and metastatic colon cancer. Eur Rev Med Pharmacol Sci, 2009. 13 Suppl 1: p. 55-62.
51. Horst, D., et al., The cancer stem cell marker CD133 has high prognostic impact but unknown functional relevance for the metastasis of human colon cancer. J Pathol, 2009. 219(4): p. 427-34.
52. Niwa, H., J. Miyazaki, and A.G. Smith, Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics, 2000. 24(4): p. 372-376.
53. Shackleton, M., et al., Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell, 2009. 138(5): p. 822-9.
54. Maugeri-Sacca, M., A. Zeuner, and R. De Maria, Therapeutic targeting of cancer stem cells. Front Oncol, 2011. 1: p. 10.
55. Signore, M., L. Ricci-Vitiani, and R. De Maria, Targeting apoptosis pathways in cancer stem cells. Cancer Lett, 2013. 332(2): p. 374-82.
56. Todaro, M., et al., CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell Stem Cell, 2014. 14(3): p. 342-56.
57. Vermeulen, L., et al., Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat Cell Biol, 2010. 12(5): p. 468-76.
58. Wang, M.L., S.H. Chiou, and C.W. Wu, Targeting cancer stem cells: emerging role of Nanog transcription factor. Onco Targets Ther, 2013. 6: p. 1207-20.
59. Bae, S.U., et al., Oncologic Outcomes of Colon Cancer Patients with Extraregional Lymph Node Metastasis: Comparison of Isolated Paraaortic Lymph Node Metastasis with Resectable Liver Metastasis. Ann Surg Oncol, 2016. 23(5): p. 1562-8.
60. Bockelman, C., et al., Risk of recurrence in patients with colon cancer stage II and III: a systematic review and meta-analysis of recent literature. Acta Oncol, 2015. 54(1): p. 5-16.
61. Kazem, M.A., A.U. Khan, and C.R. Selvasekar, Validation of nomogram for disease free survival for colon cancer in UK population: A prospective cohort study. Int J Surg, 2016. 27: p. 58-65.
62. Cheng, Y.L., et al., Increased galectin-3 facilitates leukemia cell survival from apoptotic stimuli. Biochem Biophys Res Commun, 2011. 412(2): p. 334-40.
63. Shimura, T., et al., Galectin-3, a novel binding partner of beta-catenin. Cancer Res, 2004. 64(18): p. 6363-7.
64. Kelly, K.F., et al., beta-catenin enhances Oct-4 activity and reinforces pluripotency through a TCF-independent mechanism. Cell Stem Cell, 2011. 8(2): p. 214-27.
65. Ravindran, G., et al., Association of differential beta-catenin expression with Oct-4 and Nanog in oral squamous cell carcinoma and their correlation with clinicopathological factors and prognosis. Head Neck, 2014.
66. Zeineddine, D., et al., The Oct4 protein: more than a magic stemness marker. Am J Stem Cells, 2014. 3(2): p. 74-82.
67. Bidlingmaier, S., X. Zhu, and B. Liu, The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells. J Mol Med (Berl), 2008. 86(9): p. 1025-32.
68. Shekhar, M.P., et al., Alterations in galectin-3 expression and distribution correlate with breast cancer progression: functional analysis of galectin-3 in breast epithelial-endothelial interactions. Am J Pathol, 2004. 165(6): p. 1931-41.
69. Sanjuan, X., et al., Differential expression of galectin 3 and galectin 1 in colorectal cancer progression. Gastroenterology, 1997. 113(6): p. 1906-15.
70. Chung, L.Y., et al., Galectin-3 augments tumor initiating property and tumorigenicity of lung cancer through interaction with beta-catenin. Oncotarget, 2015. 6(7): p. 4936-52.
71. Li, J., J. Li, and B. Chen, Oct4 was a novel target of Wnt signaling pathway. Mol Cell Biochem, 2012. 362(1-2): p. 233-40.
72. Li, J. and B.P. Zhou, Activation of beta-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer, 2011. 11: p. 49.
73. Dubrovska, A., et al., The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proc Natl Acad Sci U S A, 2009. 106(1): p. 268-73.
74. Lee, Y.K., et al., Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via beta-catenin/GSK-3beta modulation in colorectal carcinoma. PLoS One, 2013. 8(11): p. e82478.
75. Kuo, H.Y., et al., Galectin-3 modulates the EGFR signalling-mediated regulation of Sox2 expression via c-Myc in lung cancer. Glycobiology, 2016. 26(2): p. 155-65.
76. Takeshita, A., et al., The endocrine disrupting chemical, diethylhexyl phthalate, activates MDR1 gene expression in human colon cancer LS174T cells. J Endocrinol, 2006. 190(3): p. 897-902.