Inhibition of the Sonic Hedgehog Pathway using Small Molecule Inhibitors: Targeting Colon Cancer Stem Cells

Page: [138 - 155] Pages: 18

  • * (Excluding Mailing and Handling)

Abstract

Background: The Hedgehog (HH) pathway is a key regulator of many important processes in vertebrate embryonic development, including stem cell maintenance, cell differentiation, tissue polarity and cell proliferation. During pathway activation, Ptch no longer inhibits Smo and the full length Gli translocates to the nucleus resulting in the transcription of oncogenes. When constitutively activated, this leads to tumorigenesis in several human cancers. Cyclopamine acts as an antagonist of the HH signalling pathway by directly binding to the Smo heptahelical domain. The involvement of this pathway in metastasis, and its presence in cancer stem cells (CSCs), makes it a valid option for developing a targeted therapeutic against it.

Methods: CSC were isolated from DLD1 and HT29 cell lines using magnetic cell separation labelling the CD133 receptor. The growth patterns of isolated CSCs (CD133 positive) in comparison to non-stem cells (CD133 negative) were analysed using real-time cell impedance assays (RTCA). Thereafter, adhesion, invasion and migration assays were performed with the application of small molecule inhibitors. The expression levels of CD133 and SHH were evaluated using confocal microscopy following treatment with cyclopamine.

Results and Discussion: Growth of CSCs appeared to be slower than non-CSCs. Adhesion, invasion and cell migration were inhibited when CSCs were pharmacologically treated either with cyclopamine or SANT-2 (a synthetic analogue of cyclopamine), small molecule inhibitors of the HH pathway. Using confocal microscopy the cell surface expression of Sonic Hedgehog (SHH) was significantly decreased following treatment with cyclopamine, while the expression of CD133 remained unaffected.

Conclusion: Considering these in vitro results, small molecule inhibitors targeting the SHH pathway appear to be promising therapeutic tools for the treatment of metastatic colon CSCs.

Keywords: cancer stem cells, colon cancer, Sonic Hedgehog Pathway, cyclopamine, metastasis.

Graphical Abstract

[1]
Ballinger AB, Anggiansah C. Colorectal cancer. BMJ 2007; 335(7622): 715-8.
[http://dx.doi.org/10.1136/bmj.39321.527384.BE] [PMID: 17916855]
[2]
Madiba T, Moodley Y, Sartorius B, et al. Clinicopathological spectrum of colorectal cancer among the population of the KwaZulu-Natal Province in South Africa. Pan Afr Med J 2020; 37: 74.
[PMID: 33244337]
[3]
Xi Y, Xu P. Global colorectal cancer burden in 2020 and projections to 2040. Transl Oncol 2021; 14(10), 101174.
[http://dx.doi.org/10.1016/j.tranon.2021.101174] [PMID: 34243011]
[4]
Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 2007; 104(24): 10158-63.
[http://dx.doi.org/10.1073/pnas.0703478104] [PMID: 17548814]
[5]
Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001; 414(6859): 105-11.
[http://dx.doi.org/10.1038/35102167] [PMID: 11689955]
[6]
Dalerba P, Cho RW, Clarke MF. Cancer stem cells: Models and concepts. Annu Rev Med 2007; 58: 267-84.
[http://dx.doi.org/10.1146/annurev.med.58.062105.204854] [PMID: 17002552]
[7]
Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med 2006; 355(12): 1253-61.
[http://dx.doi.org/10.1056/NEJMra061808] [PMID: 16990388]
[8]
Pan Y, Ma S, Cao K, et al. Therapeutic approaches targeting cancer stem cells. J Cancer Res Ther 2018; 14(7): 1469-75.
[http://dx.doi.org/10.4103/jcrt.JCRT_976_17] [PMID: 30589025]
[9]
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100(7): 3983-8.
[http://dx.doi.org/10.1073/pnas.0530291100] [PMID: 12629218]
[10]
Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature 2004; 432(7015): 396-401.
[http://dx.doi.org/10.1038/nature03128] [PMID: 15549107]
[11]
Galli R, Binda E, Orfanelli U, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 2004; 64(19): 7011-21.
[http://dx.doi.org/10.1158/0008-5472.CAN-04-1364] [PMID: 15466194]
[12]
O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007; 445(7123): 106-10.
[http://dx.doi.org/10.1038/nature05372] [PMID: 17122772]
[13]
Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2007; 445(7123): 111-5.
[http://dx.doi.org/10.1038/nature05384] [PMID: 17122771]
[14]
Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 2007; 104(3): 973-8.
[http://dx.doi.org/10.1073/pnas.0610117104] [PMID: 17210912]
[15]
Gou S, Liu T, Wang C, et al. Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties. Pancreas 2007; 34(4): 429-35.
[http://dx.doi.org/10.1097/MPA.0b013e318033f9f4] [PMID: 17446842]
[16]
Gisina AM, Lupatov AY, Karalkin PA, et al. Subpopulation of colorectal adenocarcinoma cells co-expressing CD133 and cancer stem cells markers of other tumors. Bull Exp Biol Med 2012; 152(6): 739-42.
[http://dx.doi.org/10.1007/s10517-012-1620-0] [PMID: 22803178]
[17]
Zhou Y, Xia L, Wang H, et al. Cancer stem cells in progression of colorectal cancer. Oncotarget 2017; 9(70): 33403-15.
[http://dx.doi.org/10.18632/oncotarget.23607] [PMID: 30279970]
[18]
Iinuma H, Watanabe T, Mimori K, et al. Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes’ stage B and C colorectal cancer. J Clin Oncol 2011; 29(12): 1547-55.
[http://dx.doi.org/10.1200/JCO.2010.30.5151] [PMID: 21422427]
[19]
Irollo E, Pirozzi G. CD133: To be or not to be, is this the real question? Am J Transl Res 2013; 5(6): 563-81.
[PMID: 24093054]
[20]
Ingham PW, Nakano Y, Seger C. Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 2011; 12(6): 393-406.
[http://dx.doi.org/10.1038/nrg2984] [PMID: 21502959]
[21]
Scales SJ, de Sauvage FJ. Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 2009; 30(6): 303-12.
[http://dx.doi.org/10.1016/j.tips.2009.03.007] [PMID: 19443052]
[22]
Li X, Li Y, Li S, Li H, Yang C, Lin J. The role of Shh signalling pathway in central nervous system development and related diseases. Cell Biochem Funct 2021; 39(2): 180-9.
[http://dx.doi.org/10.1002/cbf.3582] [PMID: 32840890]
[23]
Rimkus TK, Carpenter RL, Qasem S, Chan M, Lo HW. Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors. Cancers (Basel) 2016; 8(2), E22.
[http://dx.doi.org/10.3390/cancers8020022] [PMID: 26891329]
[24]
Taipale J, Chen JK, Cooper MK, et al. Effects of oncogenic mutations in smoothened and patched can be reversed by cyclopamine. Nature 2000; 406(6799): 1005-9.
[http://dx.doi.org/10.1038/35023008] [PMID: 10984056]
[25]
Varnat F, Duquet A, Malerba M, et al. Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion. EMBO Mol Med 2009; 1(6-7): 338-51.
[http://dx.doi.org/10.1002/emmm.200900039] [PMID: 20049737]
[26]
Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell 2011; 144(5): 646-74.
[http://dx.doi.org/10.1016/j.cell.2011.02.013] [PMID: 21376230]
[27]
Katoh Y, Katoh M. Hedgehog signaling, epithelial-to-mesenchymal transition and miRNA. (review) Int J Mol Med 2008; 22(3): 271-5.
[PMID: 18698484]
[28]
Riaz SK, Ke Y, Wang F, Kayani MA, Malik MFA. Influence of SHH/GLI1 axis on EMT mediated migration and invasion of breast cancer cells. Sci Rep 2019; 9(1): 6620.
[http://dx.doi.org/10.1038/s41598-019-43093-x] [PMID: 31036836]
[29]
Gupta R, Bhatt LK, Johnston TP, Prabhavalkar KS. Colon cancer stem cells: Potential target for the treatment of colorectal cancer. Cancer Biol Ther 2019; 20(8): 1068-82.
[http://dx.doi.org/10.1080/15384047.2019.1599660] [PMID: 31050577]
[30]
Cooper MK, Porter JA, Young KE, Beachy PA. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 1998; 280(5369): 1603-7.
[http://dx.doi.org/10.1126/science.280.5369.1603] [PMID: 9616123]
[31]
Incardona JP, Gaffield W, Kapur RP, Roelink H. The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 1998; 125(18): 3553-62.
[http://dx.doi.org/10.1242/dev.125.18.3553] [PMID: 9716521]
[32]
Campbell V, Copland M. Hedgehog signaling in cancer stem cells: A focus on hematological cancers. Stem Cells Cloning 2015; 8: 27-38.
[PMID: 25691811]
[33]
Chen JK, Taipale J, Young KE, Maiti T, Beachy PA. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci USA 2002; 99(22): 14071-6.
[http://dx.doi.org/10.1073/pnas.182542899] [PMID: 12391318]
[34]
Li L, Neaves WB. Normal stem cells and cancer stem cells: The niche matters. Cancer Res 2006; 66(9): 4553-7.
[http://dx.doi.org/10.1158/0008-5472.CAN-05-3986] [PMID: 16651403]
[35]
Behrens J. The role of cell adhesion molecules in cancer invasion and metastasis. Breast Cancer Res Treat 1993; 24(3): 175-84.
[http://dx.doi.org/10.1007/BF01833258] [PMID: 8435473]
[36]
Omar A, Jovanovic K, Da Costa Dias B, et al. Patented biological approaches for the therapeutic modulation of the 37 kDa/67 kDa laminin receptor. Expert Opin Ther Pat 2011; 21(1): 35-53.
[http://dx.doi.org/10.1517/13543776.2011.539203] [PMID: 21110766]
[37]
Kemper K, Grandela C, Medema JP. Molecular identification and targeting of colorectal cancer stem cells. Oncotarget 2010; 1(6): 387-95.
[http://dx.doi.org/10.18632/oncotarget.173] [PMID: 21311095]
[38]
Mizrak D, Brittan M, Alison M. CD133: Molecule of the moment. J Pathol 2008; 214(1): 3-9.
[http://dx.doi.org/10.1002/path.2283] [PMID: 18067118]
[39]
Penny CBMB, Gibbon VE, Ruff P. The HT29 and DLD-1 colon cancer cell lines contain cancer stem cells. Clin Anat 2013; 26(5): 653.
[40]
López-Lázaro M. The stem cell division theory of cancer. Crit Rev Oncol Hematol 2018; 123: 95-113.
[http://dx.doi.org/10.1016/j.critrevonc.2018.01.010] [PMID: 29482784]
[41]
Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006; 66(19): 9339-44.
[http://dx.doi.org/10.1158/0008-5472.CAN-06-3126] [PMID: 16990346]
[42]
Afzali M, Vatankhah M, Ostad SN. Investigation of simvastatin-induced apoptosis and cell cycle arrest in cancer stem cells of MCF-7. J Cancer Res Ther 2016; 12(2): 725-30.
[http://dx.doi.org/10.4103/0973-1482.146127] [PMID: 27461641]
[43]
Croker AK, Goodale D, Chu J, et al. High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J Cell Mol Med 2009; 13(8B): 2236-52.
[http://dx.doi.org/10.1111/j.1582-4934.2008.00455.x] [PMID: 18681906]
[44]
Omar A, Reusch U, Knackmuss S, Little M, Weiss SF. Anti-LRP/LR-specific antibody IgG1-iS18 significantly reduces adhesion and inva-sion of metastatic lung, cervix, colon and prostate cancer cells. J Mol Biol 2012; 419(1-2): 102-9.
[http://dx.doi.org/10.1016/j.jmb.2012.02.035] [PMID: 22391421]
[45]
Magi S, Tashiro E, Imoto M. A chemical genomic study identifying diversity in cell migration signaling in cancer cells. Sci Rep 2012; 2: 823.
[http://dx.doi.org/10.1038/srep00823] [PMID: 23139868]
[46]
Thayer SP, di Magliano MP, Heiser PW, et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 2003; 425(6960): 851-6.
[http://dx.doi.org/10.1038/nature02009] [PMID: 14520413]
[47]
Wan J, Zhou J, Zhao H, et al. Sonic hedgehog pathway contributes to gastric cancer cell growth and proliferation Biores Open Access 2014; 3(2): 53-9.
[http://dx.doi.org/10.1089/biores.2014.0001] [PMID: 24804165]
[48]
Wu C, Hu S, Cheng J, Wang G, Tao K. Smoothened antagonist GDC-0449 (Vismodegib) inhibits proliferation and triggers apoptosis in colon cancer cell lines. Exp Ther Med 2017; 13(5): 2529-36.
[http://dx.doi.org/10.3892/etm.2017.4282] [PMID: 28565875]
[49]
Wang Y, Zhou Z, Walsh CT, McMahon AP. Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation. Proc Natl Acad Sci USA 2009; 106(8): 2623-8.
[http://dx.doi.org/10.1073/pnas.0812110106] [PMID: 19196978]
[50]
Piérard-Franchimont C, Hermanns-Lê T, Paquet P, Herfs M, Delvenne P, Piérard GE. Hedgehog- and mTOR-targeted therapies for advanced basal cell carcinomas. Future Oncol 2015; 11(22): 2997-3002.
[http://dx.doi.org/10.2217/fon.15.181] [PMID: 26437034]
[51]
Batsaikhan BE, Yoshikawa K, Kurita N, et al. Cyclopamine decreased the expression of Sonic Hedgehog and its downstream genes in colon cancer stem cells. Anticancer Res 2014; 34(11): 6339-44.
[PMID: 25368233]
[52]
Kumar SK, Roy I, Anchoori RK, et al. Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer. Bioorg Med Chem 2008; 16(6): 2764-8.
[http://dx.doi.org/10.1016/j.bmc.2008.01.012] [PMID: 18249125]
[53]
Bahra M, Kamphues C, Boas-Knoop S, et al. Combination of hedgehog signaling blockage and chemotherapy leads to tumor reduction in pancreatic adenocarcinomas. Pancreas 2012; 41(2): 222-9.
[http://dx.doi.org/10.1097/MPA.0b013e31822896dd] [PMID: 22076568]
[54]
Hu K, Zhou H, Liu Y, et al. Hyaluronic acid functional amphipathic and redox-responsive polymer particles for the co-delivery of doxorubicin and cyclopamine to eradicate breast cancer cells and cancer stem cells. Nanoscale 2015; 7(18): 8607-18.
[http://dx.doi.org/10.1039/C5NR01084E] [PMID: 25898852]
[55]
Zhou H, Xiong Y, Peng L, Wang R, Zhang H, Fu Z. LncRNA-cCSC1 modulates cancer stem cell properties in colorectal cancer via activation of the Hedgehog signaling pathway. J Cell Biochem 2020; 121(3): 2510-24.
[http://dx.doi.org/10.1002/jcb.29473] [PMID: 31680315]
[56]
Sari IN, Phi LTH, Jun N, Wijaya YT, Lee S, Kwon HY. Hedgehog signaling in cancer: A prospective therapeutic target for eradicating cancer stem cells. Cells 2018; 7(11), E208.
[http://dx.doi.org/10.3390/cells7110208] [PMID: 30423843]