Expression of Wnt and Notch Signaling Pathways are Increased in Cancer Stem-Like Cells in Metastatic Melanoma Cell Line (A375)

Document Type : Original Article


1 Master of Science in Animal Biology, Towards Cellular-Tactile Science.Department of Animal Biology, Tow-Transe Science, Science & Culture University, Tehran, Iran.

2 Student MSc in Animal Biology. Towards Cellular-Tangential Sciences.Department of Animal Biology, Tow-Transe Science, Science & Culture University, Tehran, Iran.

3 Group of Stem Cells and Developmental Biology.Department of Stem Cells and Developmental Biology, Cellular Research Center, Stem Cell and Tactical Biology Department, Institute of Biology and Technology of Stem Cells, University of Tehran, Tehran, Iran.

4 Senior Cell and Molecular Expert.Department of Stem Cells and Developmental Biology, Cellular Research Center, Stem Cell and Tactical Biology Department, Institute of Biology and Technology of Stem Cells, University of Tehran, Tehran, Iran.

5 Associate Professor Royan Research Group.Department of Stem Cells and Developmental Biology, Cellular Research Center, Stem Cell and Tactical Biology Department, Institute of Biology and Technology of Stem Cells, University of Tehran, Tehran, Iran.


Backgound and Objective: Cancer stem cells assume to be responsible for increasing cancer properties such as migration, and drug sensitivity. A lot of studies revealed signaling pathways are enhanced in cancer stem cells.  Melanoma is well-known as heterogeneous cancer and its severity in treatment. These can be attributed to the existence of cancer stem cells. In this study, we first aimed to separate cancer stem cells (cancer stem-like cells) by sphere formation, characterized them, and second examine the expression of Wnt and Notch signaling pathway genes related to adherent cells.
Subjects and Methods: Adherent cell were cultured in the non-adherent condition with serum-free media; spheres obtained, then sphere formation,  clonogenic assay, expression of CD133 and Nestin proteins, Nanog, NESTIN and Oct4 genes as stem related genes were assessed in comparison to adherent cells. In addition, Notch and Wnt signaling pathways genes in both adherent and spheres cells evaluated.
Results: Sphere formation, clonogenic capacity, expression of Nestin protein, but not CD133 were increased in sphere cells in comparession to adherent cells. They also overexpressed β-catenin, cyclinD1, and c-Myc as Wnt down-stream genes, Notch1, HES1 as Notch down-stream genes, Nanog, and NESTIN as stem-related genes.
Conclusion: These results suggest that sphere culture model could be a proper experimental method to separate cancer stem-like cells. Our data also support two important pathways are overactivated in melanoma cancer stem-like cells which must be considered in targeting therapy.


1-Morton DL. Melanoma: Are we there yet? Journal of Surgical Oncology. 2011;104(4):337.
2-Thumar J, Giesen E, Kluger HM. Drug targets and predictive biomarkers in the management of metastatic melanoma. Pharmacogenomics and personalized medicine. 2012;5:139-48.
3-Hamid O, Boasberg PD, Rosenthal K, O'Day SJ. Systemic treatment of metastatic melanoma: new approaches. J Surg Oncol. 2011;104(4):425-9.
4-Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, et al. Identification of cells initiating human melanomas. Nature. 2008;451(7176):345.
5-Doherty MR, Smigiel JM, Junk DJ, Jackson MW. Cancer Stem Cell Plasticity Drives Therapeutic Resistance. Cancers. 2016;8(1).
6-Civenni G, Walter A, Kobert N, Mihic-Probst D, Zipser M, Belloni B, et al. Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth. Cancer research. 2011;71(8):3098-109.
7-   Hsu M-Y, Yang MH, Schnegg CI, Hwang S, Ryu B, Alani RM. Notch3 signaling-mediated melanoma–endothelial crosstalk regulates melanoma stem-like cell homeostasis and niche morphogenesis. Laboratory Investigation. 2017;97(6):725.
8-   Mallinger A, Crumpler S, Pichowicz M, Waalboer D, Stubbs M, Adeniji-Popoola O, et al. Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen. Journal of medicinal chemistry. 2015;58(4):1717-35.
9-   Lee H-J, Bao J, Miller A, Zhang C, Wu J, Baday YC, et al. Structure-based discovery of novel small molecule Wnt signaling inhibitors by targeting the cysteine-rich domain of frizzled. Journal of Biological Chemistry. 2015;290(51):30596-606.
10-Yahyanejad S, King H, Iglesias VS, Granton PV, Barbeau LM, van Hoof SJ, et al. NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma. Oncotarget. 2016;7(27):41251.
11-Lee SH, Do SI, Lee HJ, Kang HJ, Koo BS, Lim YC. Notch1 signaling contributes to stemness in head and neck squamous cell carcinoma. Laboratory Investigation. 2016;96(5):508.
12-Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, et al. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer research. 2005;65(20):9328-37.
13-Thurber A, Douglas G, Sturm E, Zabierowski S, Smit D, Ramakrishnan S, et al. Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway. Oncogene. 2011;30(27):3036.
14-de Boo J, Hendriksen C. Reduction strategies in animal research: a review of scientific approaches at the intra-experimental, supra-experimental and extra-experimental levels. ATLA-NOTTINGHAM-. 2005;33(4):369.
15-Mo J, Sun B, Zhao X, Gu Q, Dong X, Liu Z, et al. The in-vitro spheroid culture induces a more highly differentiated but tumorigenic population from melanoma cell lines. Melanoma research. 2013;23(4):254-63.
16-Wong CE, Paratore C, Dours-Zimmermann MT, Rochat A, Pietri T, Suter U, et al. Neural crest–derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J Cell Biol. 2006;175(6):1005-15.
17-Howard CM, Valluri J, Alberico A, Julien T, Mazagri R, Marsh R, et al. Analysis of chemopredictive assay for targeting cancer stem cells in glioblastoma patients. Translational oncology. 2017;10(2):241-54.
18-Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Molecular cancer. 2006;5(1):67.
19-Pfenninger CV, Roschupkina T, Hertwig F, Kottwitz D, Englund E, Bengzon J, et al. CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells. Cancer research. 2007;67(12):5727-36.
20-Ernst A, Aigner M, Nakata S, Engel F, Schlotter M, Kloor M, et al. A gene signature distinguishing CD133hi from CD133− colorectal cancer cells: essential role for EGR1 and downstream factors. Pathology-Journal of the RCPA. 2011;43(3):220-7.
21-Rajabi Fomeshi M, Ebrahimi M, Mowla SJ, Sahraneshin Samani F. Evaluating the expression of cell surface markers CD133, CD44 and ABCG2 in melanoma cell lines and its relationship with cancer stem cells. Daneshvar. 2013;20(106):1-12.
22-Sharma BK, Manglik V, O'Connell M, Weeraratna A, McCarron EC, Broussard JN, et al. Clonal dominance of CD133+ subset population as risk factor in tumor progression and disease recurrence of human cutaneous melanoma. International journal of oncology. 2012;41(5):1570-6.
23- Perego M, Tortoreto M, Tragni G, Mariani L, Deho P, Carbone A, et al. Heterogeneous phenotype of human melanoma cells with in vitro and in vivo features of tumor-initiating cells. Journal of Investigative Dermatology. 2010;130(7):1877-86.
24- Setia N, Abbas O, Sousa Y, Garb JL, Mahalingam M. Profiling of ABC transporters ABCB5, ABCF2 and nestin-positive stem cells in nevi, in situ and invasive melanoma. Modern Pathology. 2012;25(8):1169.
25- Kanoh M, Amoh Y, Tanabe K, Maejima H, Takasu H, Katsuoka K. Nestin is expressed in HMB‐45 negative melanoma cells in dermal parts of nodular melanoma. The Journal of dermatology. 2010;37(6):505-11.
26-Fusi A, Reichelt U, Busse A, Ochsenreither S, Rietz A, Maisel M, et al. Expression of the stem cell markers nestin and CD133 on circulating melanoma cells. Journal of Investigative Dermatology. 2011;131(2):487-94.
27-Frank NY, Margaryan A, Huang Y, Schatton T, Waaga-Gasser AM, Gasser M, et al. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer research. 2005;65(10):4320-33.
28-Klein WM, Wu BP, Zhao S, Wu H, Klein-Szanto AJ, Tahan SR. Increased expression of stem cell markers in malignant melanoma. Modern pathology. 2007;20(1):102.
29-Woo T, Okudela K, Mitsui H, Yazawa T. Prognostic value of CD133 expression in stage I lung adenocarcinomas. International journal of clinical and experimental pathology. 2011;4(1):32.
30-Grudzien P, Lo S, Albain KS, Robinson P, Rajan P, Strack PR, et al. Inhibition of Notch signaling reduces the stem-like population of breast cancer cells and prevents mammosphere formation. Anticancer research. 2010;30(10):3853-67.
31-Pinnix CC, Lee JT, Liu Z-J, McDaid R, Balint K, Beverly LJ, et al. Active Notch1 confers a transformed phenotype to primary human melanocytes. Cancer research. 2009;69(13):5312-20.
32-Xu R, Shimizu F, Hovinga K, Beal K, Karimi S, Droms L, et al. Molecular and clinical effects of notch inhibition in glioma patients: a phase 0/I trial. Clinical Cancer Research. 2016;22(19):4786-96.
33-Barat S, Chen X, Cuong Bui K, Bozko P, Götze J, Christgen M, et al. Gamma‐Secretase Inhibitor IX (GSI) Impairs Concomitant Activation of Notch and Wnt‐Beta‐Catenin Pathways in CD44+ Gastric Cancer Stem Cells. Stem cells translational medicine. 2017;6(3):819-29.
34-Marotta LL, Almendro V, Marusyk A, Shipitsin M, Schemme J, Walker SR, et al. The JAK2/STAT3 signaling pathway is required for growth of CD44+ CD24–stem cell–like breast cancer cells in human tumors. The Journal of clinical investigation. 2011;121(7):2723-35.
35-Blank CU, Hooijkaas AI, Haanen JB, Schumacher TN. Combination of targeted therapy and immunotherapy in melanoma. Cancer Immunology, Immunotherapy. 2011;60(10):1359.
36-O’Connell MP, Weeraratna AT. Hear the Wnt Ror: how melanoma cells adjust to changes in Wnt. Pigment cell & melanoma research. 2009;22(6):724-39.
37-Agur Z, Kirnasovsky OU, Vasserman G, Tencer-Hershkowicz L, Kogan Y, Harrison H, et al. Dickkopf1 regulates fate decision and drives breast cancer stem cells to differentiation: an experimentally supported mathematical model. PLoS One. 2011;6(9):e24225.
38-Balint K, Xiao M, Pinnix CC, Soma A, Veres I, Juhasz I, et al. Activation of Notch1 signaling is required for β-catenin–mediated human primary melanoma progression. The Journal of clinical investigation. 2005;115(11):3166-76.
39-Delmas V, Beermann F, Martinozzi S, Carreira S, Ackermann J, Kumasaka M, et al. β-Catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development. Genes & development. 2007;21(22):2923-35.
40-Castoreno AB, Eggert US. Small molecule probes of cellular pathways and networks. ACS chemical biology. 2010;6(1):86-94.