Cancer Stem Cells with Overexpression of Neuronal Markers Enhance Chemoresistance and Invasion in Retinoblastoma

Author(s): Sekaran Balaji, Radhakrishnan Santhi, Usha Kim, Veerappan Muthukkaruppan, Chidambaranathan G. Priya, Ayyasamy Vanniarajan*

Journal Name: Current Cancer Drug Targets

Volume 20 , Issue 9 , 2020


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Graphical Abstract:


Abstract:

Background: Retinoblastoma is a sight and life-threatening embryonal tumor in children. Though chemotherapy is the main mode of therapy, evolving resistance remains a major obstacle in treatment success. The presence of cancer stem cells (CSC) is frequently reported to be responsible for chemoresistance in multiple tumors.

Objective: Our study aims to identify the molecular factors that facilitate the chemoresistance through cancer stem cells in retinoblastoma.

Methods: We developed etoposide and carboplatin resistant retinoblastoma (Y79) cell lines by stepwise drug increment treatment, validated with MTT and TUNEL assays. Colony forming and invasive ability were studied by soft-agar colony forming and transwell assays, respectively. Similar analysis in non-responsive retinoblastoma tumors were carried out by histopathology. Finally, expression of CSC/neuronal markers and ABC transporters were examined by quantitative PCR and protein expression of neuronal stem cell markers was confirmed by Western blot.

Results: Larger colony size of resistant cells in soft-agar assay provided evidence for increased selfrenewability. Histopathology in non-responsive tumors showed poorly differentiated cells predominantly. Besides, both resistant cell lines and non-responsive tumors showed increased invasion with higher expression of neuronal stem cell markers - SOX2, NANOG, OCT4 and ABC transporters - ABCB1 and ABCC3. Increased self-renewal ability and invasion along with overexpression of stemness markers in resistant cells and tumors provide evidence for stemness driving chemoresistance and invasion in retinoblastoma.

Conclusion: We have demonstrated Neuronal stem cell/CSC markers that facilitate the maintenance of cancer stem cells. Developing therapies targeting these factors will help in overcoming resistance and improving retinoblastoma treatment.

Keywords: Chemoresistance, ABC transporter, retinoblastoma, histopathology, cancer stem cell, neuronal stem cell markers.

[1]
Villegas, V.M.; Hess, D.J.; Wildner, A.; Gold, A.S.; Murray, T.G. Retinoblastoma. Curr. Opin. Ophthalmol., 2013, 24(6), 581-588.
[http://dx.doi.org/10.1097/ICU.0000000000000002] [PMID: 24100372]
[2]
Thirumalairaj, K.; Abraham, A.; Devarajan, B.; Gaikwad, N.; Kim, U.; Muthukkaruppan, V.; Vanniarajan, A. A stepwise strategy for rapid and cost-effective RB1 screening in Indian retinoblastoma patients. J. Hum. Genet., 2015, 60(9), 547-552.
[http://dx.doi.org/10.1038/jhg.2015.62] [PMID: 26084579]
[3]
Meel, R.; Radhakrishnan, V.; Bakhshi, S. Current therapy and recent advances in the management of retinoblastoma. Indian J. Med. Paediatr. Oncol., 2012, 33(2), 80-88.
[http://dx.doi.org/10.4103/0971-5851.99731] [PMID: 22988349]
[4]
Chan, H.S.L.; Thorner, P.S.; Haddad, G.; Gallie, B.L. Multidrug-resistant phenotype in retinoblastoma correlates with P-glycoprotein expression. Ophthalmology, 1991, 98(9), 1425-1431.
[http://dx.doi.org/10.1016/S0161-6420(91)32134-1] [PMID: 1682862]
[5]
Chan, H.S.; Lu, Y.; Grogan, T.M.; Haddad, G.; Hipfner, D.R.; Cole, S.P.; Deeley, R.G.; Ling, V.; Gallie, B.L. Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein. Cancer Res., 1997, 57(12), 2325-2330.
[PMID: 9192801]
[6]
Jiang, Z.S.; Sun, Y.Z.; Wang, S.M.; Ruan, J.S. Epithelial-mesenchymal transition: potential regulator of ABC transporters in tumor progression. J. Cancer, 2017, 8(12), 2319-2327.
[http://dx.doi.org/10.7150/jca.19079] [PMID: 28819436]
[7]
Masui, S.; Nakatake, Y.; Toyooka, Y.; Shimosato, D.; Yagi, R.; Takahashi, K.; Okochi, H.; Okuda, A.; Matoba, R.; Sharov, A.A.; Ko, M.S.; Niwa, H. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat. Cell Biol., 2007, 9(6), 625-635.
[http://dx.doi.org/10.1038/ncb1589] [PMID: 17515932]
[8]
Jeon, H.M.; Sohn, Y.W.; Oh, S.Y.; Kim, S.H.; Beck, S.; Kim, S.; Kim, H. ID4 imparts chemoresistance and cancer stemness to glioma cells by derepressing miR-9*-mediated suppression of SOX2. Cancer Res., 2011, 71(9), 3410-3421.
[http://dx.doi.org/10.1158/0008-5472.CAN-10-3340] [PMID: 21531766]
[9]
Lee, S.H.; Oh, S.Y.; Do, S.I.; Lee, H.J.; Kang, H.J.; Rho, Y.S.; Bae, W.J.; Lim, Y.C. SOX2 regulates self-renewal and tumorigenicity of stem-like cells of head and neck squamous cell carcinoma. Br. J. Cancer, 2014, 111(11), 2122-2130.
[http://dx.doi.org/10.1038/bjc.2014.528] [PMID: 25321191]
[10]
Ben-Porath, I.; Thomson, M.W.; Carey, V.J.; Ge, R.; Bell, G.W.; Regev, A.; Weinberg, R.A. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat. Genet., 2008, 40(5), 499-507.
[http://dx.doi.org/10.1038/ng.127] [PMID: 18443585]
[11]
Seigel, G.M.; Campbell, L.M.; Narayan, M.; Gonzalez-Fernandez, F. Cancer stem cell characteristics in retinoblastoma. Mol. Vis., 2005, 11, 729-737.
[PMID: 16179903]
[12]
Seigel, G.M.; Hackam, A.S.; Ganguly, A.; Mandell, L.M.; Gonzalez-Fernandez, F. Human embryonic and neuronal stem cell markers in retinoblastoma. Mol. Vis., 2007, 13, 823-832.
[PMID: 17615543]
[13]
Shukla, S.; Srivastava, A.; Kumar, S.; Singh, U.; Goswami, S.; Chawla, B.; Bajaj, M.S.; Kashyap, S.; Kaur, J. Expression of multidrug resistance proteins in retinoblastoma. Int. J. Ophthalmol., 2017, 10(11), 1655-1661.
[PMID: 29181307]
[14]
Busch, M.; Philippeit, C.; Weise, A.; Dünker, N. Re-characterization of established human retinoblastoma cell lines. Histochem. Cell Biol., 2015, 143(3), 325-338.
[http://dx.doi.org/10.1007/s00418-014-1285-z] [PMID: 25326674]
[15]
Liu, F. Soft–agar colony formation assay. Bio Protoc., 2012, 2(13) e220
[http://dx.doi.org/10.21769/BioProtoc.220]
[16]
Lee, S.H.; Jeyapalan, J.N.; Appleby, V.; Mohamed Noor, D.A.; Sottile, V.; Scotting, P.J. Dynamic methylation and expression of Oct4 in early neural stem cells. J. Anat., 2010, 217(3), 203-213.
[http://dx.doi.org/10.1111/j.1469-7580.2010.01269.x] [PMID: 20646110]]
[17]
Xu, X.L.; Singh, H.P.; Wang, L.; Qi, D.L.; Poulos, B.K.; Abramson, D.H.; Jhanwar, S.C.; Cobrinik, D. Rb suppresses human cone-precursor-derived retinoblastoma tumours. Nature, 2014, 514(7522), 385-388.
[http://dx.doi.org/10.1038/nature13813] [PMID: 25252974]
[18]
Nair, R.M.; Balla, M.M.; Khan, I.; Kalathur, R.K.R.; Kondaiah, P.; Vemuganti, G.K. In vitro characterization of CD133lo cancer stem cells in Retinoblastoma Y79 cell line. BMC Cancer, 2017, 17(1), 779.
[http://dx.doi.org/10.1186/s12885-017-3750-2] [PMID: 29162051]
[19]
Amaral, M.V.S. DE Sousa Portilho, A.J.; DA Silva, E.L.; DE Oliveira Sales, L.; DA Silva Maués, J.H.; DE Moraes, M.E.A.; Moreira-Nunes, C.A. Establishment of drug-resistant cell lines as a model in experimental oncology: A review. Anticancer Res., 2019, 39(12), 6443-6455.
[http://dx.doi.org/10.21873/anticanres.13858] [PMID: 31810908]
[20]
Kim, C.; Gao, R.; Sei, E.; Brandt, R.; Hartman, J.; Hatschek, T.; Crosetto, N.; Foukakis, T.; Navin, N.E. Chemoresistance evolution in triple-negative breast cancer delineated by single-cell sequencing. Cell, 2018, 173(4), 879-893.
[http://dx.doi.org/10.1016/j.cell.2018.03.041]
[21]
Wang, P.; Wan, W.W.; Xiong, S.L.; Feng, H.; Wu, N. Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer. Cell Death Discov., 2017, 3, 16105.
[http://dx.doi.org/10.1038/cddiscovery.2016.105] [PMID: 28179999]
[22]
Chen, X.; Liao, R.; Li, D.; Sun, J. Induced cancer stem cells generated by radiochemotherapy and their therapeutic implications. Oncotarget, 2017, 8(10), 17301-17312.
[http://dx.doi.org/10.18632/oncotarget.14230] [PMID: 28038467]
[23]
Mukherjee, P.; Gupta, A.; Chattopadhyay, D.; Chatterji, U. Modulation of SOX2 expression delineates an end-point for paclitaxel-effectiveness in breast cancer stem cells. Sci. Rep., 2017, 7(1), 9170.
[http://dx.doi.org/10.1038/s41598-017-08971-2] [PMID: 28835684]
[24]
Oliveira, B.R.; Figueiredo, M.A.; Trindade, G.S.; Marins, L.F. OCT4 mutations in human erythroleukemic cells: Implications for multiple drug resistance (MDR) phenotype. Mol. Cell. Biochem., 2015, 400(1-2), 41-50.
[http://dx.doi.org/10.1007/s11010-014-2260-7] [PMID: 25355160]
[25]
Bourguignon, L.Y.; Peyrollier, K.; Xia, W.; Gilad, E. Hyaluronan-CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells. J. Biol. Chem., 2008, 283(25), 17635-17651.
[http://dx.doi.org/10.1074/jbc.M800109200] [PMID: 18441325]
[26]
Cho, Y.; Lee, H.W.; Kang, H.G.; Kim, H.Y.; Kim, S.J.; Chun, K.H. Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer. Oncotarget, 2015, 6(11), 8709-8721.
[http://dx.doi.org/10.18632/oncotarget.3325] [PMID: 25909162]
[27]
Fredlund, E.; Ringnér, M.; Maris, J.M.; Påhlman, S. High Myc pathway activity and low stage of neuronal differentiation associate with poor outcome in neuroblastoma. Proc. Natl. Acad. Sci. USA, 2008, 105(37), 14094-14099.
[http://dx.doi.org/10.1073/pnas.0804455105] [PMID: 18780787]
[28]
Enane, F.O.; Saunthararajah, Y.; Korc, M. Differentiation therapy and the mechanisms that terminate cancer cell proliferation without harming normal cells. Cell Death Dis., 2018, 9(9), 912.
[http://dx.doi.org/10.1038/s41419-018-0919-9] [PMID: 30190481]
[29]
Jögi, A.; Vaapil, M.; Johansson, M.; Påhlman, S. Cancer cell differentiation heterogeneity and aggressive behavior in solid tumors. Ups. J. Med. Sci., 2012, 117(2), 217-224.
[http://dx.doi.org/10.3109/03009734.2012.659294] [PMID: 22376239]
[30]
Chen, Y.L.; Chen, P.M.; Lin, P.Y.; Hsiau, Y.T.; Chu, P.Y. abcg2 overexpression confers poor outcomes in hepatocellular carcinoma of elderly patients. Anticancer Res., 2016, 36(6), 2983-2988.
[PMID: 27272814]
[31]
Filho, J.P.; Correa, Z.M.; Odashiro, A.N.; Coutinho, A.B.; Martins, M.C.; Erwenne, C.M.; Burnier, M.N., Jr Histopathological features and P-glycoprotein expression in retinoblastoma. Invest. Ophthalmol. Vis. Sci., 2005, 46(10), 3478-3483.
[http://dx.doi.org/10.1167/iovs.04-1290] [PMID: 16186322]
[32]
Frank, N.Y.; Schatton, T.; Frank, M.H. The therapeutic promise of the cancer stem cell concept. J. Clin. Invest., 2010, 120(1), 41-50.
[http://dx.doi.org/10.1172/JCI41004] [PMID: 20051635]
[33]
Tripathy, A.; Thakurela, S.; Sahu, M.K.; Uthanasingh, K.; Behera, M.; Ajay, A.K.; Kumari, R. The molecular connection of histopathological heterogeneity in hepatocellular carcinoma: A role of Wnt and Hedgehog signaling pathways. PLoS One, 2018, 13(12) e0208194
[http://dx.doi.org/10.1371/journal.pone.0208194] [PMID: 30513115]
[34]
Baba, J.; Kioi, M.; Akimoto, K.; Nagashima, Y.; Taguri, M.; Inayama, Y.; Aoki, I.; Ohno, S.; Mitsudo, K.; Tohnai, I. atypical protein kinase c λ/ι expression is associated with malignancy of oral squamous cell carcinoma. Anticancer Res., 2018, 38(11), 6291-6297.
[http://dx.doi.org/10.21873/anticanres.12985] [PMID: 30396949]
[35]
Pereira, M.A.; Ramos, M.; Dias, A.R.; Faraj, S.F.; Cirqueira, C.D.S.; de Mello, E.S.; Zilberstein, B.; Alves, V.A.F.; Ribeiro, U., Jr Immunohistochemical expression of thymidylate synthase and prognosis in gastric cancer patients submitted to fluoropyrimidine-based chemotherapy. Chin. J. Cancer Res., 2018, 30(5), 526-536.


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Article Details

VOLUME: 20
ISSUE: 9
Year: 2020
Published on: 27 September, 2020
Page: [710 - 719]
Pages: 10
DOI: 10.2174/1568009620666200504112711
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