Molecular Assessment of Non-Muscle Invasive and Muscle Invasive Bladder Tumors: Mapping of Putative Urothelial Stem Cells and Toll-Like Receptors (TLR) Signaling

Rafael Mamprin Stopiglia¹, Wagner Eduardo Matheus^1*, Patrick Vianna Garcia², Athanase Billis³, Mariana Anteghini Castilho⁴, Vitor Hugo Figueiredo de Jesus⁴, Ubirajara Ferreira¹, Wagner José Fávaro²

¹Department of Urology, School of Medicine, University of Campinas-UNICAMP, Sao Paulo, Brazil.
²Laboratory of Urogenital Carcinogenesis and Immunotherapy, Institute of Biology, University of Campinas-UNICAMP, Sao Paulo, Brazil.
³Department of Pathology, School of Medicine, University of Campinas-UNICAMP, Sao Paulo, Brazil.
⁴Botucatu School of Medicine, Universidade Estadual Paulista-UNESP, Botucatu, Brazil.

DOI: 10.4236/jct.2015.62014                

Abstract

Purpose: The main objectives of this study were to characterize and compare the urothelial stem cells (healthy and cancer cells) and TLRs features in the urinary bladder of men without lesionsand with non-muscle-invasive and muscle invasive urothelial tumors. Materials and Methods: Thirty samples of the urinary bladder of 50 to 80-year-old men, with and without diagnosis of malignant urothelial lesions were used. The 30 samples were divided into 3 groups (n = 10 per group): Normal Group; Non-Muscle Invasive Bladder Cancer Group; Muscle Invasive Bladder Cancer Group. The samples were histopathologically and immunohistochemically analyzed. The study was conducted at teaching Hospital of the University of Campinas (UNICAMP). Results: The CD44 and CD133 immunoreactivities were significantly intense in the muscle-invasive cancer group when compared to the other groups. The ABCG2 biomarker demonstrated intense immunoreactivities in both non-muscle and muscle invasive groups, and absent immunoreactivity in the normal group. All groups showed weak CD117 immunoreactivity. Putative Healthy Stem Cells (CD44/CD133/ CD117⁺) occurred in all groups. Putative Cancer Stem Cells (CD44/CD133/ABCG2⁺) only occurred in the non-muscle and muscle invasive cancer groups. TLR2 immunoreactivity was significantly lower in the non-muscle invasive cancer group and absent in the muscle invasive cancer group. TLR4 immunoreactivity was significantly lower in both cancer groups. Conclusions: This study leads us to the conclusion that putative cancer stem cell occurrence was sensitive to the decreased in TLR2 and TLR4 immunoreactivities. Also, TLR2 and TLR4 demonstrated their involvement in the regulation of the different biomarkers for putative healthy and cancer urothelial stem cells, probably acting as negative regulators of urothelial carcinogenesis. Taken together data obtained suggest that use of TLRs agonists could be a promising alternative for the treatment of non-muscle and muscle invasive bladder tumors.

Keywords

Non-Muscle Invasive Bladder Cancer, Muscle Invasive Bladder Cancer, Toll-Like Receptors, Cancer Stem Cell, Stem Cells Biomarkers

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	[1] Bryan, R.T. (2011) Bladder Cancer and Cancer Stem Cells: Basic Science and Implications for Therapy. The Scientific World Journal, 11, 1187-1194. http://dx.doi.org/10.1100/tsw.2011.117
[2]	Hatina, J. and Schulz, W.A. (2012) Stem Cells in the Biology of Normal Urothelium and Urothelium Carcinoma. Neoplasma, 59, 728-736. http://dx.doi.org/10.4149/neo_2012_089
[3]	Zupancic, D., Zakrajsek, M., Zhou, G. and Romih, R. (2011) Expression and Localization of Four Uroplakins in Urothelial Preneoplastic Lesions. Histochemistry and Cell Biology, 136, 491-500. http://dx.doi.org/10.1007/s00418-011-0857-4
[4]	Kroft, S.H. and Oyasu, R. (1994) Urinary Bladder Cancer: Mechanisms of Development and Progression. Laboratory Investigation, 71, 158-174.
[5]	Shimada, K., Fujii, T., Anai, S., Fujimoto, K. and Konishi, N. (2011) ROS Generation via NOX4 and Its Utility in the Cytological Diagnosis of Urothelial Carcinoma of the Urinary Bladder. BMC Urology, 11, 22. http://dx.doi.org/10.1186/1471-2490-11-22
[6]	Bentivegna, A., Conconi, D., Panzeri, E., Sala, E., Bovo, G., Vigano, P., Brunelli, S., Bossi, M., Tredici, G., Strada, G. and Dalpra, L. (2010) Biological Heterogeneity of Putative Bladder Cancer Stem-Like Cell Populations from Human Bladder Transitional Cell Carcinoma Samples. Cancer Science, 101, 416-424. http://dx.doi.org/10.1111/j.1349-7006.2009.01414.x
[7]	Tang, Y., Hamburger, A.W., Wang, L., Khan, M.A. and Hussain, A. (2010) Androgen Deprivation and Stem Cell Markers in Prostate Cancers. International Journal of Clinical and Experimental Pathology, 10, 128-138.
[8]	Moltzahn, F.R., Volkmer, J.P., Rottke, D. and Ackermann, R. (2008) Cancer Stem Cells-Lessons from Hercules to Fight the Hydra. Urologic Oncology, 26, 581-589. http://dx.doi.org/10.1016/j.urolonc.2008.07.009
[9]	Gaisa, N.T., Graham, T.A., McDonald, S.A., Canadillas-Lopez, S., Poulsom, R. and Heidenreich, A. (2011) The Human Urothelium Consists of Multiple Clonal Units, Each Maintained by a Stem Cell. The Journal of Pathology, 225, 163-171. http://dx.doi.org/10.1002/path.2945
[10]	McConkey, D.J., Lee, S., Choi, W., Tran, M. and Majewski, T. (2010) Molecular Genetics of Bladder Cancer: Emerging Mechanisms of Tumor Initiation and Progression. Urologic Oncology, 28, 429-440. http://dx.doi.org/10.1016/j.urolonc.2010.04.008
[11]	Visvader, J.E. and Lindeman, G.J. (2008) Cancer Stem Cells in Solid Tumors: Accumulating Evidence and Unresolved Questions. Nature Reviews Cancer, 8, 755-768. http://dx.doi.org/10.1038/nrc2499
[12]	Richardson, G.D., Robson, C.N., Lang, S.H., Neal, D.E., Maitland, N.J. and Collins, A.T. (2006) CD133, a Novel Marker for Human Prostatic Epithelial Stem Cells. Journal of Cell Science, 117, 3539-3545. http://dx.doi.org/10.1242/jcs.01222
[13]	Akira, S., Uematsu, S. and Takeuchi, O. (2006) Pathogen Recognition and Innate Immunity. Cell, 124, 783-801. http://dx.doi.org/10.1016/j.cell.2006.02.015
[14]	Srikrishna, G. and Freeze, H.H. (2009) Endogenous Damage-Associated Molecular Pattern Molecules at the Crossroads of Inflammation and Cancer. Neoplasia, 11, 615-628.
[15]	Rakoff-Nahoum, S. and Medzhitov, R. (2009) Toll-Like Receptors and Cancer. Nature Reviews Cancer, 9, 57-63. http://dx.doi.org/10.1038/nrc2541
[16]	Epstein, J.L., Amin, M.B., Reuter, V.R. and Mostoli, F.K., Bladder Consensus Conference Committee (1998) The World Health Organization/International Society of Urological Pathology Consensus Classification of Urothelial (Transitional Cell) Neoplasms of the Urinary Bladder. American Journal of Surgical Pathology, 22, 1435-1448. http://dx.doi.org/10.1097/00000478-199812000-00001
[17]	Fávaro, W.J., Hetzl, A.C., Reis, L.O., Ferreira, U., Billis, A. and Cagnon, V.H. (2012) Periacinar Retraction Clefting in Nonneoplastic and Neoplastic Prostatic Glands: Artifact or Molecular Involvement. Pathology & Oncology Research, 18, 285-292. http://dx.doi.org/10.1007/s12253-011-9440-5
[18]	Messing, E.M. (2002) Urothelial Tumors of the Urinary Tract. In: Walsh, P.C., Retik, A.B., Vaughan Jr., E.D., et al., Eds., Campbell’s Urology, 8th Edition, Saunders, Philadelphia, 2732-2784.
[19]	Borden Jr., L.S., Clark, P.E. and Hall, M.C. (2005) Bladder Cancer. Current Opinion in Oncology, 17, 275-280. http://dx.doi.org/10.1097/01.cco.0000156985.47984.9e
[20]	Zeegers, M.P., Tan, F.E. and Dorant, E. (2000) The Impact of Characteristics of Cigarette Smoking on Urinary Tract Cancer Risk: A Meta-Analysis of Epidemiologic Studies. Cancer, 89, 630-639. http://dx.doi.org/10.1002/1097-0142(20000801)89:3<630::AID-CNCR19>3.0.CO;2-Q
[21]	Grimmer, G., Dettbarn, G. and Seidel, A. (2000) Detection of Carcinogenic Aromatic Amines in the Urine of Non-Smokers. Science of the Total Environment, 247, 81-90. http://dx.doi.org/10.1016/S0048-9697(99)00471-4
[22]	Fabian, A., Barok, M., Vereb, G. and Szollosi, J. (2009) Die Hard: Are Cancer Stem Cells the Bruce Willises of Tumor Biology? Cytometry Part A, 75, 67-74. http://dx.doi.org/10.1002/cyto.a.20690
[23]	Pardal, R., Clarke, M.F. and Morrison, S.J. (2003) Applying the Principles of Stem-Cell Biology to Cancer. Nature Reviews Cancer, 3, 895-902. http://dx.doi.org/10.1038/nrc1232
[24]	Bentivegna, A., Conconi, D., Panzeri, E., Sala, E., Bovo, G., Vigano, P., Brunelli, S., Bossi, M., Tredici, G., Strada, G. and Dalpra, L. (2010) Biological Heterogeneity of Putative Bladder Cancer Stem-Like Cell Populations from Human Bladder Transitional Cell Carcinoma Samples. Cancer Science, 101, 416-424. http://dx.doi.org/10.1111/j.1349-7006.2009.01414.x
[25]	Miki, J. (2009) Investigations of Prostate Epithelial Stem Cells and Prostate Cancer Stem Cells. International Journal of Urology, 17, 139-147. http://dx.doi.org/10.1111/j.1442-2042.2009.02438.x
[26]	Alam, T.N., O’hare, M.J., Laczko, I., Freeman, A., Al-Beidh, F., Masters, J.R. and Hudson, D.L. (2004) Differential Expression of CD44 during Human Prostate Epithelial Cell Differentiation. Journal of Histochemistry Cytochemistry, 52, 1083-1090. http://dx.doi.org/10.1369/jhc.4A6256.2004
[27]	Ugolkov, A.V., Eisengart, L.J., Luan, C. and Yang, X.J. (2010) Expression Analysis of Putative Stem Cell Markers in Human Benign and Malignant Prostate. Prostate, 71, 18-25. http://dx.doi.org/10.1002/pros.21217
[28]	Taylor, R.A. and Risbridger, G.P. (2008) The Path toward Identifying Prostatic Stem Cells. Differentiation, 76, 671-681. http://dx.doi.org/10.1111/j.1432-0436.2008.00289.x
[29]	Leong, K.G., Wang, B.E., Johnson, L. and Gao, W.Q. (2008) Generation of a Prostate from a Single Adult Stem Cell. Nature, 456, 804-808. http://dx.doi.org/10.1038/nature07427
[30]	Kasper, S. (2009) Identification, Characterization, and Biological Relevance of Prostate Cancer Stem Cells from Clinical Specimens. Urologic Oncology, 27, 301-303. http://dx.doi.org/10.1016/j.urolonc.2008.12.012
[31]	Ayari, C., Bergeron, A., LaRue, H., Menard, C. and Fradet, Y. (2011) Toll-Like Receptors in Normal and Malignant Human Bladders. Journal of Urology, 185, 1915-1921. http://dx.doi.org/10.1016/j.juro.2010.12.097
[32]	Fávaro, W.J., Nunes, O.S., Seiva, F.R., Nunes, I.S., Woolhiser, L.K., Duran, N. and Lenaerts, A.J. (2012) Effects of P-MAPA Immunomodulator on Toll-Like Receptors and p53: Potential Therapeutic Strategies for Infectious Diseases and Cancer. Infectious Agents and Cancer, 7, 14. http://dx.doi.org/10.1186/1750-9378-7-14