Exosomes of invasive urothelial carcinoma cells are characterized by a specific miRNA expression signature

Muscle-invasive bladder cancer (MIBC) represents a highly aggressive tumor type compared to non-muscle-invasive tumors. MIBC is characterized by specific molecular alterations, which may also modulate extracellular tumorigenic effects. Tumor-associated exosomes, especially exosomal miRNAs, are important regulators in the interaction between tumor cells and tumor microenvironment by affecting tumor-promoting processes in target cells. It is important to analyze whether their exosomal patterns also reflect the specific molecular characteristics of MIBC. The aim of this study was to compare the miRNA expression in secreted exosomes from urinary bladder cancer cells (UBC) with different degrees of invasiveness. By electron microscopy, nanotracking analysis and western blot we proofed a high quality of isolated exosomes. Microarray analysis identified an invasion-associated signature of 15 miRNAs, which is significantly altered in exosomes of invasive UBC compared to non-invasive counterparts. Therefrom, 9 miRNAs are consistent differently expressed in both, invasive cells and their secreted exosomes. The remaining 6 exosome-specific miRNAs are only deregulated in exosomes but not in their parental cells. MiRNA alterations were verified by qPCR in cell culture and urinary exosomes. In conclusion, we showed that exosomes from invasive UBC cells are characterized by a specific miRNA signature. Further analyses have to clarify the functional relevance of exosomal miRNAs secreted by invasive bladder cancer cells for modification of the tumor microenvironment and their putative role as molecular markers in liquid biopsies.

[1]  D. Theodorescu,et al.  Molecular genesis of non-muscle-invasive urothelial carcinoma (NMIUC) , 2010, Expert Reviews in Molecular Medicine.

[2]  Ping Yang,et al.  microRNA-99a acts as a tumor suppressor and is down-regulated in bladder cancer , 2014, BMC Urology.

[3]  A. Jemal,et al.  Cancer statistics, 2013 , 2013, CA: a cancer journal for clinicians.

[4]  Lixue Chen,et al.  Bladder cancer cell-derived exosomes inhibit tumor cell apoptosis and induce cell proliferation in vitro. , 2013, Molecular medicine reports.

[5]  M. Korpal,et al.  The emerging role of miR-200 family of MicroRNAs in epithelial-mesenchymal transition and cancer metastasis , 2008, RNA biology.

[6]  H. Geuze,et al.  Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes* , 1998, The Journal of Biological Chemistry.

[7]  You-cai Zhao,et al.  MicroRNA expression profiles in muscle-invasive bladder cancer: identification of a four-microRNA signature associated with patient survival , 2015, Tumor Biology.

[8]  J. Lozano,et al.  Using microRNA profiling in urine samples to develop a non‐invasive test for bladder cancer , 2013, International journal of cancer.

[9]  G. Goodall,et al.  The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1 , 2008, Nature Cell Biology.

[10]  Masafumi Nakamura,et al.  Purification, characterization and biological significance of tumor-derived exosomes. , 2005, Anticancer research.

[11]  Jian-Bing Fan,et al.  miR-1289 and “Zipcode”-like Sequence Enrich mRNAs in Microvesicles , 2012, Molecular therapy. Nucleic acids.

[12]  S. Jun,et al.  Expression of miRNAs and ZEB1 and ZEB2 correlates with histopathological grade in papillary urothelial tumors of the urinary bladder , 2014, Virchows Archiv.

[13]  C. Bucana,et al.  Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice. , 1995, The Journal of urology.

[14]  Jørgen Kjems,et al.  Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro. , 2009, Cancer research.

[15]  E. Messing,et al.  Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression. , 2014, The Journal of urology.

[16]  L. Du,et al.  Serum microRNA expression signatures identified from genome‐wide microRNA profiling serve as novel noninvasive biomarkers for diagnosis and recurrence of bladder cancer , 2015, International journal of cancer.

[17]  I. Fidler,et al.  The organ microenvironment and cancer metastasis. , 2002, Differentiation; research in biological diversity.

[18]  S. Vacher,et al.  microRNA expression profile in a large series of bladder tumors: Identification of a 3‐miRNA signature associated with aggressiveness of muscle‐invasive bladder cancer , 2013, International journal of cancer.

[19]  T. Orfeo,et al.  One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. , 1977, Journal of the National Cancer Institute.

[20]  P. Altevogt,et al.  Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment , 2015, Oncoimmunology.

[21]  R. Pink,et al.  Routes and mechanisms of extracellular vesicle uptake , 2014, Journal of extracellular vesicles.

[22]  Thomas Thum,et al.  Exosomes: new players in cell-cell communication. , 2012, The international journal of biochemistry & cell biology.

[23]  Wang Jing,et al.  Plasma microRNA profiles for bladder cancer detection. , 2013, Urologic oncology.

[24]  F. Reyal,et al.  Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression. , 2012, Cancer research.

[25]  F. Hamdy,et al.  An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer , 2012, British Journal of Cancer.

[26]  M. Zöller,et al.  Exosomal tumor microRNA modulates premetastatic organ cells. , 2013, Neoplasia.

[27]  K. Rieger-Christ,et al.  A non-invasive miRNA based assay to detect bladder cancer in cell-free urine. , 2015, American journal of translational research.

[28]  Luigi Biancone,et al.  Exosomes/microvesicles as a mechanism of cell-to-cell communication. , 2010, Kidney international.

[29]  T. Ørntoft,et al.  Cellular disposal of miR23b by RAB27-dependent exosome release is linked to acquisition of metastatic properties. , 2014, Cancer research.

[30]  G. Kristiansen,et al.  Circulating microRNAs in serum: novel biomarkers for patients with bladder cancer? , 2014, World Journal of Urology.

[31]  M. Loda,et al.  Identification and Prognostic Significance of an Epithelial-Mesenchymal Transition Expression Profile in Human Bladder Tumors , 2007, Clinical Cancer Research.

[32]  M. Korpal,et al.  The miR-200 Family Inhibits Epithelial-Mesenchymal Transition and Cancer Cell Migration by Direct Targeting of E-cadherin Transcriptional Repressors ZEB1 and ZEB2* , 2008, Journal of Biological Chemistry.

[33]  M. Sporn,et al.  The tumour microenvironment as a target for chemoprevention , 2007, Nature Reviews Cancer.

[34]  A. Llorente,et al.  Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells. , 2012, Biochimica et biophysica acta.

[35]  R. Blackwell,et al.  Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes , 2015, Oncogenesis.

[36]  S. Yip,et al.  Expression of microRNAs in the urine of patients with bladder cancer. , 2012, Clinical genitourinary cancer.

[37]  Lesley Cheng,et al.  Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood , 2014, Journal of extracellular vesicles.

[38]  N. Dubrawsky.  Cancer statistics , 1989, CA: a cancer journal for clinicians.

[39]  Ylva Ivarsson,et al.  Syndecan–syntenin–ALIX regulates the biogenesis of exosomes , 2012, Nature Cell Biology.

[40]  F. Sánchez‐Madrid,et al.  Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs , 2013, Nature Communications.

[41]  Weiying Zhou,et al.  Breast cancer-secreted miR-122 reprograms glucose metabolism in pre-metastatic niche to promote metastasis , 2015, Nature Cell Biology.

[42]  L. O’Driscoll,et al.  Biological properties of extracellular vesicles and their physiological functions , 2015, Journal of extracellular vesicles.

[43]  A. Valencia,et al.  The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies , 2015, BMC Genomics.

[44]  E. Cukierman,et al.  Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. , 2005, Seminars in cancer biology.

[45]  Mark Ibberson,et al.  Endogenous RNAs modulate microRNA sorting to exosomes and transfer to acceptor cells. , 2014, Cell reports.

[46]  S. Povey,et al.  Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines. , 1986, Cancer research.

[47]  P. Kuo,et al.  Characterization of Uptake and Internalization of Exosomes by Bladder Cancer Cells , 2014, BioMed research international.

[48]  Jørgen Kjems,et al.  Coordinated epigenetic repression of the miR‐200 family and miR‐205 in invasive bladder cancer , 2011, International journal of cancer.

[49]  Sascha Keller,et al.  Exosomes: from biogenesis and secretion to biological function. , 2006, Immunology letters.

[50]  Antonio Alcaraz,et al.  MicroRNA in prostate, bladder, and kidney cancer: a systematic review. , 2011, European urology.