Osteosarcoma Stem Cells Have Active Wnt/β‐catenin and Overexpress SOX2 and KLF4

Osteosarcoma is a bone tumor, displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma, a consensus on their characterization is still missing. We used a combination of functional assays (sphere‐forming, Aldefluor, and side‐population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness‐related transcription factors, quiescent nature, in vivo tumorigenicity, and Wnt/β‐catenin activation were evaluated. We show that different cancer stem cell populations may co‐exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly‐proliferating populations, overexpress SOX2, and KLF4 stemness‐related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β‐catenin signaling as evidenced by increased nuclear β‐catenin, TCF/LEF activity, and AXIN2 expression, in a subset of the cell lines. Aldefluor‐positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side‐population phenotype is correlated with ABCG2 drug‐efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. J. Cell. Physiol. 231: 876–886, 2016. © 2015 Wiley Periodicals, Inc.

[1]  I. Weissman,et al.  A role for Wnt signalling in self-renewal of haematopoietic stem cells , 2003, Nature.

[2]  Wange Lu,et al.  Klf4 Interacts Directly with Oct4 and Sox2 to Promote Reprogramming , 2009, Stem cells.

[3]  A. Assalian,et al.  MICROCAT: A Novel Cell Proliferation and Cytotoxicity Assay Based on WST-1 , 1996 .

[4]  Carl R Walkley,et al.  Cells of origin in osteosarcoma: mesenchymal stem cells or osteoblast committed cells? , 2014, Bone.

[5]  Kornelia Polyak,et al.  Heterogeneity for Stem Cell–Related Markers According to Tumor Subtype and Histologic Stage in Breast Cancer , 2010, Clinical Cancer Research.

[6]  S. Lyle,et al.  Quiescent, Slow-Cycling Stem Cell Populations in Cancer: A Review of the Evidence and Discussion of Significance , 2010, Journal of oncology.

[7]  N. Agarwal,et al.  CD 117 and Stro-1 Identify Osteosarcoma Tumor-Initiating Cells Associated with Metastasis and Drug Resistance , 2010 .

[8]  X. Zi,et al.  The Wnt signaling pathway: implications for therapy in osteosarcoma , 2011, Expert review of anticancer therapy.

[9]  B. Alman,et al.  Side population cells isolated from mesenchymal neoplasms have tumor initiating potential. , 2007, Cancer research.

[10]  Hongwei Xu,et al.  The oxidative stress response regulates DKK1 expression through the JNK signaling cascade in multiple myeloma plasma cells. , 2007, Blood.

[11]  Profiling of high-grade central osteosarcoma and its putative progenitor cells identifies tumourigenic pathways , 2009, British Journal of Cancer.

[12]  F. Chiaradonna,et al.  Energy Metabolism Characterization of a Novel Cancer Stem Cell‐Like Line 3AB‐OS , 2014, Journal of cellular biochemistry.

[13]  Edward W Scott,et al.  Stem-like cells in bone sarcomas: implications for tumorigenesis. , 2005, Neoplasia.

[14]  R. Tuan,et al.  Concise Review: The Surface Markers and Identity of Human Mesenchymal Stem Cells , 2014, Stem cells.

[15]  A. Balmain,et al.  Guidelines for the welfare and use of animals in cancer research , 2010, British Journal of Cancer.

[16]  Satoshi Tanaka,et al.  Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML , 2010, Nature Biotechnology.

[17]  A. Allan,et al.  The Role of Human Aldehyde Dehydrogenase in Normal and Cancer Stem Cells , 2011, Stem Cell Reviews and Reports.

[18]  C. Eaves,et al.  Heterogeneity of neoplastic stem cells: theoretical, functional, and clinical implications. , 2013, Cancer research.

[19]  A. Gentles,et al.  Targeting Unique Metabolic Properties of Breast Tumor Initiating Cells , 2014, Stem cells.

[20]  T. Hughes,et al.  E2F1 up-regulates the expression of the tumour suppressor axin2 both by activation of transcription and by mRNA stabilisation. , 2005, Biochemical and biophysical research communications.

[21]  A. Cleton-Jansen,et al.  Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2 , 2009, The Journal of pathology.

[22]  Choun-Ki Joo,et al.  Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway , 2002, Molecular and Cellular Biology.

[23]  A. Llombart‐Bosch,et al.  Functional characterization of osteosarcoma cell lines provides representative models to study the human disease , 2011, Laboratory Investigation.

[24]  J. Mather.  Concise Review: Cancer Stem Cells: In Vitro Models , 2012 .

[25]  Bob van de Water,et al.  Aven‐mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma , 2015, The Journal of pathology.

[26]  O. Myklebost,et al.  Molecular characterization of commonly used cell lines for bone tumor research: A trans‐European EuroBoNet effort , 2010, Genes, chromosomes & cancer.

[27]  Ian A. White,et al.  CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. , 2008, The Journal of clinical investigation.

[28]  K. Nagashima,et al.  Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma , 2012, PloS one.

[29]  T. He,et al.  Osteosarcoma Development and Stem Cell Differentiation , 2008, Clinical orthopaedics and related research.

[30]  S. Niclou,et al.  Critical appraisal of the side population assay in stem cell and cancer stem cell research. , 2011, Cell stem cell.

[31]  D. Placantonakis,et al.  Sox2 antagonizes the Hippo pathway to maintain stemness in cancer cells , 2015, Nature Communications.

[32]  S. Orkin,et al.  Sox2 maintains self-renewal of tumor initiating cells in osteosarcomas , 2011, Oncogene.

[33]  Chia-Ying Lin,et al.  Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99‐1 cells based on high aldehyde dehydrogenase activity , 2011, International journal of cancer.

[34]  T. Reya,et al.  Loss of β-Catenin Impairs the Renewal of Normal and CML Stem Cells In Vivo , 2007 .

[35]  H. Taubert,et al.  Alternate splicing of the p53 inhibitor HDMX offers a superior prognostic biomarker than p53 mutation in human cancer. , 2012, Cancer research.

[36]  A. Moussaieff,et al.  Concise Review: Energy Metabolites: Key Mediators of the Epigenetic State of Pluripotency , 2015, Stem cells.

[37]  Irving L Weissman,et al.  Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. , 2006, Cancer research.

[38]  Eduard Batlle,et al.  Metastatic stem cells: sources, niches, and vital pathways. , 2014, Cell stem cell.

[39]  A. Cleton-Jansen,et al.  Inactive Wnt/β‐catenin pathway in conventional high‐grade osteosarcoma , 2010, The Journal of pathology.

[40]  P. Chambon,et al.  Cutaneous cancer stem cell maintenance is dependent on β-catenin signalling , 2008, Nature.

[41]  A. Paiva,et al.  Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line , 2012, BMC Cancer.

[42]  Hans Clevers,et al.  Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas , 2012, Science.

[43]  N. Agarwal,et al.  CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance. , 2010, Cancer research.

[44]  J. Mather,et al.  CANCER STEM CELLS Concise Review: Cancer Stem Cells: In Vitro Models , 2012 .

[45]  G. Siegal,et al.  Osteosarcoma: anatomic and histologic variants. , 2006, American journal of clinical pathology.

[46]  S. Morrison,et al.  Prospective identification of tumorigenic breast cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Chia-Ying Lin,et al.  Characterization of stem cell attributes in human osteosarcoma cell lines , 2009, Cancer biology & therapy.

[48]  R. Weinberg,et al.  Tackling the cancer stem cells — what challenges do they pose? , 2014, Nature Reviews Drug Discovery.

[49]  E. van Marck,et al.  Does the histological subtype of high-grade central osteosarcoma influence the response to treatment with chemotherapy and does it affect overall survival? A study on 570 patients of two consecutive trials of the European Osteosarcoma Intergroup. , 2002, European journal of cancer.

[50]  Lianhong Li,et al.  Roles of Wnt/β-catenin signaling in the gastric cancer stem cells proliferation and salinomycin treatment , 2014, Cell Death and Disease.

[51]  Anne-Marie Cleton-Jansen,et al.  mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts , 2011, BMC Medical Genomics.

[52]  Cynthia Hawkins,et al.  Identification of a cancer stem cell in human brain tumors. , 2003, Cancer research.

[53]  F. Hamdy,et al.  High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer. , 2010, Cancer research.

[54]  M. Schilham,et al.  Anti-EGFR Antibody Cetuximab Enhances the Cytolytic Activity of Natural Killer Cells toward Osteosarcoma , 2011, Clinical Cancer Research.

[55]  Erika Pastrana,et al.  Eyes wide open: a critical review of sphere-formation as an assay for stem cells. , 2011, Cell stem cell.

[56]  A. Shiau,et al.  Oct-3/4 expression reflects tumor progression and regulates motility of bladder cancer cells. , 2008, Cancer research.

[57]  Diana Anderson.  In Vitro Models , 1990, Drug safety.

[58]  W. Quint,et al.  Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification , 1993, Applied and environmental microbiology.

[59]  Megan F. Cole,et al.  Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells , 2005, Cell.

[60]  C. Fontes-Ribeiro,et al.  Sensitizing osteosarcoma stem cells to doxorubicin-induced apoptosis through retention of doxorubicin and modulation of apoptotic-related proteins. , 2015, Life sciences.

[61]  Goberdhan P Dimri,et al.  A Positive Feedback Loop Regulates the Expression of Polycomb Group Protein BMI1 via WNT Signaling Pathway* , 2012, The Journal of Biological Chemistry.