WT1 expression in breast cancer disrupts the epithelial/mesenchymal balance of tumour cells and correlates with the metabolic response to docetaxel

WT1 is a transcription factor which regulates the epithelial-mesenchymal balance during embryonic development and, if mutated, can lead to the formation of Wilms’ tumour, the most common paediatric kidney cancer. Its expression has also been reported in several adult tumour types, including breast cancer, and usually correlates with poor outcome. However, published data is inconsistent and the role of WT1 in this malignancy remains unclear. Here we provide a complete study of WT1 expression across different breast cancer subtypes as well as isoform specific expression analysis. Using in vitro cell lines, clinical samples and publicly available gene expression datasets, we demonstrate that WT1 plays a role in regulating the epithelial-mesenchymal balance of breast cancer cells and that WT1-expressing tumours are mainly associated with a mesenchymal phenotype. WT1 gene expression also correlates with CYP3A4 levels and is associated with poorer response to taxane treatment. Our work is the first to demonstrate that the known association between WT1 expression in breast cancer and poor prognosis is potentially due to cancer-related epithelial-to-mesenchymal transition (EMT) and poor chemotherapy response.

[1]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[2]  J. Klijn,et al.  Low-risk susceptibility alleles in 40 human breast cancer cell lines , 2009, BMC Cancer.

[3]  I. Fabregat,et al.  Snail blocks the cell cycle and confers resistance to cell death. , 2004, Genes & development.

[4]  土肥 聡.  WT1 expression correlates with angiogenesis in endometrial cancer tissue , 2012 .

[5]  Z. Dvořák,et al.  Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene. , 2010, Biochemical pharmacology.

[6]  H. Nevanlinna,et al.  Tenascin-C expression in invasion border of early breast cancer: a predictor of local and distant recurrence. , 1998, British Journal of Cancer.

[7]  M. Franco-Molina,et al.  RNAi silencing of the WT1 gene inhibits cell proliferation and induces apoptosis in the B16F10 murine melanoma cell line , 2007, Melanoma research.

[8]  G. Fraizer,et al.  Regulation of vascular endothelial growth factor, VEGF, gene promoter by the tumor suppressor, WT1. , 2007, Frontiers in bioscience : a journal and virtual library.

[9]  S M Hewitt,et al.  Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1. , 1995, Cancer research.

[10]  J. Green,et al.  Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis , 2008, Oncogene.

[11]  Musaffe Tuna,et al.  Downregulation of Wilms' tumor 1 protein inhibits breast cancer proliferation. , 2002, Biochemical and biophysical research communications.

[12]  J. Verweij,et al.  Effect of Cytochrome P450 3A4 Inhibition on the Pharmacokinetics of Docetaxel , 2004, Clinical pharmacology and therapeutics.

[13]  D. Spector,et al.  The Noncoding RNA MALAT 1 Is a Critical Regulator of the Metastasis Phenotype of Lung Cancer Cells , 2013 .

[14]  Y. Kaneda,et al.  The Transcription Factors Tbx18 and Wt1 Control the Epicardial Epithelial-Mesenchymal Transition through Bi-Directional Regulation of Slug in Murine Primary Epicardial Cells , 2013, PloS one.

[15]  C. B. Patel,et al.  Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation. , 2001, Cancer research.

[16]  T. Kafri,et al.  A trans-lentiviral packaging cell line for high-titer conditional self-inactivating HIV-1 vectors. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[17]  G. Moreno-Bueno,et al.  Correlation of Snail expression with histological grade and lymph node status in breast carcinomas , 2002, Oncogene.

[18]  Julia Schüler,et al.  The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs , 2009, Nature Cell Biology.

[19]  Peter Hohenstein,et al.  The many facets of the Wilms' tumour gene, WT1. , 2006, Human molecular genetics.

[20]  E. Gilson,et al.  The Wilms’ tumour suppressor Wt1 is a major regulator of tumour angiogenesis and progression , 2014, Nature Communications.

[21]  A. Chávez-Reyes,et al.  HER2/neu increases the expression of Wilms' Tumor 1 (WT1) protein to stimulate S-phase proliferation and inhibit apoptosis in breast cancer cells , 2005, Oncogene.

[22]  S. Ramaswamy,et al.  Twist, a Master Regulator of Morphogenesis, Plays an Essential Role in Tumor Metastasis , 2004, Cell.

[23]  K. Wagner,et al.  The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo , 2008, Oncogene.

[24]  T. Kudoh,et al.  Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis. , 1996, Blood.

[25]  Jason I. Herschkowitz,et al.  Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer , 2010, Breast Cancer Research.

[26]  A. C. Williams,et al.  Genomic imprinting at the WT1 gene involves a novel coding transcript (AWT1) that shows deregulation in Wilms' tumours. , 2003, Human molecular genetics.

[27]  R. Coombes,et al.  Detection of Wilms' Tumor Antigen–Specific CTL in Tumor-Draining Lymph Nodes of Patients with Early Breast Cancer , 2006, Clinical Cancer Research.

[28]  S. Y. Kim,et al.  Prognostic significance of RSPO1, WNT1, P16, WT1, and SDC1 expressions in invasive ductal carcinoma of the breast , 2013, World Journal of Surgical Oncology.

[29]  Stephen T. C. Wong,et al.  EMT is not required for lung metastasis but contributes to chemoresistance , 2015, Nature.

[30]  D. Spector,et al.  The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. , 2013, Cancer research.

[31]  J. Davies,et al.  A wt1-controlled chromatin switching mechanism underpins tissue-specific wnt4 activation and repression. , 2011, Developmental cell.

[32]  D. Lubahn,et al.  Hypermethylation of the Wilms' tumor suppressor gene CpG island in human breast carcinomas , 1999, Breast Cancer Research and Treatment.

[33]  Rodney J Scott,et al.  Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value , 2015, Nature Communications.

[34]  I. Kawase,et al.  Immunohistochemical detection of WT1 protein in a variety of cancer cells , 2006, Modern Pathology.

[35]  Toshimichi Yoshida,et al.  Tenascin C induces epithelial-mesenchymal transition-like change accompanied by SRC activation and focal adhesion kinase phosphorylation in human breast cancer cells. , 2011, The American journal of pathology.

[36]  C. Ren,et al.  Tumour-initiating capacity is independent of epithelial–mesenchymal transition status in breast cancer cell lines , 2014, British Journal of Cancer.

[37]  Y. Hiasa,et al.  Wilms’ tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma , 2013, Journal of Gastroenterology.

[38]  C. Roberts,et al.  Altered expression of the WT1 wilms tumor suppressor gene in human breast cancer. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[39]  S. Kyo,et al.  Immunohistochemical detection of WT1 protein in endometrial cancer. , 2009, Anticancer research.

[40]  M. Ladomery,et al.  WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing. , 2011, Cancer cell.

[41]  H. Scholz,et al.  Wilms’ tumour protein Wt1 stimulates transcription of the gene encoding vascular endothelial cadherin , 2010, Pflügers Archiv - European Journal of Physiology.

[42]  E. Raso,et al.  WT1 expression in angiogenic tumours of the skin , 2005, Histopathology.

[43]  D. Faratian,et al.  KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals , 2013, Oncogene.

[44]  A. Gill,et al.  Nuclear accumulation of e-cadherin correlates with loss of cytoplasmic membrane staining and invasion in pituitary adenomas. , 2009, The Journal of clinical endocrinology and metabolism.

[45]  Abdelkader Essafi,et al.  Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin , 2009, Nature Genetics.

[46]  C. Jackson-Cook,et al.  Tumorigenicity of SV40 T antigen immortalized human prostate epithelial cells: Association with decreased epidermal growth factor receptor (EGFR) expression , 1994, International journal of cancer.

[47]  M. Minden,et al.  Wilms' Tumor 1 Suppressor Gene Mediates Antiestrogen Resistance via Down-Regulation of Estrogen Receptor-α Expression in Breast Cancer Cells , 2008, Molecular Cancer Research.

[48]  C. Yue,et al.  Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two-hit hypothesis of tumor suppressor gene , 2001, Oncogene.

[49]  Charis Eng,et al.  Direct evidence for epithelial-mesenchymal transitions in breast cancer. , 2008, Cancer research.

[50]  X. Li,et al.  Semaphorin 5A and plexin-B3 regulate human glioma cell motility and morphology through Rac1 and the actin cytoskeleton , 2012, Oncogene.

[51]  Y. Miyoshi,et al.  Prediction of response to docetaxel by CYP3A4 mRNA expression in breast cancer tissues , 2002, International journal of cancer.

[52]  G. Soma,et al.  WT1 expression correlates with angiogenesis in endometrial cancer tissue. , 2010, Anticancer research.

[53]  R. Crescitelli,et al.  WT1 protein is a transcriptional activator of the antiapoptotic bag3 gene , 2010, Leukemia.

[54]  G. Dressler,et al.  Expression of the PAX2 oncogene in human breast cancer and its role in progesterone-dependent mammary growth , 2002, Oncogene.

[55]  Thomas Kirchner,et al.  Migrating cancer stem cells — an integrated concept of malignant tumour progression , 2005, Nature Reviews Cancer.

[56]  D. Loeb,et al.  Isoforms of Wilms’ tumor suppressor gene (WT1) have distinct effects on mammary epithelial cells , 2007, Oncogene.

[57]  P. Smith,et al.  A WT1 antisense oligonucleotide inhibits proliferation and induces apoptosis in myeloid leukaemia cell lines. , 1996, Oncogene.

[58]  J. Licht,et al.  WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak. , 2005, Cancer research.

[59]  M. Sporn,et al.  Repression of the transforming growth factor-beta 1 gene by the Wilms' tumor suppressor WT1 gene product. , 1994, Molecular endocrinology.

[60]  N. Hastie,et al.  The role of Wt1 in regulating mesenchyme in cancer, development, and tissue homeostasis. , 2012, Trends in genetics : TIG.

[61]  M Ingelman-Sundberg,et al.  Cytochrome P450 pharmacogenetics and cancer , 2006, Oncogene.

[62]  L. Esserman,et al.  A genomic predictor of response and survival following taxane-anthracycline chemotherapy for invasive breast cancer. , 2011, JAMA.

[63]  T. Kudoh,et al.  Growth Inhibition of Human Leukemic Cells by WTl ( Wilms Tumor Gene ) Antisense Oligodeoxynucleotides : Implications for the Involvement of WT 1 in Leukemogenesis , 2002 .

[64]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[65]  Zhaoyi Wang,et al.  The Wilms' tumor suppressor WT1 inhibits malignant progression of neoplastigenic mammary epithelial cells. , 2008, Anticancer research.

[66]  V. LeBleu,et al.  EMT Program is Dispensable for Metastasis but Induces Chemoresistance in Pancreatic Cancer , 2015, Nature.

[67]  R. Sutherland,et al.  Wilms' tumor protein 1: an early target of progestin regulation in T-47D breast cancer cells that modulates proliferation and differentiation , 2008, Oncogene.

[68]  Yoshifumi Yamamoto,et al.  Overexpression of the Wilms' tumor gene WT1 in head and neck squamous cell carcinoma , 2003, Cancer science.

[69]  H. Nevanlinna,et al.  Tenascin-C expression in invasion border of early breast cancer — A predictor of local and distant recurrence , 1998 .

[70]  C. Garrett,et al.  Detection of a Novel Truncated WT1 Transcript in Human Neoplasia , 2000, Molecular Diagnosis.

[71]  Y. Oji,et al.  Wilms' tumor gene WT1-shRNA as a potent apoptosis-inducing agent for solid tumors. , 2008, International journal of oncology.

[72]  J. Pelletier,et al.  Functional characterization of WT1 binding sites within the human vitamin D receptor gene promoter. , 2001, Physiological genomics.

[73]  Jing Chen,et al.  ToppGene Suite for gene list enrichment analysis and candidate gene prioritization , 2009, Nucleic Acids Res..

[74]  Y. Miyoshi,et al.  High expression of Wilms' tumor suppressor gene predicts poor prognosis in breast cancer patients. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.