mRNA expression profiling of phyllodes tumours of the breast: identification of genes important in the development of borderline and malignant phyllodes tumours

The aim of this study was to identify genes involved in the development of borderline and malignant phyllodes tumours of the breast (PTs). Expression profiling of 23 PTs (12 benign, 11 borderline/malignant) was performed using Affymetrix U133A GeneChips. mRNA expression in the borderline/malignant PTs was compared to the benign PTs. A group of 162 genes was over‐expressed in the borderline/malignant group with a fold change > 2 and FDR < 0.1. Four of these genes were chosen for further investigation: PAX3, SIX1, TGFB2 and HMGA2. Over‐expression was validated in a separate set of formalin‐fixed, paraffin‐embedded (FFPE) tumours, using either in situ hybridization or immunohistochemistry. This confirmed that expression of PAX3, SIX1, TGFB2 and HMGA2 in the stromal component of PTs was associated with the borderline/malignant phenotypes (p = 8.7 × 10−5, p = 0.05, p = 0.009, p = 0.003, respectively; Fisher's exact test). The functional consequences of down‐regulating these genes were studied using siRNA in short‐term cultures and cell lines established from PTs. mRNA ‘knock‐down’ of PAX3 resulted in significantly decreased cell proliferation in both a malignant and a borderline PT cell culture. mRNA ‘knock‐down’ of SIX1 and HMGA2 resulted in decreased cell proliferation only in the malignant PT cell line, and ‘knock‐down’ of TGFB2 resulted in decreased cell proliferation only in the borderline PT cell culture. This study shows that these four genes are involved in the development of borderline/malignant PTs. SIX1 over‐expression was most marked in the highly malignant PTs, with particularly high expression in one case of metastatic PT. PAX3, TGFB2 and HMGA2 were expressed predominantly in borderline/malignant PTs, but showed some expression in benign tumours; they may be important in the transition from the benign to borderline/malignant phenotype. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

[1]  E. Sawyer,et al.  A comprehensive genetic profile of phyllodes tumours of the breast detects important mutations, intra‐tumoral genetic heterogeneity and new genetic changes on recurrence , 2008, The Journal of pathology.

[2]  J. Coindre,et al.  Gains and complex rearrangements of the 12q13‐15 chromosomal region in ordinary lipomas: The “missing link” between lipomas and liposarcomas? , 2007, International journal of cancer.

[3]  H. Ford,et al.  Six1 overexpression in ovarian carcinoma causes resistance to TRAIL-mediated apoptosis and is associated with poor survival. , 2007, Cancer research.

[4]  A. Vincent-Salomon,et al.  Phyllodes tumors of the breast segregate in two groups according to genetic criteria , 2007, Modern Pathology.

[5]  A. Bosserhoff,et al.  An imbalance between Smad and MAPK pathways is responsible for TGF-beta tumor promoting effects in high-grade gliomas. , 2007, International journal of oncology.

[6]  F. Barr,et al.  Fusions involving PAX and FOX genes in the molecular pathogenesis of alveolar rhabdomyosarcoma: recent advances. , 2007, Current molecular medicine.

[7]  C. Mayanil,et al.  Regulation of Murine TGFβ2 by Pax3 during Early Embryonic Development* , 2006, Journal of Biological Chemistry.

[8]  Y. Okada,et al.  Misexpression of full-length HMGA2 induces benign mesenchymal tumors in mice. , 2006, Cancer research.

[9]  G. Hamard,et al.  Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo , 2005, Development.

[10]  H. Ford,et al.  Gene amplification is a mechanism of Six1 overexpression in breast cancer. , 2005, Cancer research.

[11]  S. Der,et al.  Cell-type-specific regulation of distinct sets of gene targets by Pax3 and Pax3/FKHR , 2005, Oncogene.

[12]  N. Carter,et al.  Array-CGH analysis of microsatellite-stable, near-diploid bowel cancers and comparison with other types of colorectal carcinoma , 2005, Oncogene.

[13]  C. Parker,et al.  Expression of PAX 3 alternatively spliced transcripts and identification of two new isoforms in human tumors of neural crest origin , 2004, International journal of cancer.

[14]  Javed Khan,et al.  Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators , 2004, Nature Medicine.

[15]  N. Carter,et al.  DNA microarrays for comparative genomic hybridization based on DOP‐PCR amplification of BAC and PAC clones , 2003, Genes, chromosomes & cancer.

[16]  I. Ellis,et al.  The Wnt pathway, epithelial–stromal interactions, and malignant progression in phyllodes tumours , 2002, The Journal of pathology.

[17]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[18]  I. Skerjanc,et al.  Pax3 Is Essential for Skeletal Myogenesis and the Expression of Six1 and Eya2* , 2001, The Journal of Biological Chemistry.

[19]  D. Rifkin,et al.  Tamoxifen and Estrogen Effects on TGF-β Formation: Role of Thrombospondin-1, αvβ3, and Integrin-Associated Protein , 2001 .

[20]  M. Molls,et al.  Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH. , 2000, Mutation research.

[21]  I. Ellis,et al.  Molecular analysis of phyllodes tumors reveals distinct changes in the epithelial and stromal components. , 2000, The American journal of pathology.

[22]  H. Beug,et al.  TGFβ signaling is necessary for carcinoma cell invasiveness and metastasis , 1998, Current Biology.

[23]  R. Poulsom,et al.  Expression and distribution of TGFβ mRNA isoforms in a small group of human breast cancers examined by in situ hybridization , 1995 .

[24]  M. Sporn,et al.  Type beta transforming growth factor: a bifunctional regulator of cellular growth. , 1985, Proceedings of the National Academy of Sciences of the United States of America.