NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells

BMC Cancer. 2009 Oct 19:9:371. doi: 10.1186/1471-2407-9-371.

Abstract

Background: Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs.

Methods: Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses.

Results: Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3 and HEY1 was detected in primary TLX1/3 positive T-ALL cells corresponding to the cell line data.

Conclusion: Identification and analysis of MSX2 in hematopoietic cells implicates a modulatory role via NOTCH3-signaling in early T-cell differentiation. Our data suggest that reduction of NOTCH3-signaling by physiological downregulation of MSX2 expression during T-cell development is abrogated by ectopic expression of oncogenic NKLs, substituting MSX2 function.

MeSH terms

  • Cell Differentiation
  • Cell Line, Tumor
  • Cells, Cultured
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Humans
  • Leukemia, T-Cell / genetics
  • Leukemia, T-Cell / metabolism*
  • Leukemia, T-Cell / physiopathology
  • Receptor, Notch3
  • Receptors, Notch / genetics
  • Receptors, Notch / metabolism*
  • Signal Transduction*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / metabolism

Substances

  • Homeodomain Proteins
  • MSX2 protein
  • NOTCH3 protein, human
  • Receptor, Notch3
  • Receptors, Notch