Homeobox transcription factor muscle segment homeobox 2 (Msx2) correlates with good prognosis in breast cancer patients and induces apoptosis in vitro

Abstract

Introduction: The homeobox-containing transcription factor muscle segment homeobox 2 (Msx2) plays an important role in mammary gland development. However, the clinical implications of Msx2 expression in breast cancer are unclear. The aims of this study were to investigate the potential clinical value of Msx2 as a breast cancer biomarker and to clarify its functional role in vitro.

Methods: Msx2 gene expression was first examined in a well-validated breast cancer transcriptomic dataset of 295 patients. Msx2 protein expression was then evaluated by immunohistochemistry in a tissue microarray (TMA) containing 281 invasive breast tumours. Finally, to assess the functional role of Msx2 in vitro, Msx2 was ectopically expressed in a highly invasive breast tumour cell line (MDA-MB-231) and an immortalised breast cell line (MCF10a), and these cell lines were examined for changes in growth rate, cell death and cell signalling.

Results: Examination of Msx2 mRNA expression in a breast cancer transcriptomic dataset demonstrated that increased levels of Msx2 were associated with good prognosis (P = 0.011). Evaluation of Msx2 protein expression on a TMA revealed that Msx2 was detectable in both tumour cell nuclei and cytoplasm. Cytoplasmic Msx2 expression was associated with low grade tumours (P = 0.012) and Ki67 negativity (P = 0.018). Nuclear Msx2 correlated with low-grade tumours (P = 0.015), estrogen receptor positivity (P = 0.038), low Ki67 (P = 0.005) and high cyclin D1 expression (P = 0.037). Increased cytoplasmic Msx2 expression was associated with a prolonged breast cancer-specific survival (P = 0.049), recurrence-free survival (P = 0.029) and overall survival (P = 0.019). Ectopic expression of Msx2 in breast cell lines resulted in radically decreased cell viability mediated by induction of cell death via apoptosis. Further analysis of Msx2-expressing cells revealed increased levels of p21 and phosphorylated extracellular signal-regulated kinase (ERK) and decreased levels of Survivin and the 'split ends' (SPEN) protein family member RBM15.

Conclusions: We conclude that increased Msx2 expression results in improved outcome for breast cancer patients, possibly by increasing the likelihood of tumour cell death by apoptosis.

Figure 1

Analysis of muscle segment homeobox 2 (Msx2) expression at the mRNA level. Kaplan-Meier estimates of overall survival (OS) stratified by Msx2 expression within the Van de Vijver dataset of 295 breast tumours.

Figure 2

Validation of Msx2 antibody specificity. (a) Western blot analysis of breast cell lines for Msx2 expression, showing Msx2 migrating at approximately 37 kDa. β-actin levels were used to evaluate protein loading. (b) Immunohistochemical staining for Msx2 on formalin-fixed, paraffin-embedded (FFPE) breast cell lines (×20 magnification). (c) Immunohistochemical staining for Msx2 on breast tumour tissue microarray (TMA) cores, showing examples of tumours with scores from 0 to 3 for nuclear and cytoplasmic staining (×20 magnification; scale bar represents 10 μm).

Figure 3

Kaplan-Meier estimates of survival in 281 breast tumours on a TMA. (a) Breast cancer-specific survival (BCSS), (b) recurrence-free survival (RFS), (c) OS stratified according to cytoplasmic Msx2 expression. (d) BCSS, (e) RFS, and (f) OS stratified according to nuclear Msx2 expression.

Figure 4

Characterisation of Msx2-overexpressing breast cell lines. (a) Light microscopic images of MDA-MB-231 and MCF10a cell lines, transduced with EV or Msx2 expression vector, showing increased cell death in Msx2-overexpressing cells. (b) Immunofluorescence microscopy detecting Msx2 localisation in MDA-MB-231 and MCF10a cell lines with ectopic Msx2 expression. Scale bar represents 10 μm. Images are representative of three experiments. (c) Examination of a number of cell cycle and Msx2-interacting proteins by Western blot analysis in empty vector and Msx2-overexpressing MDA-MB-231 and MCF10a cell lines. Images are representative of three replicates.

Figure 5

Functional analysis of Msx2-overexpressing breast cell lines. (a) Measurement of cell proliferation rates by MTT assay. Significance was determined by comparing the slopes of the lines of best fit. (b) Measurement of colony forming ability of cellsby clonogenic assays. (c) Measurement of caspase-3 and -7 activation as an indicator of apoptosis. Values are relative to control levels. (d) Cell cycle analysis by propidium iodide staining. Values represent the percentage of cells within sub-G1 gate. For all assays, values denote the mean readings of three independent replicates (n = 3 within each experiment), and error bars represent SD within replicate experiments. Unpaired, two-tailed Student's t-tests were used to determine the significance of difference between samples.