HOX transcription factors are potential therapeutic targets in non-small-cell lung cancer (targeting HOX genes in lung cancer)

Abstract

The HOX genes are a family of homeodomain-containing transcription factors that determine the identity of cells and tissues during embryonic development. They are also known to behave as oncogenes in some haematological malignancies. In this study, we show that the expression of many of the HOX genes is highly elevated in primary non-small-cell lung cancers (NSCLCs) and in the derived cell lines A549 and H23. Furthermore, blocking the activity of HOX proteins by interfering with their binding to the PBX co-factor causes these cells to undergo apoptosis in vitro and reduces the growth of A549 tumours in vivo. These findings suggest that the interaction between HOX and PBX proteins is a potential therapeutic target in NSCLC.

Figure 1

HOX gene expression in primary lung NSCLC tumours, normal adjacent tissue (NAT) and derived cell lines. The expression of each HOX gene was determined by Q-PCR and is presented as a ratio to the amount of β-actin transcript ( × 1000). Error bars show the s.e.m.

Figure 2

HXR9 blocks HOX/PBX dimer formation in NSCLC cells. (A) HXR9 uptake by A549 cells. A fluorescent micrograph of A549 cells incubated with 15 μM HXR9-FITC (green) for 2 h, combined with the corresponding phase-contrast image. HXR9 is primarily located in the nucleus. (B) HOXA2 expression is maintained in part through a HOX/PBX-binding site in its promoter. Correspondingly, Q-PCR analysis of H23 cells treated with 60 μM HXR9 for 2 h reveals that HOXA2 expression is significantly reduced upon HXR9 treatment. (C) ChIP analysis of the HOXA2 promoter. Chromatin was extracted from HXR9- or CXR9-treated cells. After cleavage, fragments were immunoprecipitated using an anti-HOXA2 or an anti-PBX antibody, and the number of copies of the HOXA2 promoter region recovered was determined by Q-PCR. A reduced number of HOXA2 promoter sequences is recovered from HXR9-treated cells when immunoprecipitating with anti-HOXA2 or anti-PBX antibodies, whereas no significant difference is apparent when equal amounts of chromatin are used without selection.

Figure 3

HXR9 triggers apoptosis in A549 and H23 NSCLC cells. Flow cytometric analysis of Annexin-V-PE-stained A549 and H23 cells demonstrated induction of apoptosis in cultures treated with HXR9. Data are the mean±s.e.m. of four independent experiments. ^*P<0.05, significantly different from CXR9-treated cells; ^**P<0.005, highly significantly different from CXR9-treated cells; ^***P<0.0001, extremely significantly different from CXR9-treated cells. (A) Example plots of flow cytometric data for untreated and HXR9-treated A549 cells. x axis – Annexin-V staining indicating degree of apoptotic changes in the cell membrane; y axis – 7-AAD vital dye counterstaining as a measure of cell permeability. (B) Summary of flow cytometry data for A549 cells and (C) for H23 cells. Error bars represent the s.d. (n=4); ‘V’ – viable cells not in apoptosis, ‘EA’ – cells in early apoptosis, ‘LA’ – cells in late apoptosis and ‘N’ – necrotic cells.

Figure 4

HXR9 causes transcriptional changes. (A) Subsequent to treatment with CXR9 or HXR9, RNA was extracted from A549 cells and analysed by microarray. There was no significant change in the transcription of the majority of genes, although five of them showed an increase, and eight a decrease, of five-fold or greater. (B) Semi-quantitative PCR was used to confirm the microarray findings. Gene expression was determined relative to β-actin expression and results are presented as a ratio with the expression of each gene in untreated cells. Error bars represent the s.e. of the mean.

Figure 5

HXR9 blocks tumour growth in vivo. (A) Thymic nude mice were inoculated subcutaneously with 2.5 × 10⁶ A549 cells. When tumours reached volumes of approximately 100 mm³, the animals received an initial dose of 100 mg kg⁻¹ of CXR9 or HXR9 either in to the peritoneum or intratumorally, with subsequent dosing of 10 mg kg⁻¹ twice weekly. (B) Structural changes in HXR9-treated tumours. Tumours were excised from mice after the 18-day treatment cycle (twice weekly intraperitoneal administration of 10 mg kg⁻¹ HXR9), embedded and sectioned. Distinct differences in tumour architecture are apparent in HXR9-treated mice, most notably in areas where extensive cell death has occurred (arrow heads). Scale bar: 100 μm.