HOX transcription factors are potential targets and markers in malignant mesothelioma

The cell lines used in this study are listed in Table 2. They were obtained from the ATCC through LGC Standards Ltd (UK), and were cultured according to the instructions on the LGC Standards website.

HXR9 is an 18 amino acid peptide consisting of the previously identified hexapeptide sequence that can bind to PBX and nine C-terminal arginine residues (R9) that facilitate cell entry. The N-terminal and C-terminal amino bonds are in the D-isomer conformation, which has previously been shown to extend the half-life of the peptide to 12 h in human serum [14]. CXR9 is a control peptide that lacks a functional hexapeptide sequence but which includes the R9 sequence. The sequences of these peptides have been published previously [13]. All peptides were synthesized using conventional column based chemistry and purified to at least 80 % (Biosynthesis Inc., USA).

Cells were plated in 6-well plates using 2 ml of medium and allowed to recover for at least 24 h. When approximately 60 % confluent, cells were treated with the active peptide HXR9 (60 μM) or the control peptide CXR9 (60 μM) for 3 h.

Expression of HOXA4, HOXA9, and HOXB4 in mesothelioma and normal mesothelium tissue was investigated using 3 μm-thick, formalin fixed, paraffin embedded tissue array sections (MS081, US Biomax, Rockville, MD, USA). Immunohistochemical analysis was performed using a monoclonal rabbit anti-HOXB4 antibody (ab676093, 1:100 dilution, Abcam, Cambridge, UK), a polyclonal rabbit anti-HOXA4 antibody (ab131049, 1:500 dilution, Abcam, Cambridge, UK), and a polyclonal rabbit anti-HOXA9 antibody (ab191178, 1:75 dilution, Abcam, Cambridge, UK). The ABC detection method with peroxidase block (DakoCytomation) was used for all of these primary antibodies. Antigen retrieval was performed using pH 9.0 Tris/EDTA buffer (DakoCytomation) and heating in a microwave for 23 min.

Cells were treated with HXR9 or CXR9 as described above. Cell viability was assessed using the MTS assay (Promega) according to the manufacturer’s instructions. Cells were harvested by incubating in trypsin-EDTA (Sigma) at 37 °C until detached and dissociated. Apoptotic cells were identified using flow cytometry (Beckman Coulter Epics XL Flow) and the Annexin V-PE apoptosis detection kit (BD Pharmingen) as described by the manufacturer’s protocol. Caspase-3 activity was measured using the EnzCheck Caspase-3 Assay Kit (Molecular Probes), using the protocol defined by the manufacturer.

Total RNA was isolated from cells using the RNeasy Plus Mini Kit (Qiagen) by following the manufacturer’s protocol. The RNA was denatured by heating to 65 °C for 5 min. cDNA was synthesized from RNA using the Cloned AMV First Strand Synthesis Kit (Invitrogen) according to the manufacturer’s instructions.

Quantitative PCR was performed using the Stratagene MX3005P real-time PCR machine and the Brilliant SYBR Green QPCR Master Mix (Stratagene). The following primers were designed to facilitate the unique amplification of β-actin, c-Fos, and each HOX gene:

HsBeta-ActinF: 5′ ATGTACCCTGGCATTGCCGAC 3′

HsBeta-ActinR: 5′ GACTCGTCATACTCCTGCTTG 3′

HscFos1F: 5′ CCAACCTGCTGAAGGAGAAG 3′

HscFos1R: 5′ GCTGCTGATGCTCTTGACAG 3′

HsHOXA1F: 5′ CTGGCCCTGGCTACGTATAA 3′

HsHOXA1R: 5′ TCCAACTTTCCCTGTTTTGG 3′

HsHOXA4F: 5′ CCCTGGATGAAGAAGATCCA 3′

HsHOXA4R: 5′ AATTGGAGGATCGCATCTTG 3′

HsHOXA5F: 5′ CCGGAGAATGAAGTGGAAAA 3′

HsHOXA5R: 5′ ACGAGAACAGGGCTTCTTCA 3′

HsHOXA9F: 5′ AATAACCCAGCAGCCAACTG 3′

HsHOXA9R: 5′ ATTTTCATCCTGCGGTTCTG 3′

HsHOXB3F: 5′ TATGGCCTCAACCACCTTTC 3′

HsHOXB3R: 5′ AAGCCTGGGTACCACCTTCT 3′

HsHOXB4F: 5′ TCTTGGAGCTGGAGAAGGAA 3′

HsHOXB4R: 5′ GTTGGGCAACTTGTGGTCTT 3′

HsHOXB5F: 5′ AAGGCCTGGTCTGGGAGTAT 3′

HsHOXB5R: 5′ GCATCCACTCGCTCACTACA 3′

HsHOXB6F: 5′ ATTTCCTTCTGGCCCTCACT 3′

HsHOXB6R: 5′ GGAAGGTGGAGTTCACGAAA 3′

HsHOXB9F: 5′ TAATCAAAGACCCGGCTACG 3′

HsHOXB9R: 5′ CTACGGTCCCTGGTGAGGTA 3′

HsHOXC4F: 5′ CGCTCGAGGACAGCCTATAC 3′

HsHOXC4R: 5′ GCTCTGGGAGTGGTCTTCAG 3′

HsHOXC8F: 5′ CTCAGGCTACCAGCAGAACC 3′

HsHOXC8R: 5′ TTGGCGGAGGATTTACAGTC 3′

Athymic nude mice were inoculated subcutaneously with a suspension of 2.5 × 10⁶ MSTO-211H cells in culture media (100 μl). Once tumors reached volumes of approximately 100 mm³, mice were injected IP with PBS or 25 mg/Kg HXR9 in PBS (injection volume 100 μl), every 4 days. The mice were sacrificed after 36 days and the tumors were excised for RNA extraction, as previously described [12]. Each treatment group contained ten mice. The mice were monitored carefully for signs of distress, including behavioral changes and weight loss.

Primary mesothelioma samples were obtained from 16 male and five female patients. The median patient age at diagnosis was 63.9 years (range, 38.2–79.53 years) and median survival was 9.04 months (range, 0.23–81.85 months). Recruitment was via a specialized multidisciplinary thoracic oncology clinic, involving thoracic surgeons, radiation oncologists, and medical oncologists. Histopathology and imaging review was undertaken for all patients. Patients underwent tumor resection at the Department of Thoracic Surgery, Guy’s & St Thomas’ NHS Foundation Trust. Tumor samples were confirmed as mesothelioma by pathological examination and categorized as a sarcomatoid, biphasic, or epithelial type using an antibody panel that included BerEP4, CEA, TTF1, Calretinin, WT1, CK5, MNF116, and EMA. Pseudoanonymised tissues and data were collected by the KHP Cancer Biobank, and subsequently released for this study in accordance with NHS REC approval number 07/H0804/91. Written informed consent was obtained from patients when they agreed to their tissue samples being included in the Biobank, it was not required for the specific use of these tissues in this project.

All values are given as the mean of three independent experiments and error bars show the standard error of the mean. Categorical variables were compared using Student’s t-test or a one-way ANOVA. Survival curves were generated using the Kaplan-Meier method and compared using the log-rank test. A p value < 0.05 was considered to be significant.

In order to assess the expression of HOX genes in mesothelioma we used QPCR to measure RNA levels in four cell lines derived from this malignancy: NCI-H28, NCI-H2052, NCI-H226, and MSTO-211H, together with Met-5A which is derived from non-malignant mesothelium cells (Table 2). HOX gene expression was also studied in primary mesothelioma tumors. The expression of HOX genes within each cell line and between cell lines varied considerably, with MSTO-211H and Met-5A generally having far higher expression than the other cell lines. The only HOX genes expressed uniquely by a single cell line were HOXC12 and HOXD12, in Met-5A. Analysis of HOX genes that are known to have oncogenic or tumor suppressive functions (Table 1) likewise reveals considerable variation, although Met-5A showed higher expression of the potential tumor suppressor genes HOXA4 and HOXA5 compared to the malignant cell lines (Fig. 1a). We also assessed the expression of these HOX genes in 21 primary tumors using RT-QPCR, as well the protein expression of the three most strongly expressed, HOXA4, HOXA9, and HOXB4 at the protein level using immunohistochemistry (Fig. 1b).

We looked for associations between the RNA expression levels of the different HOX genes and patient survival. The tumors of patients surviving less than 6 months had a significantly higher expression of HOXB4 (p = 0.0166; Fig. 1c), and likewise a Kaplan-Meier analysis of overall survival (OS) showed that high HOXB4 tumor expression was associated with a significantly shorter OS (p = 0.041; Fig. 1d).

Given the high level of HOX expression in the mesothelioma cell lines, we treated cells with the HOX / PBX inhibitor HXR9 that has previously been shown to block HOX / PBX interactions and trigger apoptosis in a number of other cancers [11‐17]. Use of a fluorescently labeled version of HXR9 demonstrated that it can be taken up by the cell lines studied here (Fig. 2a), and the MTS assay for cell viability revealed that HXR9 is cytotoxic in all five cell lines (Fig. 2b,c; Table 2). The non-malignant line Met-5A is amongst the least sensitive with an IC50 of 98 μM, whilst the NCI-H28 cell line is the most sensitive with an IC50 of 18 μM (Fig. 2c, Table 2).

Previous studies have suggested that the mechanism of cell death when HOX function is blocked by HXR9 is primarily through apoptosis [11‐17]. To establish whether this is also the case of the mesothelioma derived cell lines, a standard FACS based assay for apoptosis-associated cell membrane changes was used. This involves the use of Annexin V that binds to membrane components usually located on the cytoplasmic side but which relocate to the external surface during apoptosis [19], and a fluorescent dye (7AAD) which binds to DNA but can only enter cells when membrane integrity has been lost. This assay revealed that all the mesothelioma cell lines underwent apoptosis when treated with HXR9 at the relevant IC50 (Fig. 3), with the non-malignant cell line Met-5A showing the lowest level of apoptosis and NCI-H2052 the highest (Fig. 3c).

The induction of apoptosis by HXR9 is thought to depend, at least in part, upon a rapid increase in cFos expression [14], and QPCR analysis of the HXR9 treated cells correspondingly showed a significant increase in cFos in all of the cell lines, with the smallest increase in Met-5A and the largest increase in the most sensitive cell line, NCI-H28 (Fig. 4a). Correspondingly NCI-H28 also showed the greatest increase in Caspase 3 activity (a protease involved in the apoptotic pathway; Fig. 4b), whilst Met-5A failed to show any significant increase in caspase activity (Fig. 4c).

The expression of HOX genes with previously identified oncogenic or tumor suppressor properties (Table 1; Fig. 1), raises the possibility that the expression profile of these genes could determine the sensitivity of cells to HXR9. To assess this we divided HOX genes into two groups – those with potential oncogenic functions, and those with possible tumor suppressor functions. An expression ratio was obtained by dividing the total expression of genes in the former group with that in the latter (‘O/S ratio’). This revealed that the most sensitive cell line, NCI-H28, has the highest O/S ratio, whilst Met-5a and the least sensitive malignant line, NCI-H226, have the lowest O/S ratios (Fig. 5a). Plotting these ratios against the IC50 for each cell line suggest a positive correlation between the O/S ratio and sensitivity (Fig. 5b). Furthermore, the calculated O/S ratios for the primary mesothelium tumors indicate that these cells could also be sensitive HXR9 (Fig. 5b).

In order to determine whether HXR9 could also block tumor growth in vivo, we established a xenograft mouse flank model using the MSTO-211H cell line. Mice were injected IP with either PBS or 25 mg/Kg HXR9 in PBS every 4 days after tumors had grown to a mean volume of 100 mm³. HXR9 significantly retarded tumor growth compared to PBS alone (Fig. 6a). In tumors from mice injected with PBS only, we found a significant, linear relationship between the expression of HOXB4 and final tumor size (r² = 0.8278; p = 0.0321; Fig. 6b).