Young-Bem Se and Seung Hyun Kim contributed equally to this work.
Homeobox (HOX) genes are essential developmental regulators that should normally be in the silenced state in an adult brain. The aberrant expression of HOX genes has been associated with the prognosis of many cancer types, including glioblastoma (GBM). This study examined the identity and role of HOX genes affecting GBM prognosis and treatment resistance.
The full series of HOX genes of five pairs of initial and recurrent human GBM samples were screened by microarray analysis to determine the most plausible candidate responsible for GBM prognosis. Another 20 newly diagnosed GBM samples were used for prognostic validation.
The underexpression of HOXA11 was identified as a consistent signature for a poor prognosis among the HOX genes. The overall survival of the GBM patients indicated a significantly favorable prognosis in patients with high HOXA11 expression (31±15.3 months) compared to the prognoses in thosewith low HOXA11 expression (18±7.3 months, p=0.03). When HOXA11 was suppressed in the GBM cell lines, the anticancer effect of radiotherapy and/or temozolomide declined. In addition, five candidate mediators (
The treatment resistance induced by the underexpression of HOXA11 can contribute to a poor prognosis in GBM. Further investigation will be needed to confirm the value of HOXA11 as a potential target for overcoming the treatment resistance by developing chemo- or radiosensitizers.
Homeobox (HOX) genes are essential developmental regulators that control a wide range of processes, including apoptosis, differentiation, motility, and angiogenesis [
Aberrantly expressed HOX genes in cancer cells have multicapacity functions, including metastasis, tumor growth, anti-apoptosis, and differentiation suppression [
HOX genes were previously reported to be the genes of interest related to GBM recurrence and treatment resistance [
In the present study, this research was extended to another HOX gene,
Fresh frozen tumor tissue samples of five GBM patients, in whom of pairs of initial and recurrent samples were available for screening, and another 20 newly diagnosed GBM patients for validation were used in this study. All patients were managed using a standard GBM treatment protocol of concurrent radiotherapy and TMZ treatment, followed by adjuvant TMZ as a primary treatment. The tumor tissues were obtained during surgery, snap-frozen in liquid nitrogen, and stored at −80°C prior to use. The study was approved by an institutional review committee.
The human glioma U251, U373, and LN18 cell lines were purchased from the American Type Culture Collection (Manassas, VA), and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5% antibiotics (streptomycin) in a humidified atmosphere containing 5% CO2 and 95% air at 37°C.
The cell lines were lysed with TRIzol (Life Technologies, Carlsbad, CA), and RNA isolation was performed using an RNeasy Mini Kit (#74104, Qiagen, Valencia, CA). The total RNA was treated with DNase and then quantified by spectrophotometry. In addition, cDNA was synthesized from 1 μg of the total RNA using a reverse transcription kit (#205311, Qiagen) according to the manufacturer’s protocol. The primers used were designed using an online primer-BLAST tool (
The whole protein extracts from the tissue samples were prepared using a PRO-PREP lysis buffer (#17081, iNtRon Biotechnology, Seongnam, Korea), and the protein concentrations were determined using a bicinchoninic acid protein assay (#23227, Thermo Fisher Scientific, Waltham, MA). The proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by blotting onto nitrocellulose membranes, and probing with the antibodies against HOXA11 (#SC-48542, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). The blotted membranes were then incubated with the goat anti-rabbit IgG secondary antibody for 1 hour. Subsequently, the membranes were incubated in an Amersham ECL-prime solution (#RPN2232, GE Healthcare Life Sciences, Pittsburgh, PA) in the dark for 1 minute and exposed under FluorChemHD2 (Cell Biosciences, Santa Clara, CA) for visualization. Only the samples with a consistent result from repeated experiments were selected for analysis. The densities of the bands were measured using free image analyzer software (ImageJ V1.8x, National Institutes of Health,
To knock down HOXA11 expression in cells, small interfering RNA (siRNA) experiments were performed using commercially available sequences targeting HOXA11 (#SASI-Hs01-00110410, #SASI-Hs01-00110413, and #SASI-Hs01-00110417, Sigma Aldrich, St. Louis, MO) as well as with the non-targeting control siRNA (#D-001610-01-05, Dharmacon, Lafayette, CO). When the cells reached 70%-80% confluence, they were transfected with siRNA under the most efficient transfection condition, as determined by the NEON Transfection system (#MPK5000, Life Technologies). The cells were cultured in media without antibiotics to increase the siRNA transfection efficiency for 24 hours.
The control and transfected cells were grown on 96-well plates at a density of 4×103 cells per well for 24 hours. Subsequently, the cells were either treated with TMZ (#ALX-420-044-M100, Enzo Life Sciences, Farmingdale, NY) to a final concentration of 300 μg/mL for 24 hours or irradiated with 4 MV X-rays from a linear accelerator (Clinac 4/100, Varian Medical Systems, Palo Alto, CA) at a dose rate of 10.0 Gy/min. For the combination treatment, the cells were irradiated first and then treated with TMZ.
Cell viability analysis was performed using a Colorimetric Cell Counting Kit-8 (CCK; Dojindo Molecular Technologies, Kumamoto, Japan). The number of viable cells was quantified according to the manufacturer’s instructions by reading the ultraviolet absorption spectra at 450 nm on a microplate 2 hours after adding 10 μL of a CCK solution per well. All experiments were conducted in triplicate.
For tissue sample analysis, the total RNA was extracted from the tissue samples using the mirVana miRNA Isolation Kit (#AM1560, Ambion, Austin, TX) for microarray analysis after quantification and qualification. The total RNA quality was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The cut off RNA integrity number for RNA used in RNA amplification was 7.0 or above. The cRNA was produced using an Illumina TotalPrep RNA Amplification Kit (#IL1791, Ambion) according to the provided protocol. The cRNA was used for hybridization to a human HT12-v4 Illumina Beadchip gene expression array (Illumina, San Diego, CA) according to the manufacturer’s protocol. The arrays were scanned and the fluorescence signals were obtained using an Illumina BeadArray Reader (BeadStation 500GXDW, Illumina). The signal obtained from the scanned beadchip was transformed to intensity raw data using GenomeSortudio software (ver. 2009.1, Illumina) and was used for further data analysis. The raw data were normalized by applying a log2 transformation, quantile normalization, and gene and array centering. All data processing was performed using the R/Bioconductor packages (ver. 2.14,
To determine the changes in gene expression before and after HOXA11 knockdown, the total RNA extracted from the LN18 cells transduced with siHOXA11 or control siRNA were analyzed by Affymetrix GeneChip Human Gene 1.0ST Arrays (Affymetrix, Santa Clara, CA). The RNA was amplified and labeled using a GeneChip WT Sense Target Labeling and Control Reagents Kit (Affymetrix). The cDNA was synthesized, labeled, and hybridized to the GeneChip array according to the manufacturer's protocol. The GeneChips were washed and stained using the GeneChip Fluidics Station 450 (Affymetrix), and then scanned using a GeneChip Scanner 3000 7G (Affymetrix). The expression data were normalized using the robust multi-array average method. Affymetrix Expression Console ver. 1.1 (Affymetrix) was used to compare the group signals, and the data were logtransformed (base 2) for parametric analysis. The differentially expressed genes (DEGs) were identified by significance analysis of the microarrays method in the R package ‘samr’ (R 2.11.1).
The data from the experiments were tested for their significance using an unpaired two-tailed Student's t test. An ANOVA and Student's t test were used to identify the significant differences in cell death rates. Kaplan-Meier curve analysis was used to generate the overall survival curves. The differences between the survival curves were analyzed using a log-rank test. The results were analyzed using IBM SPSS Statistics software ver. 19.0 (IBM Co., Armonk, NY). The data are presented as mean±standard deviation for three or more separate experiments. A p-value of 0.05 or lower was considered significant.
For microarray analyses, the false discovery rates (FDRs) were calculated using three GenePattern software modules (
The relative changes in HOX gene expression in five pairs of primary and recurrent GBM samples were assessed using microarray analysis (
To confirm the effects of HOXA11 expression on the resistance to the current standard treatment protocol for GBM, three malignant glioma cell lines (U251, U373, and LN18) were transduced with HOXA11 siRNA to assess the cell viability. The viable cell fractions were analyzed 72 hours after treatment with either single or combination applications of radiation treatment (RT) and TMZ. A significant increase in the number of cells with HOXA11 suppression after either RT or TMZ was observed (
To identify the genes under the regulation of HOXA11, the microarray expression profiling data of the control LN18 cells was compared with that of the HOXA11-silenced cells with HOXA11 siRNA. LN18 was chosen because it showed the most significant decrease in HOXA11 expression after siRNA transduction among the three cell lines tested. After normalization of the values and DEG analysis, 11 up-regulated and 51 down-regulated genes that exhibited more than two-fold changes after HOXA11 suppression were identified (
The functional annotation tools within DAVID Bioinformatics Resources (
HOX genes, which are a cluster of master regulators of embryogenesis, are expressed temporarily during the developmental phase in vertebrates, but they should be silenced in the adult central nervous system [
The present study proposes a tumor suppressor function of HOXA11 in GBM based on the results from both
Treatment resistance induced by HOXA11 down-regulation, as detected by
The treatment resistance induced by the underexpression of HOXA11 can contribute to a poor prognosis in GBM. Further investigation will be needed to confirm the value of HOXA11 as a potential target for overcoming treatment resistance by developing chemo- or radio-sensitizers.
Supplementary materials are available at Cancer Research and Treatment website (
Conflict of interest relevant to this article was not reported.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015-R1D1A1A09057171) in Korea and Seoul National University Hospital Research Fund (04-2010-0470).
Relative changes in the expression of homeobox (HOX) genes among five pairs of primary and recurrent glioblastoma (GBM) samples as determined by microarray analysis. (A) Heatmap and hierarchical clustering analysis showed inconsistent results in sequential HOX gene expression changes between the primary and recurrent samples, except for HOXA11. (B) HOXA11 gene is the only HOX gene that was consistently down-regulated in the recurrent GBM samples compared to primary samples for all five sample pairs.
(A) Results of western blot analysis for normalized HOXA11 expression. The patients were grouped according to the HOXA11 expression macroscopically, and the difference in expression level was confirmed by intensity measurement of the bands. (B) The overall survival of a separate cohort of glioblastoma patients by HOXA11 expression. The survival was significantly longer in patients with high HOXA11 expression (31±15.3 months) than in those with low HOXA11 expression (18±7.3 months, p=0.037).
Treatment resistance of glioma cell lines (U251, U373, and LN18) assessed by cell viability tests after the inhibition of HOXA11 and presented as relative viable cell fractions. Direct inhibition of HOXA11 by small interfering RNA (siRNA) results in a significant increase in cell survival, 72 hours after single or combination applications of radiation (RT) and temozolomide (TMZ) treatments (*p < 0.05).
Identification of the HOXA11-regulated genes in LN18 cells. (A) Reverse transcription polymerase chain reaction results from LN18 cells after HOXA11 knockdown by siRNA. (B) HOXA11-silencing siRNA versus control siRNA transduced cells compared using Affymetrix GeneChip Human Gene 1.0ST Arrays. The probe sets with fold changes greater than 2-fold are shown. After HOXA11 suppression, 11 up-regulated and 51 down-regulated genes were identified. (C) MA plots showing the distribution of regulated genes. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IQR, interquartile range; siRNA, small interfering RNA.
Coexpression network incorporating selected gene sets drawn from the functional gene annotation enrichment analysis of differentially expressed genes after HOXA11 suppression. The network was constructed based on coexpression interactions using GeneMANIA software (ver. 3.1.2.8). The key hub regulators associated with HOXA11 suppression are expressed as black solid circles.
Differences in the survival fraction after the direct inhibition of HOXA11 by siRNA compared with the control in cells 72 hours after treatment with single or combination applications of RT and TMZ treatments
RT (%) | TMZ (%) | RT/TMZ (%) | |
---|---|---|---|
U251 | 10.6 | 19.7 | 45.5 |
U373 | 36.8 | 34.6 | 55.8 |
LN18 | 7.0 | 24.6 | 29.1 |
siRNA, small interfering RNA; RT, radiation; TMZ, temozolomide.
Enriched gene ontology categories in the genes expressed differently after HOXA11 suppression in LN18 cells
Gene ontology category | EASE score | iHOXA11/Control |
Gene accession No. | Gene symbol | Gene description | Cytoband | |
---|---|---|---|---|---|---|---|
Log2 ratio | Absolute fold change | ||||||
GO:0040008 Regulation of growth | 0.0390089 | –1.01254 | 2.0174599 | NM_004879 | Etoposide induced 2.4 mRNA | 11q24 | |
–1.0791 | 2.1127177 | NM_016441 | Cysteine rich transmembrane BMP regulator 1 (chordin-like) | 2p21 | |||
–1.105641 | 2.1519447 | NM_024544 | Mitochondrial E3 ubiquitin protein ligase 1 | 1p36.12 | |||
–1.645323 | 3.1281789 | NM_001024847 | Transforming growth factor, beta receptor II (70/80kDa) | 3p22 | |||
GO:0005739 Mitochondrion | 0.0436296 | 1.06418 | 2.0909811 | NM_006472 | Thioredoxin interacting protein | 1q21.1 | |
–1.10251 | 2.1472795 | NM_003165 | Syntaxin binding protein 1 | 9q34.1 | |||
–1.105641 | 2.1519447 | NM_024544 | Mitochondrial E3 ubiquitin protein ligase 1 | 1p36.12 | |||
–1.191529 | 2.2839467 | NM_005230 | ELK3, ETS-domain protein (SRF accessory protein 2) | 12q23 | |||
–1.229528 | 2.3449026 | NM_001865 | Cytochrome coxidase subunit VIIa polypeptide 2 (liver) | 6q12 | |||
–1.43432 | 2.7025475 | NM_002524 | Neuroblastoma RAS viral (v-ras) oncogene homolog | 1p13.2 | |||
–1.627447 | 3.0896577 | NM_001386 | Dihydropyrimidinase-like 2 | 8p22-p21 | |||
GO:0004157 Dihydropyrimidinase activity | 0.0101296 | –1.017135 | 2.0238958 | NM_001313 | Collapsin response mediator protein 1 | 4p16.1 | |
–1.627447 | 3.0896577 | NM_001386 | Dihydropyrimidinase-like 2 | 8p22-p21 |