Cancer Res Treat.  2017 Apr;49(2):387-398. 10.4143/crt.2016.106.

Underexpression of HOXA11 Is Associated with Treatment Resistance and Poor Prognosis in Glioblastoma

• Se YB
• Kim SH
• Kim JY
• Kim JE
• Dho YS
• Kim JW
• Kim YH
• Woo HG
• Kim SH
• Kang SH
• Kim HJ
• Kim TM
• Lee ST
• Choi SH
• Park SH
• Kim IH
• Kim DG
• Park CK

• KMID: 2378110
• DOI: http://doi.org/10.4143/crt.2016.106

Abstract

PURPOSE
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.
MATERIALS AND METHODS
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. In vitro experiments were performed to confirm the role of HOX in treatment resistance. Mediators involved in HOX gene regulation were searched using differentially expressed gene analysis, gene set enrichment tests, and network analysis.
RESULTS
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 (TGFBR2, CRIM1, TXNIP, DPYSL2, and CRMP1) that may confer an oncologic effect after HOXA11 suppression were identified.
CONCLUSION
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.

Keyword

Homeobox genes; HOXA11; Glioblastoma; Treatment resistance

MeSH Terms

Adult
Brain
Cell Line
Genes, Homeobox
Glioblastoma*
Humans
In Vitro Techniques
Microarray Analysis
Prognosis*
Radiotherapy

Figure

• 

  Fig. 1. 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.

• 

  Fig. 2. (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).

• 

  Fig. 3. 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).

• 

  Fig. 4. 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.

• 

  Fig. 5. 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.

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