Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

Abstract

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Figure 1

Hierarchical cluster diagram of differential homeobox gene expression in OSCC samples. Gene expression levels in non-neoplastic margins were used as baseline. Data are visualized colorimetrically with heat plots, ‘red’ representing elevated gene expression and ‘green’ decreased gene expression.

Figure 2

Normalized expression of HOXA5, HOXD10 and HOXD11 transcripts by qRT-PCR in OSCC samples and non-tumoral margins. The full line corresponds to the median value for each group. Asterisk indicates statistically significant difference between OSCC and non-tumoral samples (p<0.05, Wilcoxon)..

Figure 3

Relative expression ratio (log₂) of HOXA5, HOXD10 and HOXD11 mRNA expression analysis by qRT-PCR in HaCAT and OSCC cell lines (SCC4, 9, 15, 25). Significant up-regulation of these genes was observed in all cell lines compared with that in NOK (p<0.001, one-way ANOVA).

Figure 4

Survival proportions (log-rank test) of OSCC patients according to HOXA5 (A), HOXD10 (B) and HOXD11 (C) expression, as well as to perineural invasion (D), lymphatic invasion (E) and pathological grade (F). (LI, lymphatic invasion; PI, perineural invasion; WD, well differentiated; MD, moderately differentiated).