HOXD Regulation of Neuroblastoma Differentiation Figure 1. RA induction of HOXD genes in BE(2)-C cells. (A) Genomic structure of the human HOXD cluster on chromosome 2q31.1, adapted from the UCSC Genome Browser. Arrows indicate the transcriptional direction of HOXD genes. (B) qRT-PCR analysis of HOXD mRNA levels at various time points following RA (10 mM) treatment. The HOXD mRNA levels in BE(2)-C cells immediately before RA treatment (RA 0h) were designated as 1.0 (dashed line). The data were from two independent samples with each being assayed in triplicates and analyzed using two-tailed Student’s t-test with the p values indicated. Error bars, standard deviation (SD). doi:10.1371/journal.pone.0040728.g001 varied in their ability to induce neuronal differentiation in BE(2)-C cell. HOXD3 was able to induce neurite outgrowth and down- regulation of PHOX2B, but failed to upregulate NEFM expression (Figure 4). In fact, HOXD3 induction resulted in a marked downregulation of NEFM expression (Figure 4 B-D). On the other hand, induction of HOXD4, HOXD11 or HOXD13, while having no significant effect on morphologic differentiation and PHOX2B expression, was able to markedly upregulate NEFM expression (Figure 4). Taken together, these findings suggest distinct roles of HOXD proteins in the control of morphologic differentiation and neuronal marker expression in neuroblastoma cells. HOXD8 is a mediator of RA action Previous studies have revealed a marked induction of HOXD1 and HOXD8 following RA treatment in several human neuro- blastoma cell lines examined including SK-N-BE, CHP-134, SK- N-SH, LA-N-1 and LA-N-5 [14,15]. Since induction of HOXD8, but not of HOXD1, was able to recapitulate the phenotype of G1 arrest and neuronal differentiation induced by RA (Figures 2, 3, 4), we investigated the possibility of HOXD8 as a mediator of RA action. We generated two BE(2)-C-derived cell lines with inducible expression of short hairpin RNA (shRNA) sequences against different regions of the human HOXD8 cDNA in the absence of doxycycline. Both sequences were highly effective in knockdown of HOXD8 expression, with HOXD8sh-1 reducing HOXD8 expression by 78% (Figure 5 A) and HOXD8sh-2 by 62% (Figure S3 A) relative to the control BE(2)-C cells with inducible expression of an shRNA sequence against the firefly luciferase gene (Luc-sh). We examined the effect of HOXD8 knockdown on the ability of RA to induce G1 arrest and neuronal differentiation primarily with the BE(2)-C cells expressing HOXD8sh-1, which showed a more pronounced inhibition of HOXD8 expression compared to the cells expressing HOXD8sh-2 (Figure 5 A and Figure S3 A). BE(2)-C cells with HOXD8 knockdown were markedly resistant to RA-induced neuronal differentiation and G1 arrest, as determined by morphology (Figure 5 B) and cell cycle analysis (Figure 5 C-D). Consistent with these observations, knockdown of HOXD8 significantly abrogated the ability of RA to downregulate cell cycle-promoting genes and to upregulate RET, the receptor for glial cell-derived neurotrophic factor (Figure 5 E) and an RA- responsive gene [25]. We obtained similar results with the BE(2)-C cells expressing HOXD8sh-2, which also showed marked re- sistance to RA-induced G1 arrest (Figure S3 B). The finding that two different HOXD8 shRNA sequences had a similar effect on RA action provided evidence that the observed RA resistant phenotype resulted from HOXD8 knockdown. Together, these findings suggest that HOXD8 is a key mediator of the RA-induced differentiation program. In addition, we investigated the role of HOXD9 in mediating RA action in BE(2)-C cells given the functional similarity between HOXD9 and HOXC9, which has recently been identified as a key effector of RA action in neuroblastoma cells [25]. We generated PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e40728