The retinoid anticancer signal: mechanisms of target gene regulation

Abstract

Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARbeta2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARbeta2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARbeta2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16.

Figure 1

Induction of target gene expression by RA in neuroblastoma (SH-SY5Y, BE(2)-C), lung (SK-MES-1, Calu-6) and breast (MDA-MB-231, T47D) cancer cell lines, and neuroblastoma tissues. cDNA samples from cultured cells treated with 10 μM aRA (A and D), or 13-cis-RA (B) or solvent control at various time points, and duplicate cDNA samples from neuroblastoma arising in MYCN transgenic mice treated with 13-cis-RA or control (C) were subjected to independent competitive RT–PCR analyses using trans-intron PCR primers, together with housekeeping gene β2M primers. An equal aliquot of PCR product was then electrophoretically size-fractionated on a polyacrylamide gel as shown (A, B and C). Fold induction of a target gene by RA in RA-treated samples was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control-treated samples (D).

Figure 2

The effect of demethylation and acetylation on aRA-induced RARβ2 and CYP26A1 expression in RA-resistant cancer cells. RARβ2 and CYP26A1 expression was determined by noncompetitive RT–PCR, with expression of housekeeping gene β2M as an internal control. (A) MDA-MB-231 and SK-MES-1 cells were treated with control, aRA, and/or various concentration of aza-CdR for 3 days. (B) The cells were treated with control, aRA, and/or 0.3 μM TSA for 3 days. The last lane of each gel (+VE) contained a positive control from RA-sensitive Calu-6 cells treated with 10 μM aRA for 3 days.

Figure 3

Liganded RARβ2 modulates CYP26A1 expression. RARβ2 gene expression was analysed with the standard competitive RT–PCR, and CYP26A1 expression was analysed by competitive RT–PCR in RA-sensitive SH-SY5Y, Calu-6, and T47D cells and noncompetitive RT–PCR in RA-resistant cells, with housekeeping gene β2M as an internal control. (A, B) CYP26A1 transcription was determined in MDA-MB-231 (A) or SK-MES-1 cells (B) transiently transfected with RARβ2 cDNA plasmid or vector plasmid and treated with control or 10 μM aRA for 24 h (Lanes 2 and 5) or 3 days (lanes 3 and 6). Last lane in (B) contained the positive control from Calu-6 cells treated with 10 μM aRA for 3 days. (C) RARβ2 and CYP26A1 expression was analysed in SH-SY5Y, Calu-6, and T-47D cells transfected with scramble siRNA or RARβ2 siRNA and treated with 10 μM aRA for 48 h.

Figure 4

Synchronous expression of both DUSP6 and RGS16 contributed to RA-induced growth inhibition. (A) DUSP6 and RGS16 gene expression was analysed with competitive RT–PCR with the housekeeping gene β2M as an internal control with samples from SH-SY5Y cells transfected with DUSP6 or RGS16 siRNA or scrambled siRNA and treated with 10 μM aRA for 48 h. ^*Indicates the siRNAs of choice for protein and functional studies. (B) DUSP6 and RGS16 protein was analysed by Western blot with samples from SH-SY5Y cells transfected with scrambled siRNA, DUSP6, or RGS16 siRNA and treated with 10 μM aRA or control solvent for 48 h. β-Actin protein was used as a loading control. (C) Phosphorylated ERK1/2 was analysed by Western blot with samples from SH-SY5Y cells transfected with scrambled, DUSP6, RGS16 siRNA, or siRNA combinations and treated with 10 μM aRA for 60 h. Total ERK1/2 protein was used as a loading control. (D) BrdU incorporation into proliferating cells was analysed in SH-SY5Y cells after transfection with scrambled or target gene siRNAs plus treatment with 10 μM aRA for 64 h. BrdU-positive cells treated with vehicle solvent and transfected with scrambled siRNA were artificially set as 100%. Error bar represented standard error. ^**Indicated statistical significant difference (P<0.05).