Transcriptome profiling and genome-wide DNA binding define the differential role of fenretinide and all-trans RA in regulating the death and survival of human hepatocellular carcinoma Huh7 cells

Abstract

Fenretinide is significantly more effective in inducing apoptosis in cancer cells than all-trans retinoic acid (ATRA). The current study uses a genome-wide approach to understand the differential role fenretinide and ATRA have in inducing apoptosis in Huh7 cells. Fenretinide and ATRA-induced gene expressions and DNA bindings were profiled using microarray and chromatin immunoprecipitation with anti-RXRα antibody. The data showed that fenretinide was not a strong transcription regulator. Fenretinide only changed the expressions of 1 093 genes, approximately three times less than the number of genes regulated by ATRA (2 811). Biological function annotation demonstrated that both fenretinide and ATRA participated in pathways that determine cell fate and metabolic processes. However, fenretinide specifically induced Fas/TNFα-mediated apoptosis by increasing the expression of pro-apoptotic genes i.e., DEDD2, CASP8, CASP4, and HSPA1A/B; whereas, ATRA induced the expression of BIRC3 and TNFAIP3, which inhibit apoptosis by interacting with TRAF2. In addition, fenretinide inhibited the expression of the genes involved in RAS/RAF/ERK-mediated survival pathway. In contrast, ATRA increased the expression of SOSC2, BRAF, MEK, and ERK genes. Most genes regulated by fenretinide and ATRA were bound by RXRα, suggesting a direct effect. This study revealed that by regulating fewer genes, the effects of fenretinide become more specific and thus has fewer side effects than ATRA. The data also suggested that fenretinide induces apoptosis via death receptor effector and by inhibiting the RAS/RAF/ERK pathway. It provides insight on how retinoid efficacy can be improved and how side effects in cancer therapy can be reduced.

Fig 1. Comparison of differential gene expression, RXRα binding genes, and binding motif between fenretinide- and ATRA-treated Huh7 cells

(A) Comparison of fenretinide and all-trans RA based on gene expression. F: fenretinide; R: all-trans RA. (B) Comparison of RXRα binding genes between fenretinide and all-trans RA. (C) Comparison of genes bound by RXRα and regulated by fenretinide and all-trans RA. (D) Motif analysis for genes bound by RXRα and regulated by fenretinide. (E) Motif analysis for genes bound by RXRα and regulated by all-trans RA. For gene expression analysis, total RNA was extracted from Huh7 cells after 12 hrs of fenretinide (10 μM), ATRA (10 μM) or DMSO (control) treatment, followed by microarray analyses. For RXRα binding and motif analysis, Huh7 cells were treated with fenretinide (10 μM) or ATRA (10 μM) for 3 hrs and then subjected to ChIP-Seq assay using IgG (negative control) or anti-RXRα antibody. Global profiling of RXRα binding motifs in Huh-7 cells were predicted by HiddenMarkov Model. DR: direct repeat; ER: everted repeat; IR: inverted repeat.

Fig 2. The effect of fenretinide and all-trans RA on apoptosis-related genes

(A) Common apoptosis-related genes regulated by fenretinide and all-trans RA in Huh7 cell. (B) The apoptosis-related genes regulated by fenretinide. (C) The apoptosis-related genes regulated by all-trans RA. Total RNA was extracted from Huh7 cells treated with fenretinide (10 μM), ATRA (10 μM) or DMSO for 12 hrs and subjected to microarray assay.

Fig 3. The effect of fenretinide and all-trans RA on proliferation-related genes

(A) Common proliferation-related genes regulated by both fenretinide and all-trans RA. (B) The proliferation-related genes regulated by all-trans RA. Total RNA was extracted from Huh7 cells treated with fenretinide (10 μM), ATRA (10 μM) or DMSO for 12 hrs and subjected to microarray assay.

Fig 4. The effect of fenretinide and all-trans RA on cell cycle-related genes

(A) Common cell cycle-related genes regulated by both fenretinide and all-trans RA. (B) The cell cycle-related genes preferentially regulated by all-trans RA. Total RNA was extracted from Huh7 cells treated with fenretinide (10 μM), ATRA (10 μM) or DMSO for 12 hrs and subjected to microarray assay.

Fig 5. The effect of fenretinide and all-trans RA on anti-apoptosis genes (A) and pro-apoptosis genes (B) by qRT-PCR

Total RNA was extracted from Huh7 cells treated with fenretinide (10 μM), ATRA (10 μM), or DMSO for 30 min, 1, 2, 3, 6, and 12 hrs, and the gene expressions were studied by real-time RT-PCR and normalization to that of GAPDH. Data were expressed as mean ± SD from three independent experiments.

Fig 6. The effect of fenretinide and all-trans RA on protein levels of HSPA1A/B, caspase 8, caspase 4, HERPUD1, and DDIT3

Huh7 cells were treated with fenretinide (10 μM), ATRA (10 μM), or DMSO for 6, 12, 18, and 24 hrs. The protein levels were analyzed by western blot using specific antibodies as described in Section 2. β-actin level was used for loading control. Representative data are shown from three independent experiments. Arrow indicated the position of cleaved caspase 4.

Fig 7. The effect of fenretinide and all-trans RA on CYP26A1 (A) and CYP26B1 (B) gene expression

Huh7 cells were treated with fenretinide (10 μM) or ATRA (10 μM) for 30 min, 1, 2, 3, 6, and 12 hrs. Data were expressed as mean ± SD from three independent experiments.

Fig 8. Schematic presentations for the differential effects of fenretinide and all-trans RA in regulating apoptosis and survival of Huh7 cells

(A) Regarding the Fas/TNFα-mediate apoptosis pathway, fenretinide up-regulated the expression of three pro-apoptotic genes including DEDD, CASP8, and CASP4, which subsequently triggered the pathway; Additionally, fenretinide also induced apoptosis by up-regulating pro-apoptotic genes HSPA1A/B, which is activated by ER stress and promotes TNF-mediated apoptosis. In contrast, ATRA induced the expression of two anti-apoptotic genes BIRC3 and TNFAIP3, which negatively regulated the Fas/TNFα-mediate signaling pathway, and eventually inhibited Fas/TNFα-mediated apoptosis. The repressed expression of PCSK9, which play an important role in caspase-3 activation, also decreased ATRA-mediated apoptosis. (B) Regarding the RAS/RAF/ERK-medicated survival pathway, fenretinide repressed the expression of BRAF, and therefore decreased the expression levels of MEK1 and ERK2, which finally inhibited cell survival. Conversely, ATRA up-regulated the expression of SOSC2, successively increased the expression of three key genes: BRAF, MEK1 and ERK2, and therefore induced the RAS/RAF/ERK-mediated survival pathway. Additionally, ATRA promoted the NFkB-dependent survival pathway by repressing CITE2 gene expression. “→” indicates induction and “⊥” indicates repression. The genes marked in green circle indicate pro-apoptosis genes. The genes marked in red circle indicate anti-apoptosis genes. The numbers in bracket represent the fold-change of gene expression changed by fenretinide or ATRA at corresponding time-point.