Destabilization of ERBB2 transcripts by targeting 3' untranslated region messenger RNA associated HuR and histone deacetylase-6

Abstract

In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies.

Figure 1

ERBB2 expression is regulated by the transcript stability factor, HuR, which binds a conserved U-rich element in the 3′UTR of ERBB2 mRNA. A. Near the poly-A tail of the ERBB2 3′ UTR is an evolutionarily conserved U-rich region (nt 465–505). B. In vitro binding assay using SKBR3 cytosol showing that HuR, but not AUF1, is capable of binding to a radiolabelled probe consisting of the conserved U-rich element of the ERBB2 3′ UTR mRNA. HuR antibody induces a supershift in the HuR-probe complex with loss of the higher mobility probe-bound HuR band. C. Immunoblots showing downregulation of cytosolic AUF1 and HuR levels after siRNA treatment (48 h) of SKBR3 cells, with resultant effects on ERBB2 levels normalized to β-actin.

Figure 2

Association of cytosolic HuR with ERBB2 mRNA and HDAC enzymatic activity, and the co-precipitation and co-localization of HuR with HDAC6 in SKBR3 cells. A. Immunoprecipitation of cytoplasmic HuR by specific (or isotype control) antibody followed by RT-PCR assessment of co-precipitated ERBB2 and GAPDH transcripts. PCR primer pairs were chosen to amplify a 137 nt sequence within the ERBB2 3′ UTR or a 183 nt sequence within GAPDH transcripts. B. Following control or HuR immunoprecipitations from SKBR3 cytosols +/− RNase treatment, a fluorogenic deacetylation assay was used to monitor co-precipitating total HDAC enzymatic activity. C. SKBR3 cytosolic lysates subjected to immunoprecipitation (IP) followed by Western blotting (WB), demonstrating HuR IP enriched with HDAC6 and HDAC6 IP enriched with HuR (also compared to β-actin co-precipitation). Longer WB exposures (not shown) confirmed equivalent lane loading by IgG heavy and light chain band intensities. D. Immunofluorescence microscopic imaging (40x objective with 4x digital zoom) showing major nuclear and minor cytoplasmic abundance of HuR (green) and major cytoplasmic and minor nuclear abundance of HDAC6 (red), and their nuclear and cytoplasmic co-localization (yellow), in cultured SKBR3 cells.

Figure 3

ERBB2 effects of pan-HDAC enzymatic inhibitors (HDACi) are independent of HuR levels and associated with transcript binding to HDAC6, but dissimilar to that of HDAC6 protein downregulation by siRNA. A. ERBB2 protein inhibition by 16–20 h of SKBR3 treatment with TSA (0.4 μM), with no effect on total HuR expression. B. Effect of AUF1 or HuR downregulation by siRNA treatment (48 h, as described in Figure 1) followed by 6 h treatment with LAQ824 (10 μM) on SKBR3 ERBB2 mRNA levels (normalized to 28S and 18S rRNA). C. Immunoprecipitation of cytoplasmic HDAC6 followed by RT-PCR assessment of co-precipitated ERBB2 and GAPDH transcripts (as described in Figure 2) from control (untreated) and 3 hr LAQ824 (HDACi) treated SKBR3 cells. Untreated and HDACi RT samples were PCR amplified for 24 and 26 cycles respectively. D. HDAC6 knockdown by siRNA (72 h) in SKBR3 cells and resultant effects on ERBB2 and acetylated α-tubulin levels, normalized to total α-tubulin.

Figure 4

Structure and HDAC6-selective activity of three newly synthesized hydroxamic acids. A. Chemical structures of ST17, ST80, and ST71. B. Selective in vitro micromolar activity (IC₅₀, 50% μM inhibitory concentration) of inhibitors against deacetylase activity of affinity purified FLAG-tagged HDAC1 and HDAC6 subtypes, measured as described in Materials and Methods. C. Differential levels of acetylated α-tubulin (α-AcTubulin) and Histone H4 K12 (AcH4K12) in whole cell lystes from SKBR3 cells treated for 24 h with 0.5 μM LAQ824 or 50 μM of ST71, ST80 and ST17. Control (C) lysate from vehicle treated SKBR3 cells as shown. Densitometry determined ratios for α-AcTubulin/AcH4K12 were normalized by the value from LAQ824 treatment.

Figure 5

HDAC6-selective inhibitor ST71 fails to repress intracellular ERBB2 promoter activity but inhibits culture growth of ERBB2-positive SKBR3 cells. A. Dose-response comparison against ERBB2 promoter-reporting (luciferase expressing) MCF7/R06pGL-4 cells following exposure to HDAC6-specific inhibitor ST71 or pan-HDAC inhibitor LAQ824, showing 24 h effects (% control) on endogenous luciferase expression (blue, solid circles) and MTT cell viability (red, solid squares). B. Dose-response comparison against ERBB2-positive SKBR3 cells after exposure to ST71 or LAQ824, with loss of MTT cell viability as a measure of growth inhibition following 72 h treatment (% control).

Figure 6

HDAC6 knockdown offsets ERBB2 mRNA decay induced by pan-HDACi and HDAC6-selective inhibitors that comparably reduce SKBR3 ERBB2 mRNA and protein levels. A. Northern blot showing the influence of 3 h treatment with 0.5 μM LAQ824, 50 μM ST80 or vehicle control (C) on ERBB2 mRNA levels in SKBR3 cells pre-transfected for 72 h with HDAC6 or control (C) siRNA. Densitometry determined ratios of ERBB2/GAPDH band intensities were normalized by the value obtained from vehicle treated control siRNA transfectants (C). B. Western blots showing 24 h treatment effects of indicated drug and concentration on SKBR3 ERBB2 and β-actin protein levels; densitometry measured band intensities were used to calculate ERBB2/β-actin protein ratios, normalized such that untreated control (C) ratio = 1.