The Proteasome Inhibitor Bortezomib Induces an Inhibitory Chromatin Environment at a Distal Enhancer of the Estrogen Receptor-α Gene

<div><p>Expression of the estrogen receptor-α (ERα) gene, <i>ESR1</i>, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating <i>ESR1</i> gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of <i>ESR1</i> gene expression in breast cancer cells. Expression of <i>ESR1</i> mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ∼400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of <i>ESR1</i> gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2γ. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of <i>ESR1</i> mRNA expression that highlights the importance of the chromatin environment at the −150 kb distal enhancer in regulation of basal expression of <i>ESR1</i> in breast cancer cells.</p></div

Multi-drug loaded micelles delivering chemotherapy and targeted therapies directed against HSP90 and the PI3K/AKT/mTOR pathway in prostate cancer.

BACKGROUND:Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy. METHODS:This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy. RESULTS:In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts. CONCLUSION:These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy

Proteasome inhibition decreases transcription factor occupancy on the <i>ESR1</i> enhancer and promoter.

<p>MCF7 cells were treated with vehicle (nt) or 30 nM bortezomib (B) for 24 hours and ChIP analyses were performed to examine occupancy at regions of <i>ESR1</i> depicted in the diagram in Fig. 2A. Data representing a minimum of three independent experiments are presented as percent of input sample for <i>A</i>) GATA3, <i>B</i>) FOXA1, <i>C</i>) AP2γ, <i>D</i>) RNA PolII, and <i>E</i>) IgG (control) on the indicated regions. Data are shown as mean ± SEM. Statistical analysis comparing untreated and bortezomib-treated groups were performed using a Student’s paired t-test. Statistically significant differences of p<0.05 are indicated with an asterisk (*).</p

The effects of proteasome inhibition on ERα expression are reversible.

<p><i>A</i>) MCF7 cells were treated with bortezomib for 24 hours and harvested immediately (t = 0) or at the indicated time from 4 to 48 hours post bortezomib removal. <i>ESR1</i> mRNA levels were measured by qRT-PCR. Data are shown relative to an untreated control before bortezomib-treatment, which was set at 1. Results are representative of three independent experiments and are shown as mean ± SEM. For statistical analysis, ANOVA was performed using the ΔCt values followed by a post hoc Tukey’s test to compare each point to the 0 hour. Statistically significant values (p<0.05) are indicated with an *. <i>B</i>) Western blots were performed on whole cell lysates from cells that were treated with vehicle (−) or bortezomib (B) for 24 hours (24 h). After 24 hours, bortezomib was removed by washing cells twice with PBS and replacing media. Cells were then harvested at the indicated times between 4 and 48 hours after bortezomib removal. Blots were probed with antibodies for ERα, with actin serving as a loading control.</p

Proteasome inhibition decreases p300 occupancy on the <i>ESR1</i> enhancer.

<p>MCF7 cells treated with vehicle (nt) or 30 nM bortezomib (B) for 24 hours and ChIP assays were performed to assess occupancy of p300 at the indicated region. Immunoprecipitation was performed using antibodies for <i>A</i>) p300 and <i>B</i>) IgG, as a control. Data are presented as percentage of the input sample. The data represent the average of a six independent experiments ± SEM. Statistical analysis was performed to compare untreated and bortezomib-treated samples using a Student’s paired t-test. Statistically significant differences of p<.05 are indicated with an asterisk (*).</p

DNase sensitivity is reduced in the <i>ESR1</i> enhancer region with proteasome inhibition.

<p><i>A</i>) Schematic of <i>ESR1</i> promoter structure depicting the location of primers used for detecting DNase sensitivity and ChIP analysis <i>B)</i> Nuclei were isolated from MCF7 cells treated for 24 hours with vehicle or 30 nM bortezomib. Isolated nuclei were either left undigested or digested with DNase for 5 minutes, followed by incubation with Proteinase K to stop the DNase reaction, as described in Materials and Methods. DNA was purified and q-PCR was performed with primers to the indicated regions. Data are shown as the 2^{∧(Ct cut–Ct uncut)} and presented as mean ± SEM. Statistical analysis was performed using a Student’s t-test. No statistically significant differences were observed (p>0.05).</p

Proteasome inhibition decreases histone acetylation and increases H4K20me3 on the distal <i>ESR1</i> enhancer.

<p>MCF7 cells were treated for 24 hours with vehicle (nt) or 30 nM bortezomib (B), and ChIP was performed as described in methods using antibodies to <i>A</i>) pan acetylated histone 3 (AcH3), <i>B</i>) pan acetylated histone 4 (AcH4), <i>C</i>) histone 4 lysine 20 tri methylation (H4K20me3), and <i>D</i>) IgG as control. Data are presented as percentage of input sample and represent a minimum of three independent experiments. Error bars indicate the SEM, and an asterisk (*) indicates a statistically significant difference between vehicle and bortezomib-treated samples using a Student’s paired t-test. * p<0.05, ** p<.01.</p

Dose response of prostate cancer cells to individual drugs.

<p>(A-B) Cells were grown in either control regular media or media supplemented with DHT of increasing concentrations. Cell growth was assessed using Alamar Blue assay as described in Materials and Methods. Results are representative of 3 independent experiments. (C) Immunoblotting was performed from equal amount of total protein using the phospho-AKT, AKT, and β-actin antibodies as described in Materials and Methods. (D-I) Cytotoxic effects of increasing doses of docetaxel (D, G), rapamycin (E, H), and 17-AAG (F, I) in PTEN-P2 cells (D, E, F) and PTEN-CaP2 cells (G, H, I). Cells were exposed to the indicated concentration of drug-loaded micelle or empty micelle for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. * indicates statistically significant differences compared to micelle control (ANOVA, p < 0.05). Results are representative of 3 independent experiments.</p

Dose response of prostate cancer cells to multi-drug loaded micelles.

<p>Cytotoxic effects of DR17 in PTEN-P2 (A, C, E) and PTEN-CaP2 cells (B, D, F). Dose responses of PTEN-P2 (A, C) and PTEN-CaP2 (B, D) to DR17 free form or DR17 delivered by micelle (DR17). Cells were exposed to the indicated concentrations of free form DR17 or DMSO, or DR17 loaded micelle or empty micelle for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. E-F, Comparison of cytotoxic effects of three-drug combination DR17 to individual drug loaded micelles (C, D). For C and D, DR17 was added to the media to the final concentration of 105μM. Final concentrations of docetaxel, rapamycin, 17-AAG were calculated based on molarity ratio of each individual drug in the DR17 formula [docetaxel:rapamycin:17-AAG (1:1:8.5)], which are 12.38μM, 10.94μM, and 85.37μM, respectively. Cells were exposed to the indicated drug conditions for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. * indicates statistically significant differences compared to DR17 treatment (ANOVA, p < 0.05). Results are representative of 3 independent experiments.</p

Histology of DR17 and micelle control treated prostates.

<p>Pten het N = 9 (A, B, E, F, I, J) and Pten null N = 6 (C, D, G, H, K, L) mice were treated with empty micelles (Ctrl) or DR17 loaded micelles for 3 weeks. Animals were sacrifice two days after the last injection. Prostates were dissected and fixed in formalin. Prostate sections were used for H&E staining to assess overall morphology (A-D). Immunohistochemistry was performed to examine stromal smooth muscle actin (SMA, E-H) and epithelial cytokeratin (I-L) markers. Pictures were taken at 40X magnification. The black bar in each picture represents 30 microns.</p