Class I Histone Deacetylase Inhibitor Entinostat Suppresses Regulatory T Cells and Enhances Immunotherapies in Renal and Prostate Cancer Models

Background: Immunosuppressive factors such as regulatory T cells (Tregs) limit the efficacy of immunotherapies. Histone deacetylase (HDAC) inhibitors have been reported to have antitumor activity in different malignancies and immunomodulatory effects. Herein, we report the Tregs-targeting and immune-promoting effect of a class I specific HDAC inhibitor, entinostat, in combination with either IL-2 in a murine renal cell carcinoma (RENCA) model or a survivinbased vaccine therapy (SurVaxM) in a castration resistant prostate cancer (CR Myc-CaP) model. Methods and Results: RENCA or CR Myc-CaP tumors were implanted orthotopically or subcutaneously, respectively. Inoculated mice were randomized into four treatment groups: vehicle, entinostat, cytokine or vaccine, and combination. Tregs in the blood were assessed by FACS analysis. Real time quantitative PCR and Western blot analysis of isolated T cell subpopulations from spleen were performed to determine Foxp3 gene and protein expression. The suppressive function of Tregs was tested by T cell proliferation assay. Low dose (5 mg/kg) entinostat reduced Foxp3 levels in Tregs and this was associated with enhanced tumor growth inhibition in combination with either IL-2 or a SurVaxM vaccine. Entinostat downregulated Foxp3 expression transcriptionally and blocked Tregs suppressive function without affecting T effector cells (Teffs). In vitro low dose entinostat (0.5 mM) induced STAT3 acetylation and a specific inhibitor of STAT3 partially rescued entinostat-induced down-regulation of Foxp3, suggesting that STAT3 signaling is involved in Foxp3 down-regulation b

Dietary protein restriction inhibits tumor growth in human xenograft models of prostate and breast cancer

Purpose: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer. Experimental Design: To test our hypothesis we assessed the inhibitory effect of protein diet restriction on prostate and breast cancer growth, serum PSA and IGF-1 concentrations, mTOR activity and epigenetic markers, by using human xenograft cancer models. Results: Our results showed a 70% inhibition of tumor growth in the castrate-resistant LuCaP23.1 prostate cancer model and a 56% inhibition in the WHIM16 breast cancer model fed with a 7% protein diet when compared to an isocaloric 21% protein diet. Inhibition of tumor growth correlated, in the LuCaP23.1 model, with decreased serum PSA and IGF-1 levels, down-regulation of mTORC1 activity, decreased cell proliferation as indicated by Ki67 staining, and reduction in epigenetic markers of prostate cancer progression, including the histone methyltransferase EZH2 and the associated histone mark H3K27me3. In addition, we observed that modifications of dietary protein quality, independently of protein quantity, decreased tumor growth. A diet containing 20% plant protein inhibited tumor weight by 37% as compared to a 20% animal dairy protein diet. Conclusions: Our findings suggest that a reduction in dietary protein intake is highly effective in inhibiting tumor growth in human xenograft prostate and breast cancer models, possibly through the inhibition of the IGF/AKT/mTOR pathway and epigenetic modifications

Concurrent HDAC and mTORC1 inhibition attenuate androgen receptor and hypoxia signaling associated with alterations in microRNA expression.

Specific inhibitors towards Histone Deacetylases (HDACs) and Mammalian Target of Rapamycin Complex 1 (mTORC1) have been developed and demonstrate potential as treatments for patients with advanced and/or metastatic and castrate resistant prostate cancer (PCa). Further, deregulation of HDAC expression and mTORC1 activity are documented in PCa and provide rational targets to create new therapeutic strategies to treat PCa. Here we report the use of the c-Myc adenocarcinoma cell line from the c-Myc transgenic mouse with prostate cancer to evaluate the in vitro and in vivo anti-tumor activity of the combination of the HDAC inhibitor panobinostat with the mTORC1 inhibitor everolimus. Panobinostat/everolimus combination treatment resulted in significantly greater antitumor activity in mice bearing androgen sensitive Myc-CaP and castrate resistant Myc-CaP tumors compared to single treatments. We identified that panobinostat/everolimus combination resulted in enhanced anti-tumor activity mediated by decreased tumor growth concurrent with augmentation of p21 and p27 expression and the attenuation of angiogenesis and tumor proliferation via androgen receptor, c-Myc and HIF-1α signaling. Also, we observed altered expression of microRNAs associated with these three transcription factors. Overall, our results demonstrate that low dose concurrent panobinostat/everolimus combination therapy is well tolerated and results in greater anti-tumor activity compared to single treatments in tumor bearing immuno-competent mice. Finally, our results suggest that response of selected miRs could be utilized to monitor panobinostat/everolimus in vivo activity

The HDAC inhibitor, entinostat, enhances peptide vaccine therapy in a castration resistant prostate cancer model.

<p>A and B, Entinostat enhances the antitumor effect of the survivin vaccine, SurVaxM. FVB mice were castrated and subcutaneously inoculated with small castration resistant tumor pieces. Approximately 7 days after inoculation when tumors reached an average size of 50 mm², mice were treated three weeks with vehicle, entinostat (5 mg/kg, 1 dose/day, 5 days/wk), survivin vaccine SurVaxM (100 µg/dose, 1 dose/wk), or combination. A, Single tumor growth graph lines were generated by serial caliper measurements. B, Tumors were harvested at the end of treatment and weighed (combination vs. survivin vaccine, <i>p</i> = 0.003). C, Entinostat treatment reduced Foxp3 levels in Tregs from CR Myc-CaP tumor-bearing mice. After 5 days of treatment, peripheral blood cells were collected from mice, stained for CD4 and Foxp3, and subjected to FACS analysis. Quantitation of Foxp3 mean fluorescence intensity (Foxp3 MFI) (vehicle vs. entinostat, <i>p</i> = 0.0002; combination vs. survivin, <i>p</i> = 0.0004).</p

Class I inhibition, not class II inhibition suppresses Foxp3 expression in Tregs.

<p>Splenocytes were isolated from BALB/c mice and put in culture with different treatment conditions as indicated for 24 hours. Cells were harvested, stained for surface markers and intracellular Foxp3, and subjected to FACS analysis. Plot gating and parameter indication were described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030815#pone-0030815-g001" target="_blank">Figure 1</a>. Histograms show quantification of Foxp3 levels in Tregs. A, Both class I HDAC inhibitors, entinostat and MGCD0103, inhibit Foxp3 expression in Tregs. When comparing entinostat with MGCD0103, PE conjugated anti-Foxp3 antibody was used. B, Class I HDAC inhibitor, entinostat, and a pan inhibitor, panobinostat, but not class II HDAC inhibitors, MC1568 and MC1575, inhibit Foxp3 expression in Tregs. When comparing entinostat with class II HDAC inhibitors and the pan inhibitor, panobinostat, the Alexa 700 conjugated anti-Foxp3 antibody was used since other fluorochrome channels are interfered by the autofluorescence of the class II HDAC inhibitors. ** represents <i>p</i><0.01 for marked treatment vs. vehicle.</p

Enhancement of IL-2 therapy by entinostat is associated with inhibition of Tregs.

<p>Mice with orthotopic inoculation of RENCA cells were treated for 5 days. Blood was drawn from mice on the fifth day and stained for antibodies specific for CD4, CD25, and Foxp3. Tumor weights were measured at the end of two weeks of treatment. A and B, Effect of entinostat on Tregs in tumor bearing mice. A, Effects of vehicle, IL-2, entinostat, or combination treatment on Tregs Foxp3 expression. Cells were stained and subjected to flow cytometry analysis. The dot plots were gated for CD4⁺ cells. The rectangular area encloses the CD4⁺Foxp3⁺ cells, the numbers on the top represent the percentage of Foxp3⁺ cells. The numbers on the bottom in the area represent the mean fluorescence intensity (MFI) of Foxp3 PE staining of CD4⁺Foxp3⁺ cells. B, Quantification of Tregs percentage in CD4 population (left panel) and Foxp3 levels (MFI) in Tregs (right panel) by FACS analysis. Values are means and error bars represent S.D. for 5–7 samples per group. In right panel, <i>p</i> = 0.0011 for IL-2 vs. vehicle; <i>p</i> = 0.000009 for entinostat vs. vehicle. Results are representative of three separate experiments. C, Tumor weight measurements. Columns, mean grams of tumor; Bars, S.D. n = 5–7. <i>p</i> = 0.0209 for entinostat vs. vehicle; <i>p</i> = 0.0077 for combination vs. vehicle; <i>p</i> = 0.0272 for combination vs. entinostat. Results are representative of three separate experiments. D, Entinostat enhanced IFN-γ type immune response induced by IL-2 treatment. Splenocytes (1×10⁶ cells) were harvested and stimulated with PMA (20 ng/ml) and Ionomycin (1 µg/ml) for 5 hours in the presence of Brefeldin A. Cells were then stained for surface markers and intracellular IFN-γ. Histograms show percentage of IFN-γ expressing cells in CD8 population. <i>p</i> = 0.01 for combination vs. IL-2. E, Entinostat reduced tumor infiltration of Tregs. Tumor sections were stained with anti-Foxp3 antibody to show infiltration of Tregs. Histogram shows average numbers of stained Tregs in random 20× resolution bright fields (Tregs number in each field was obtained by blinded count). F, Entinostat treatment induced H3 histone acetylation in splenocytes. BALB/c mice were treated with vehicle (0.5% methocel) or 5 mg/kg/day entinostat by gavage for 5 days. Cells were harvested from spleens and subjected to Western blot analysis for acetylated-H3 histone.</p

Survivin vaccine induces antigen-specific CD8 cells and entinostat enhances IFN-γ induction.

<p>A. Tetramer analysis of splenocytes obtained from mice immunized with SurVaxM. Splenocytes were stained with anti CD8 antibodies and survivin-specific tetramers for flow cytometric analysis. Results are based upon gating of CD8+ T cells and indicate the percent of double labeled cells (CD8+/Tetramer+) with respect to specific tetramer. B, Combination treatment led to CD8⁺ IFN-γ⁺ cells induction. Mice were treated as indicated. Splenocytes were stimulated and intracellular IFN-γ staining was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030815#pone-0030815-g001" target="_blank">Figure 1D</a>. <i>p</i><0.01 for combination vs. vehicle.</p

Antitumor activity of Tregs depletion antibody PC61 in RENCA tumor.

<p>A, Plots show effect of vehicle, PC61, entinostat or combination treatment on CD25 and Foxp3 levels. Quantification of Tregs percentage, Foxp3 expression by FACS analysis is shown as histograms. Values are means and error bars represent S.D. for 6–7 samples per group. B, Tumor weight measurements from PC61 depletion experiment. Columns, mean grams of tumor; Bars, S.D.. n = 6–7. * represents <i>p</i><0.05 for marked point vs. vehicle; ** represents <i>p</i><0.01 for marked point vs. vehicle.</p

STAT3 signaling is involved in down-regulation of Foxp3 by entinostat.

<p>A and B, Entinostat treatment induces STAT3 acetylation. HepG2 cells or splenocytes were treated for 6 hours, harvested, and lyzed for Western immunobloting or immunoprecipitation. A, Entinostat induced STAT3 acetylation in HepG2 cells. Upper panel: Cell lysates were analyzed directly and blotted with anti-STAT3 and actin. Bottom panel, Cell lysates were immunoprecipitated with anti-STAT3 antibody and then blotted with anti-STAT3 antibody and with anti-acetylated lysine antibody. B, Entinostat induced STAT3 acetylation in splenocytes. IP and Western blot were performed as described in A. C and D, Down-regulation of Foxp3 is inhibited by blocking STAT3 signaling. Splenocytes were harvested from BALB/c mice and put in culture. Cultures were treated with vehicle or 0.5 mM specific STAT3 peptide inhibitor for one hour, followed by vehicle or 0.5 µM entinostat treatment for 23 hours. Cells were harvested and analyzed by flow cytometry using fluorescence-conjugated antibodies specific to CD4 and Foxp3. Fixable live/dead dye was used to stain cells and live cells were gated. Cell culture was in absence (panel C) or in presence (panel D) of IL-2. In each condition, histograms show quantification of Foxp3 expression in Tregs by FACS analysis. Graph shows means, error bars represent standard deviations. In C, <i>p</i> = 0.0002 for entinostat vs. STAT3 inhibitor+entinostat. In D, <i>p</i> = 0.00015 for entinostat vs. STAT3 inhibitor+entinostat. Results are representative of three separate experiments.</p