HDAC 1 and 6 modulate cell invasion and migration in clear cell renal cell carcinoma

Indexación: Web of ScienceBackground: Class I histone deacetylases (HDACs) have been reported to be overexpressed in clear cell renal cell carcinoma (ccRCC), whereas the expression of class II HDACs is unknown. Methods: Four isogenic cell lines C2/C2VHL and 786-O/786-OVHL with differential VHL expression are used in our studies. Cobalt chloride is used to mimic hypoxia in vitro. HIF-2 alpha knockdowns in C2 and 786-O cells is used to evaluate the effect on HDAC 1 expression and activity. Invasion and migration assays are used to investigate the role of HDAC 1 and HDAC 6 expression in ccRCC cells. Comparisons are made between experimental groups using the paired T-test, the two-sample Student's T-test or one-way ANOVA, as appropriate. ccRCC and the TCGA dataset are used to observe the clinical correlation between HDAC 1 and HDAC 6 overexpression and overall and progression free survival. Results: Our analysis of tumor and matched non-tumor tissues from radical nephrectomies showed overexpression of class I and II HDACs (HDAC6 only in a subset of patients). In vitro, both HDAC1 and HDAC6 over-expression increased cell invasion and motility, respectively, in ccRCC cells. HDAC1 regulated invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, hypoxia stimulation in VHL-reconstituted cell lines increased HIF isoforms and HDAC1 expression. Presence of hypoxia response elements in the HDAC1 promoter along with chromatin immunoprecipitation data suggests that HIF-2 alpha is a transcriptional regulator of HDAC1 gene. Conversely, HDAC6 and estrogen receptor alpha (ER alpha) were co-localized in cytoplasm of ccRCC cells and HDAC6 enhanced cell motility by decreasing acetylated alpha-tubulin expression, and this biological effect was attenuated by either biochemical or pharmacological inhibition. Finally, analysis of human ccRCC specimens revealed positive correlation between HIF isoforms and HDAC. HDAC1 mRNA upregulation was associated with worse overall survival in the TCGA dataset. Conclusions: Taking together, these results suggest that HDAC1 and HDAC6 may play a role in ccRCC biology and could represent rational therapeutic targets.http://bmccancer.biomedcentral.com/articles/10.1186/s12885-016-2604-

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

EZH2 modifies sunitinib resistance in renal cell carcinoma by kinome reprogramming

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represent a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft (PDX) model that is intrinsically resistant to the RTKI sunitinib but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its anti-angiogenic and anti-metastatic activity but lost its direct anti-tumor effects due to kinome reprogramming, which resulted in suppression of pro- apoptotic and cell cycle regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTK, restoring the anti-tumor effects of sunitnib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease.This research was funded by Roswell Park Cancer Institute’s Cancer Center Support Grant from National Cancer Institute, NIH P30CA016056 (RP) and a generous donation by Richard and Deidre Turner (RP). This investigation was conducted in-part in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR020128-01 from the National Center for Research Resources, National Institutes of Health

Tasquinimod Modulates Suppressive Myeloid Cells and Enhances Cancer Immunotherapies in Murine Models

A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206(+)). CD11b(+) myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8(+) T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFN gamma production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies. (C) 2014 AACR

Dll4 Blockade Potentiates the Anti-Tumor Effects of VEGF Inhibition in Renal Cell Carcinoma Patient-Derived Xenografts

<div><p>Background</p><p>The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC).</p><p>Methods and Results</p><p>Severe combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36–62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38–54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72–80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model.</p><p>Conclusions</p><p>Overall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.</p></div

Effects of REGN1035 and/or ziv-aflibercept on tumor cell proliferation in RP-R-01 ccRCC xenograft.

<p>Mice were treated with vehicle, ziv-aflibercept and/or REGN1035. Tumors were harvested, processed, and tissue sections were stained for differential expression of Ki67. (A) Representative images. (B) Quantitative analysis was done in a blinded manner. Results are expressed as mean percentage positive nuclei ± S.E. *<i>p</i><0.05 using adjusted t-test analysis.</p

Anti-tumor efficacy of anti-Dll4 (REGN1035) and anti-VEGF (ziv-aflibercept) in a sunitinib resistant RP-R-01 ccRCC patient-derived xenograft model.

<p>Mice with established subcutaneous tumors were treated with sunitinib for 4 weeks until tumor tissue was no longer responsive to treatment (tumor volume was ∼6 times that of pretreatment volume). At which time, mice were treated with either sunitinib, ziv-aflibercept, REGN1035 or the combination of ziv-aflibercept plus REGN1035 for a period of four weeks. (A) Tumor growth curve of average tumor volume (mm³) ± S.E. (B) End point tumor weights (g). *<i>p</i><0.05, as compared to combination group using adjusted t-test analysis. <i>Effect of REGN1035 and/or anti-VEGF (ziv-aflibercept) on sunitinib resistant RP-R-01 tumor vasculature</i>. Sunitinib resistant tumors from treated mice were harvested, processed, and tissue sections were stained for the differential expression of CD31. (C) Representative pictures. (D) Blind quantitative analysis of CD31. Results are expressed as mean percentage positive stained area ± S.E. *<i>p</i><0.05, as compared to combination group using t-test analysis.</p

Anti-tumor efficacy of anti-Dll4 (REGN1035) and/or anti-VEGF (sunitinib or ziv-aflibercept) in RP-R-01 and RP-R-02 ccRCC patient derived xenografts.

<p>Mice (8 mice/group) were treated with vehicle, sunitinib, or ziv-aflibercept and/or REGN1035. Left panel: Tumor growth curves. (A) RP-R-01 REGN1035 and/or sunitinib treatment groups (B) RP-R-01 REGN1035 and/or ziv-aflibercept treatment groups (C) RP-R-02 REGN1035 and/or sunitinib treatment groups. Each line represents the average tumor volume (mm³) of each treatment group ± S.E. (D, E, F) End point tumor weights (g). * <i>p</i><0.05 using adjusted t-test analysis.</p

Effect of REGN1035 and/or anti-VEGF (sunitinib or ziv-aflibercept) on (A, B) RP-R-01 and (C) RP-R-02 tumor vasculature.

<p>Tumors from treated mice were harvested, processed, and tissue sections were stained for the differential expression of CD31 (red) for visualization of endothelial cells (left panels). (D, E, F) Blinded quantitative analysis of CD31 (right panels). Results are expressed as mean percentage positive stained area ± S.E. *<i>p</i><0.05 using adjusted t-test analysis.</p