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-

Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in Translocation Renal Cell Carcinoma

Purpose: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease. Experimental Design: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models. Results: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3–tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation. Conclusions: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3–tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3–tRCC

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

Therapeutic Targeting of Sunitinib-Induced AR Phosphorylation in Renal Cell Carcinoma

Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array, we discovered increased expression of AR in an RCC patient–derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro. An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2. Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in a RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC

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

Suppression of Serum Interferon-γ Levels as a Potential Measure of Response to Ustekinumab Treatment in Patients With Systemic Lupus Erythematosus

Objective: In a previously reported phase II randomized, placebo-controlled, interventional trial, we demonstrated that treatment with ustekinumab, an anti–interleukin-12 (IL-12)/IL-23 p40 neutralizing monoclonal antibody, improved global and organ-specific measures of disease activity in patients with active systemic lupus erythematosus (SLE). Utilizing the biomarker data from this phase II clinical study, we sought to determine whether modulation of the expression of IL-12, IL-23, or both cytokines by ustekinumab is associated with clinical efficacy in patients with SLE. Methods: This phase II randomized, placebo-controlled study enrolled 102 patients with autoantibody-positive SLE whose disease remained active despite standard-of-care therapy. Patients were randomized at a 3:2 ratio to receive ~6 mg/kg ustekinumab intravenously or placebo at week 0, followed by subcutaneous injections of 90 mg ustekinumab or placebo every 8 weeks, with placebo crossover to 90 mg ustekinumab every 8 weeks. The SLE Responder Index 4 (SRI-4) at week 24 was used to determine which patients could be classified as ustekinumab responders and which could be classified as nonresponders. In addition to measurements of p40 and IL-23, serum levels of interferon-γ (IFNγ), IL-17A, IL-17F, and IL-22, as a proxy for the IL-12 and IL-23 pathways, were quantified by immunoassay. Results: Changes in the serum levels of IL-17A, IL-17F, and IL-22 at different time points after treatment were not consistently significantly associated with an SRI-4 clinical response to ustekinumab in patients with SLE. In contrast, an SRI-4 response to ustekinumab was significantly associated (P < 0.01) with durable reductions in the serum IFNγ protein levels at several time points relative to baseline, which was not observed in ustekinumab nonresponders or patients who received placebo. Conclusion: While not diminishing a potential role of IL-23, these serum biomarker assessments indicate that IL-12 blockade has an important role in the mechanism of action of ustekinumab treatment in patients with SLE