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BackgroundBreast cancer (BC) is considered to be one of the primary causes of cancer deaths in women. Cuproptosis was suggested to play an important role in tumor proliferation and tumor immune microenvironment. Therefore, an investigation was conducted to identify the relationship between cuproptosis-related long non-coding RNAs (lncRNAs) and BC prognosis.MethodBased on The Cancer Genome Atlas (TCGA), nine cuproptosis-related lncRNAs were identified by Pearson’s analysis and Cox regression analysis to create a cuproptosis-related lncRNA signature. Subsequently, patients with BC were divided into high-risk and low-risk groups. The Kaplan–Meier curves and a time-dependent receiver operating characteristic (ROC) analysis were employed to elucidate the predictive capability of the signature. After that, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted by Gene Set Enrichment Analysis (GSEA), and the lncRNA–mRNA co-expression network was established by Cytoscape software. Furthermore, the ESTIMATE score was calculated, and the immune cell type component analysis was conducted. Eventually, immunotherapy response analysis was applied to identify the predictive power of cuproptosis-related lncRNAs to tumor immunotherapy response, including immune checkpoint gene expression levels, tumor mutational burden (TMB), and microsatellite instability (MSI).ResultsPatients with BC in the low-risk groups showed better clinical outcomes. The KEGG pathways in the high-risk groups were mainly enriched in immune response and immune cell activation. Furthermore, the ESTIMATE scores were higher in the low-risk groups, and their immune cell infiltrations were dramatically different from those of the high-risk groups. The low-risk groups were shown to have higher infiltration levels of CD8+ T cells and TMB-high status, resulting in better response to immunotherapies.ConclusionThe findings of this study revealed that the nine-cuproptosis-related lncRNA risk score was an independent prognostic factor for BC. This signature was a potential predictor for BC immunotherapy response. What we found will provide novel insight into immunotherapeutic treatment strategies in BC.

BackgroundDysregulated non-coding RNAs exhibit critical functions in various cancers. Nonetheless, the levels and corresponding functions of cirCSNX14 in esophageal squamous cell carcinoma (ESCC) yet remain to be elucidated.MethodsInitially, the aberrant low levels of lncRNA-LET within ESCC tissues are validated via qRT-PCR observations. Moreover, the effects of lncRNA-LET upregulation on cell proliferation in vitro are determined. In addition, a series of assays determining the mechanistic views related to metabolism is conducted. Furthermore, the effects of lncRNA-LET in affecting tumor growth are investigated in vivo in a mouse model. Moreover, the interactions between lncRNA-LET and its networks are predicted and determined by RNA immunoprecipitation-assisted qRT-PCR as well as luciferase reporter assays.ResultsThe downregulation of lncRNA-LET is correlated to the poor prognosis of ESCC patients. Moreover, the upregulated expression of lncRNA-LET could have reduced the cell viability. In vivo tumor inhibition efficacy assays showed that an increase of lncRNA-LET presented excellent inhibitory effects on cancer proliferation as reflected by tumor weight and volume in mice. Finally, the mechanistic views regarding the effects of miR-106b-5p or miR-93-5p and SOCS4 on ESCC are related to the feedback of lncRNA-LET.ConclusionCollectively, this study suggested that lncRNA-LET miR-93-5p or the miR-106b-5p–SOCS4 axis may provide great potential in establishing ESCC therapy.

BackgroundPyroptosis is a novel identified form of inflammatory cell death that is important in the development and progression of various diseases, including malignancies. However, the relationship between pyroptosis and triple-negative breast cancer (TNBC) is still unclear. Therefore, we started to investigate the potential prognostic value of pyroptosis-associated genes in TNBC.MethodsThirty-three genes associated with pyroptosis were extracted from previous publications, 30 of which were identified in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort. On the basis of the 30 pyroptosis-related genes, patients with TNBC were divided into three subtypes through unsupervised cluster analysis. The prognostic value of each pyroptosis-associated gene was assessed, and six genes were selected by univariate and LASSO Cox regression analysis to establish a multigene signature. According to the median value of risk score, patients with TNBC in the training and validation cohorts were separated to high- and low-risk sets. The enrichment analysis was conducted on the differentially expressed genes (DEGs) of the two risk sets using R clusterProfiler package. Moreover, the ESTIMATE score and immune cell infiltration were calculated by the ESTIMATE and CIBERSORT methods. After that, the correlation among pyroptosis-associated risk score and the expression of immune checkpoint-associated genes as well as anti-cancer drugs sensitivities were further analyzed.ResultsIn the training and validation cohorts, patients with TNBC in the high-risk set were found in a lower survival rate than those in the low-risk set. Combined with the clinical characteristics, the pyroptosis-related risk score was identified as an independent risk factor for the prognosis of patients with TNBC. The enrichment analysis indicated that the DEGs between the two risk groups were mainly enriched by immune responses and activities. In addition, patients with TNBC in the low-risk set were found to have a higher value of ESTIMATE score and a higher rate of immune cell infiltration. Finally, the expression levels of five genes [programmed cell death protein 1 (PD-1); cytotoxic t-lymphocyte antigen-4 (CTLA4); lymphocyte activation gene 3 (LAG3); T cell immunoreceptor with Ig and ITIM domains (TIGIT)] associated with immune checkpoint inhibitors were identified to be higher in the low-risk sets. The sensitivities of some anti-cancer drugs commonly used in breast cancer were found closely related to the pyroptosis-associated risk model.ConclusionThe pyproptosis-associated risk model plays a vital role in the tumor immunity of TNBC and can be applied to be a prognostic predictor of patients with TNBC. Our discovery will provide novel insight for TNBC immunotherapies.

Purpose Cabazitaxel has been demonstrated to improve the overall survival for men with metastatic castrate-resistant prostate cancer. The purpose of this study was to determine the maximum tolerated dose for concurrent cabazitaxel with androgen deprivation and intensity modulated radiation therapy in men with high-risk prostate cancer. Methods and Materials Twenty men were enrolled in this institutuional review board–approved phase I clinical trial using a 3 + 3 design. Patients were followed prospectively for safety, efficacy, and health-related quality of life (Expanded Prostate Index Composite). Efficacy was assessed biochemically using the Phoenix definition. Results With a median follow-up time of 56 months, the maximum tolerated dose of concurrent cabazitaxel was 6 mg/m2. The 5-year biochemical disease-free survival was 73%, despite 75% of patients having very high risk prostate cancer per the National Comprehensive Cancer Network guidelines. Four patients were unable to complete chemotherapy owing to dose-limiting toxicities (eg, rectal bleeding, diarrhea, and elevated transaminase). There was no significant minimally important difference in Expanded Prostate Index Composite patient-reported outcomes for either the urinary or bowel domains; however, there was a significant decrease in the sexual domain. Conclusions This is the first clinical trial of prostate cancer to report on the combination of cabazitaxel and radiation therapy. The maximum tolerated dose of concurrent cabazitaxel with radiation and androgen deprivation therapy was determined to be 6 mg/m2. Despite the aggressive nature of the disease, robust biochemical control was observed.

BackgroundAs a kind of small membrane vesicles, exosomes are secreted by most cell types from multivesicular endosomes, including tumor cells. The relationship between exosomes and immune response plays a vital role in the occurrence and development of tumors. Nevertheless, the interaction between exosomes and the microenvironment of tumors remains unclear. Therefore, we set out to study the influence of exosomes on the triple-negative breast cancer (TNBC) microenvironment.MethodOne hundred twenty-one exosome-related genes were downloaded from ExoBCD database, and IVL, CXCL13, and AP2S1 were final selected because of the association with TNBC prognosis. Based on the sum of the expression levels of these three genes, provided by The Cancer Genome Atlas (TCGA), and the regression coefficients, an exosome risk score model was established. With the median risk score value, the patients in the two databases were divided into high- and low-risk groups. R clusterProfiler package was employed to compare the different enrichment ways between the two groups. The ESTIMATE and CIBERSORT methods were employed to analyze ESTIMATE Score and immune cell infiltration. Finally, the correlation between the immune checkpoint-related gene expression levels and exosome-related risk was analyzed. The relationship between selected gene expression and drug sensitivity was also detected.ResultsDifferent risk groups exhibited distinct result of TNBC prognosis, with a higher survival rate in the low-risk group than in the high-risk group. The two groups were enriched by immune response and biological process pathways. A better overall survival (OS) was demonstrated in patients with high scores of immune and ESTIMATE rather than ones with low scores. Subsequently, we found that CD4+-activated memory T cells and M1 macrophages were both upregulated in the low-risk group, whereas M2 macrophages and activated mast cell were downregulated in the low-risk group in patients from the TCGA and GEO databases, respectively. Eventually, four genes previously proposed to be targets of immune checkpoint inhibitors were evaluated, resulting in the expression levels of CD274, CTLA4, LAG3, and TIM3 being higher in the low-risk group than high-risk group.ConclusionThe results of our study suggest that exosome-related risk model was related to the prognosis and ratio of immune cell infiltration in patients with TNBC. This discovery may make contributions to improve immunotherapy for TNBC.

Despite androgen dependence in a majority of castration-resistant prostate cancers, some cancer cells are independent of androgen receptor (AR) function, a feature of heterogeneity in prostate cancer. One of the aggressive variants of prostate cancer that are AR independent is neuroendocrine prostate cancer (NEPC). This manuscript will focus on the new finding of human one cut domain family member 2 (ONECUT2) transcription factor and its role in castration resistance, especially in NEPC.

Objectives There is no study published regarding the benefit of radiation therapy (RT) in combination with immune checkpoint inhibitors (ICIs) for the treatment of metastatic renal cell cancer (mRCC). This report is part of an exploratory study aiming to determine the immunomodulatory activity of RT alone or in combination with pembrolizumab in solid tumors. Materials and methods mRCC patients were treated with a combination of RT (8 Gy×1 or 4 Gy×5) followed by pembrolizumab with or without lead-in dose of pembrolizumab. Treatment response was measured based on the modified Response Evaluation Criteria in Solid Tumors criteria. Adverse events were monitored and graded. Pre-RT and post-RT tumor biopsies were obtained to evaluate programmed death-ligand 1 expression. Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. Results Twelve mRCC patients who progressed on prior antiangiogenic therapy were enrolled. Half had 2 lines of prior therapy. Two patients (16.7%) had partial responses and were on study for 12.4 and 14.5 months. Three patients had stable disease for a period ranging from 4.2 to 10.4 months, whereas 7 patients had progressive disease. Median progression-free survival was 8.6 months and median overall survival was 32.3 months. Three patients had grade ≥3 events (hyperglycemia, thrombocytopenia, transaminitis). Biopsied tissue programmed death-ligand 1 expression and tumor-infiltrating lymphocytes were numerically higher in responders comparing to nonresponders (Modified Proportion Score 45% vs. 30.45%; tumor-infiltrating lymphocytes odds ratio 4.92). Conclusion Combining RT with pembrolizumab in pretreated mRCC is well-tolerated and appears to have comparable efficacy with single-agent nivolumab.

It is known that variations in the concentrations of certain elements in humans may be an indication of cancers. In this work, a method for the quantitative analysis of 22 elements in non-tumor and esophageal squamous cell carcinoma (ESCC) tissues from the same individual is reported. Based on the optimized platform combined with multivariate analysis, diagnostic models of ESCC were established using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), showing excellent classification of cancerous and non-cancerous group by metallomic profiling. Elemental concentrations of 10 elements (Mn, Se, Cu, Ti, Mg, Fe, Co, Zn, Sr, Ca) showed significant difference (p 0.001) in tumor and non-tumor tissues, in which Mn, Se, Cu and Ti are the top 4 elements of statistical significance and a shift towards higher concentration levels has also been observed in the tumor samples. These results confirm the considerable potential of elemental studies for biomedical purposes. To our knowledge, previous studies on elemental concentration in esophageal cancer were performed in serum or plasma levels; and this is the first study to evaluate the association of tissue elemental concentrations with ESCC.

In our previous study we have found that miR-548k has oncogenic roles in esophageal squamous cell carcinoma (ESCC) via repressing long noncoding RNA (lncRNA)-LET and further upregulating nuclear factor 90 (NF90). However, the upstream factors controlling miR-548k expression are still unknown. In this study, we found NF90 directly binds pri-miR-548k, increases the stability of pri-miR-548k, and upregulates the expression of pri-miR-548k and miR-548k. Therefore, NF90, miR-548k and lncRNA-LET forms a feedback loop. Gain-of-function and loss-of-function assays demonstrated that in accordance with the roles of miR-548k, NF90 also promotes ESCC cell proliferation and migration. Furthermore, we verified the regulatory feedback loop between NF90, miR-548k, and lncRNA-LET. We found NF90 upregulated miR-548k and downregulated lncRNA-LET. miR-548k downregulated lncRNA-LET and upregulated NF90. lncRNA-LET downregulated NF90 and miR-548k. Through the reciprocal regulations between each other, the NF90/miR-548k/lncRNA-LET feedback loop controls the expressions of NF90 targets (HIF-1α and VEGF), miR-548k targets (KLF10 and EGFR), and lncRNA-LET target (p53). Further functional assays demonstrated that activation of the NF90/miR-548k/lncRNA-LET feedback loop via simultaneously overexpressing NF90 and miR-548k and simultaneously depleting lncRNA-LET significantly promotes ESCC cell proliferation and migration in vitro and ESCC tumor growth in vivo. Targeting the NF90/miR-548k/lncRNA-LET feedback loop via simultaneously depleting NF90 and miR-548k and simultaneously overexpressing lncRNA-LET significantly inhibits ESCC cell proliferation and migration in vitro and ESCC tumor growth in vivo. In summary, our findings identified a crucial oncogenic NF90/lncRNA-LET/miR-548k feedback amplification loop, which may be promising therapeutic targets for ESCC.

Lessons Learned Entinostat at the selected dose levels in combination with a standard dose of enzalutamide showed a promising safety profile in this small phase I study Background Entinostat inhibits prostate cancer (PCa) growth and suppresses Treg cell function in vitro and in vivo. Methods This was a phase I study to explore the safety and preliminary efficacy of entinostat (3 and 5 mg orally per week) in combination with enzalutamide in castration resistant PCa (CRPC). The study was carried out in an open-label two-cohort design. Patients who had developed disease progression on or were eligible for enzalutamide were enrolled in the study. The safety profile of the combination therapy, Prostate specific antigen (PSA) levels, the pharmacokinetics of enzalutamide after entinostat administration, peripheral T-cell subtype (including Treg quantitation), and mononuclear cell (PBMC) histone H3 acetylation were analyzed. Results Six patients with metastatic CRPC were enrolled. There was no noticeable increment of fatigue related to entinostat. Toxicities possibly or probably related to entinostat or the combination therapy included grade 3 anemia 1/6 (17%), grade 2 white blood cell (WBC) decrease 1/6 (17%), and other self-limiting grade 1 adverse events (AEs). Median duration of treatment with entinostat was 18 weeks. Entinostat did not affect the steady plasma concentration of enzalutamide. Increased PBMC histone H3 acetylation was observed in blood samples. No evident T-cell subtype changes were detected, including in Treg quantitation. Conclusion Entinostat 5 mg weekly in combination with enzalutamide showed an acceptable safety profile in this small phase I study. A planned phase II part of the trial was terminated because of sponsor withdrawal.

Background：Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in the world, and its mechanism is related to tumor microenvironment. Immune / stromal score can be used to identify differential expressed genes (DEGs) of cancer, but it has not been reported in ESCC.Methods: The expression profile and clinicopathological information of patients with ESCC were downloaded from The Cancer Genome Atlas (TCGA) database. The immune score and stromal score of patients with ESCC were calculated by ESTIMATE algorithm. The relationship between immune / stromal score and clinicopathological characteristics and survival prognosis of patients with ESCC was analyzed. DEGs were identified according to the score, followed by gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) analysis. The DEGs with prognostic value were revealed by overall survival analysis. Protein-protein interaction (PPI) network analysis was performed on these DEGs. Finally, the DEGs were verified by Chinese Cancer Genomic Database-Esophageal Squamous-Cell Carcinoma (CCGD-ESCC) database. Results: Immune and stromal scores were related to the prognosis of patients with ESCC. 223 DEGs were screened according to immune / stromal scores. GO and KEGG analysis showed that the selected DEGs was mainly related to plasma membrne, immune response and inflammatory response. Overall survival analysis showed that 68 genes were associated with prognosis. PPI network analysis showed that the core genes were CD86, LCP2, TLR7, CSF1R, C1QA, IRF8, CTLA4 and so on. A total of 17 genes with prognostic value were verified by CCGD-ESCC database. Conclusions: we screened and verified tumor microenvironment-related genes that have prognostic value and are expected to be used as therapeutic targets for patients with ESCC.

BackgroundThyroid carcinoma (TC) is the most common malignant endocrine tumor worldwide. Several studies have documented that male patients with TC have a higher rate of metastasis and disease recurrence than female patients. However, the mechanism underlying this observation is not completely clear. The goal of our research was to investigate the potential key candidate genes and pathways related to TC progression in male patients at the molecular level.MethodsA total of 320 samples were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Hub genes were screened out using weighted gene coexpression network analysis (WGCNA) and a protein–protein interaction (PPI) network analysis. Survival analysis was used to identify hub genes associated with disease-free survival (DFS) rates. Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression (ESTIMATE) data were used to assess the relationship between hub genes and immune cell infiltration. The molecular mechanism and biological functions of hub genes were explored using RT-qPCR, Western blot, Cell Counting Kit-8 Assay, flow cytometry, Transwell assays, and scratch assays.ResultsForty-seven hub genes were identified, and the survival analysis demonstrated that anti-silencing function 1B (ASF1B) was the sole independent risk factor for poor DFS in male TC patients. Possible associations between the results from the ESTIMATE analysis showed that the ASF1B expression level was related to the ESTIMATE score, immune score, and T-cell regulatory (Treg) infiltration level. Through in vitro cell function experiments, we verified that knockdown of ASF1B inhibited KTC-1 cell proliferation, promoted cell apoptosis, and blocked cell cycle. The silencing of ASF1B reduced protein kinase B (AKT), phospho-AKT (p-AKT), and forkhead box p3 (FOXP3) in KTC-1 cells. Moreover, FOXP3 overexpression markedly restored the cell migration, invasion, and proliferation abilities repressed by ASF1B knockdown.ConclusionsOur results indicate that ASF1B can be considered a prognostic marker, therapeutic target, and predictor of immunotherapy response in male thyroid cancer patients. However, further in-depth studies are required to validate this finding.

Dual specificity phosphatase 4 (DUSP4) is a prognostic marker and potential target of papillary thyroid carcinoma (PTC); however, the molecular mechanism underlying DUSP4-regulated PTC carcinogenesis is unknown. DUSP4 is a negative regulator of the autophagy promoter, JNK. This study explored the relationship between DUSP4 and JNK-mediated autophagic cell death in PTC, and the roles of DUSP4 in PTC using gain-of-function and loss-of-function assays. In addition, we further identified the significance of the JNK–BCL2–Beclin1–autophagy signaling pathway on DUSP4-regulated PTC carcinogenesis by combining knockdown of DUSP4 with a JNK-specific inhibitor (SP600125). We found that knockdown of DUSP4 promoted the phosphorylation of JNK and BCL2 in PTC cells, and enhanced the release of Beclin1 from the BCL2–Beclin1 complex. Knockdown of DUSP4 promoted autophagy and the death of PTC cells. The death and autophagy enhanced by knockdown of DUSP4 was reversed by the JNK inhibitor. We further extended the in-vitro experiments by subcutaneously injecting nude mice with K1 cells transfected with DUSP4-silencing vector. In-vivo assays showed that knockdown of DUSP4 not only inhibited tumor growth, but also promoted the phosphorylation of JNK and BCL2 and the expression of LC3II. In conclusion, DUSP4 inhibits BCL2–Beclin1–autophagy signaling by negatively regulating JNK activity, thus inhibiting PTC oncogenesis. The data from this study contribute to the prevention and cure of PTC.

Inhibition of androgen receptor (AR) signaling is initially effective in treating metastatic prostate cancers (mCRPC). However, these treatments are not curative, and the emergence of castration-re...

Dysregulated noncoding RNAs have been observed in diverse cancers. MIR458K is frequently amplified in esophageal squamous cell carcinoma (ESCC). However, the expression, clinical significances, and action mechanisms of miR-548k in ESCC are still unclear. In this study, we found that miR-548k is significantly up-regulated in ESCC tissues and cell lines. Up-regulated miR-548k expression is significantly correlated with advanced invasion depth, lymph node metastasis, advanced TNM stage, and poor overall survival. Gain-of- and loss-of-function assays demonstrated that miR-548k promotes the proliferation and migration of ESCC cells in vitro and tumor growth in vivo. Mechanistically, we found that miR-548k directly targets and represses the expression of long noncoding RNA-LET (lncRNA-LET), and further down-regulates p53 and up-regulates NF90. In addition, we found that lncRNA-LET is down-regulated and inversely correlated with miR-548k in ESCC. Down-regulated lncRNA-LET also indicated poor overall survival of ESCC patients. Functional assays demonstrated that lncRNA-LET inhibits the proliferation and migration of ESCC cells, and the effects of miR-548k on ESCC are dependent on the negative regulation of lncRNA-LET. In summary, our data revealed the critical roles of miR-548k-lncRNA-LET regulation axis in ESCC and suggested that the miR-548k-lncRNA-LET regulation axis may be promising prognostic biomarkers and therapeutic targets for ESCC.

As a unique member of the cadherin superfamily, T-cadherin (T-cad) has been demonstrated to be associated with gastric cancer (GC) prognosis. To elucidate the function of T-cad in GC in vitro, the present study firstly examined T-cad protein expression in normal and gastric cancer tissues and cell lines, and it was demonstrated to be significantly downregulated in gastric cancer samples compared with normal samples. Control and T-cad expression vectors were then transfected into the MGC8-03 and AGS GC cell lines. Utilizing MTT, clonogenic, flow cytometry, wound healing and Transwell invasion assays in addition to Western blotting, the present study demonstrated that the overexpression of T-cad suppressed GC cell growth and colony formation via cell cycle arrest at the G0/G1 phase via downregulating the expression of cyclin dependent kinase 4 and Cyclin D1. In addition, overexpression of T-cad significantly inhibited GC cell migration and invasion by increasing E-cadherin and decreasing Vimentin expression. These findings suggest T-cad may be important in GC cell proliferation and metastasis and serve as a promising target for the treatment of GC in the future.

Gastric cancer (GC) is among the most common types of human cancer and is associated with recurrence and metastasis, despite comprehensive surgical and medical treatment. Previous studies observed downregulation of T-cadherin expression in GC tissues, suggesting that this protein may act as an oncosuppressor. The current study investigated the activity of T-cadherin in GC tissues. In a follow-up study of 81 patients with GC, a Kaplan-Meier analysis of overall survival revealed a strong association of T-cadherin overexpression with increased overall survival (P

96 Background: Entinostat (SNDX-275, MS-275) is an oral histone deacetylase (HDAC) inhibitor with selectivity towards class I and IV HDACs. Entinostat inhibits prostate cancer (PCa) growth and suppresses Treg cell function in vitro and in vivo. The primary objective of this study was to determine the safe dose of Entinostat in combination with Enzalutamide in prostate cancer patients (PCa). Methods: Phase I, 2 cohorts "3+3", dose-escalation study to explore the safety and preliminary efficacy of Entinostat in combination with Enzalutamide in castration-resistant PCa (CRPC). The planned dose level of Entinostat was 3 and 5 mg orally per week. CRPC Patients progressed on Enzalutamide or eligible for Enzalutamide, able to tolerate 160 mg daily dose (in the initial run-in phase if Enzalutamide naïve), ECOG 0-1, and acceptable organ functions were enrolled in the study. The safety profile of the combination therapy, PSA, pharmacokinetics of Enzalutamide post-Entinostat administration, and peripheral T cell subtype (including Treg), mononuclear cell (PBMC) histone 3 acetylation were analyzed. Results: Total 6 mCRPC patients were enrolled. There was no dose limiting toxicity related to Entinostat in these patients. No obvious increased fatigue related to Entinostat. Toxicities possibly or probably related to Entinostat or the combination therapy included G3 anemia 1/6 (17%), G2 WBC decrease 1/6 (17%), All other toxicities were grade 1 only (Nausea 2/6, anorexia 1/6, emesis 1/6, constipation 1/6, headache 1/6, platelet count decrease 1/6, hypokalemia 1/6, hypoalbuminemia 1/6, hypermagnesemia 1/6). The median duration of treatment with Entinostat was 18 weeks. For patients already progressed on Enzalutamide there was no PSA response after Entinostat was added. Entinostat did not affect the steady plasma concentration of Enzalutamide. Increased PBMC H3 acetylation was observed (up to 3.6 fold from baseline) in the tested samples. No evident T cell subtype, including Treg, changes from these sample analysis. Phase II part of the trial was terminated because of sponsor withdrawal. Conclusions: Entinostat at the selected dose levels in combination with standard dose of enzalutamide showed promising safety profile in this small phase I study. Clinical trial information: NCT03829930.

// Wenyi Wu 1, * , Liting Zhang 2, * , Jianqing Lin 1 , Hanwei Huang 3 , Bai Shi 1 , Xingong Lin 1 , Zhongxin Huang 1 , Chaoyang Wang 1 , Jianlong Qiu 4 , Xiaolong Wei 5 1 Department of general surgery, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian, China 2 Endocrine Department, The 180th Military Hospital of Chinese Peoples, Liberation Army, Quanzhou, Fujian, China 3 Endocrine Department, Affiliated Zhongshan Hospital of Guangdong Medical College, Zhongshan, Guangdong, China 4 Department of Pathology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian, China 5 Department of Pathology, Cancer Hospital of Shantou University Medical College, Shantou, Guangdong, China * These authors contributed equally to this work Correspondence to: Xiaolong Wei, email: weixiaolonghh@126.com Wenyi Wu, email: wwyii8522858@163.com Keywords: HIC1, SIRT1, papillary thyroid carcinoma, promoter hypermethylation Received: April 21, 2016 Accepted: October 21, 2016 Published: October 26, 2016 ABSTRACT Hypermethylation leading to the loss of hypermethylated in cancer-1 (HIC1) gene expression occurs in many different types of human cancer. HIC1 is a transcriptional repressor that directly binds to the promoter region of NAD-dependent deacetylase sirtuin-1 (SIRT1). SIRT1 functions in cell growth, is anti-apoptotic, protect neurons, functions in senescence, and regulates energy restriction. Epigenetic modification and dysregulation affecting the HIC1/SIRT1 axis is potentially important for the development of malignancies. However, the importance of HIC1 expression in the development of papillary thyroid carcinoma, especially in Chinese patients, is uncertain. Therefore, we assessed the level of methylation in the HIC1 promoter and the mRNA and protein expression levels of HIC1 and SIRT1 in human thyroid papillary carcinoma and tumor adjacent control tissues. The demethylation reagent 5-aza-2′-deoxyctidine (5-aza-dc) and an HIC1 overexpression plasmid were used to manipulate the HIC1/SIRT1 pathway, and the effects on cell senescence, apoptosis, and cell cycle progression were assessed. Compared to normal thyroid tissue, thyroid tumors had lower expression of HIC1 and higher SIRT1 expression. The level of HIC1 methylation was also higher in thyroid carcinoma tissues than adjacent tissues. HIC1 expression was closely correlated with patient age and tumor progression. Restoration of HIC1 expression through an overexpression plasmid or 5-aza-dC treatment reduced SIRT1 expression and cell proliferation, and led to senescence, cell cycle arrest, and apoptosis. Aberrant expression of HIC1/SIRT1 and hypermethylation of the HIC1 promoter may be critical for the development and progression of papillary thyroid cancer.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing oxidoreductase that catalyzes the initial and rate-limiting step in the breakdown of non-dietary tryptophan. The biology and immunomodulatory role for IDO is discussed in this review with a focus on its interaction with immune cells and its potential therapeutic target in the clinic. IDO has been revealed to be a central regulator of immune responses in a broad variety of physiological and pathological settings, mostly serving as a multifaceted negative feedback mechanism, to self-regulate immune responses. IDO is considered a therapeutic target in cancer and the use of IDO inhibitors as single agent or in combination with other treatment modalities are under active investigation.

BACKGROUND Rab7 belongs to the Ras oncogene family. Many studies have shown that its dysfunction is associated with many types of malignant tumors, but its effect on the pathogenesis of gastric cancer (GC) is still unknown. Therefore, we investigated the effect and mechanism of Rab7 in GC. MATERIAL AND METHODS The expression of Rab7 in GC and adjacent tissues was detected by immunohistochemistry, Western blot analysis, and qRT-PCR. The relationship of Rab7 with clinicopathological parameters and prognosis was analyzed. The expressions of Rab7, PI3K, and AKT in GC cells were assessed by Western blot. Overexpressed and silenced GC cell lines were constructed and AGS cells were treated with LY294002. The proliferation capacity of GC cells was detected by CCK8 assay, cell cycle changes were detected by flow cytometry, and the invasion and migration abilities of GC cells were assessed by transwell assay. RESULTS The expression of Rab7 was upregulated in the samples and cells, and was positively correlated with lymph node metastasis but negatively correlated with histological differentiation and clinical prognosis. In cell function experiments, overexpression of Rab7 induced the transition from S phase to G2 phase and promoted the proliferation, invasion, and migration of GC cells. Our assessment of the molecular mechanism showed that Rab7 promoted the phosphorylation of PI3K and AKT in GC cells. Incubation with the PI3K inhibitor Ly294002 impaired the enhanced effect of Rab7 overexpression on proliferation, migration, and invasion abilities of GC cells. These results show that the Rab7 affects GC cell progression by modulating the PI3K/AKT pathway. CONCLUSIONS Rab7 could be a prognostic biomarker and therapeutic target of the PI3K/AKT pathway in GC.

Radium-223 is a first-in-class radiopharmaceutical recently approved for the treatment of castration-resistant prostate cancer in patients with symptomatic bone metastases. Initial studies investigating Radium-223 primarily used nonsteroidal first-generation antiandrogens. Since that time, newer antiandrogen therapies have demonstrated improved survival in patients with castration-resistant prostate cancer. It has been suggested that the rational combination of these newly approved agents with Radium-223 may lead to improved response rates and clinical outcomes. Currently, there is lack of information regarding the safety of concurrent administration of these agents with radiopharmaceuticals. Here, we report on hematologic toxicity findings from our institution in patients receiving concurrent Radium-223 and next-generation antiandrogen therapies with either enzalutamide or abiraterone.In a retrospective study, we analyzed patients who received Radium-223 as part of an early-access trial, and following FDA approval in May 2013, patients receiving Radium-223 as part of standard care. Radium-223 was given at standard dosing of 50 kBq/kg each month for 6 total cycles. Complete blood counts were performed before treatment monthly and following each injection. Blood counts from patients receiving Radium alone and concurrently with next-generation antiandrogens were compared. To date, 25 total patients were analyzed, with a median of 5 monthly doses received per patient. Fourteen patients received concurrent therapy during monthly Radium-223 with either enzalutamide (n=8) or abiraterone (n=6).Six patients expired due to disease progression. Two patients discontinued treatment due to grade 3 myelosuppression. For patients receiving either Radium alone and with concurrent next-generation antiandrogen therapy, there did not appear to be any statistically significant differences between initial and nadir blood counts. Mean change from initial neutrophil count to nadir was 1.9×10/L in patients receiving Radium alone, versus 2.3×10/L in patients receiving concurrent therapy (P=0.77). Mean change from initial hemoglobin value to nadir was 1.5 g/L in patients receiving Radium alone, versus 1.8 g/L in patients receiving concurrent therapy (P=0.31). Mean change from initial platelet count to nadir was 52.3×10 cells/L in patients receiving Radium alone versus 70.6×10 cells/L in patients receiving concurrent therapy (P=0.39). Individual blood counts for each measured laboratory are included in the supplemental data. PSA was stable or decreased in 22% of patients receiving Radium alone versus 35% of patients receiving combination treatment (P=0.24).Concurrent administration of Radium-223 and next-generation antiandrogen therapies appears to be well tolerated with similar toxicities to standard administration of Radium-223 alone. This particular cohort of patients represents a high-risk, heavily pretreated group of patients with advanced metastatic disease and significant marrow burden. Despite these risk factors, hematologic toxicity was modest and was in the range expected for this risk group based on previous trials. To date, this is the first study investigating the toxicity of combination treatment. Further studies investigating the safety and efficacy of combination treatments are warranted.

Multiple phase-2 trials in men with biochemically-recurrent prostate cancer (BRPC) have assessed the impact of nonhormonal agents on PSA kinetics. We have previously demonstrated that changes in PSA kinetics correlate with metastasis-free survival; however, it is unknown whether these changes also correlate with overall survival (OS).We performed a combined retrospective analysis of 146 men with BRPC treated on phase-2 trials using one of four investigational drugs: lenalidomide (n=60), marimastat (n=39), ATN-224 (n=22) and imatinib (n=25). We examined factors influencing OS, including within-subject changes in PSA kinetics (PSA slope, PSA doubling time and PSA velocity), before and 6 months after treatment initiation.After a median follow-up of 83.1 months, 49 of 146 men had died. In univariate Cox regression analysis, two factors were associated with OS: baseline PSA velocity and change in PSA velocity on therapy. In a landmark multivariable model, stratified by study (which controlled for age, Gleason score, type of local therapy and use of androgen-deprivation therapy prior to metastases), baseline PSA velocity and increase in PSA velocity on therapy remained independent predictors of OS. Median OS for men with an increase in PSA velocity on treatment was 115.4 months and was not reached for men with a decrease in PSA velocity (hazard ratio=0.47, 95% confidence interval 0.25-0.88; P=0.02).This hypothesis-generating study suggests that within-subject changes in PSA velocity after initiation of nonhormonal therapy may correlate with OS in men with BRPC. If validated in prospective trials, change in PSA velocity may represent a reasonable intermediate end point for screening new agents in these patients.

115 Background: Incidence of metastatic prostate cancer in U.S. males has increased over the past ten years, but it is unknown how this trend varies over time within different racial and ethnic populations. Methods: We identified all men first diagnosed with prostate cancer from 2005-2015 in the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute, which monitors 18 population-based cancer registries. Yearly cancer diagnosis frequency from 2005 to 2015 was categorized and analyzed by stage (in situ/localized, regional, and distant), race/ethnicity [White, Asian American/Pacific Islander (AAPI), Black], and age group (45-54, 55-69, 70-75). Chi-square tests, multivariable logistic regression models were used for data analysis with p < 0.05 considered significant. Results: In the 10-year study period, the proportion of regional-stage prostate cancer increased from 14.2% to 16.6% of cases (p < .0001) and distant-stage increased from 3.3% to 5.8% (p < .0001). The odds of being diagnosed with regional-stage prostate cancer in 2013-2015 compared to 2005-2008 were 1.3 times higher for Black men (95% CI: 1.2-1.5), 1.3 times higher for AAPI men (95% CI: 1.1-1.5), and 1.2 times higher for White men (95% CI: 1.2-1.3). The odds of being diagnosed with distant-stage prostate cancer in 2013-2015 compared to 2005-2008 were 1.6 times higher for Black men (95% CI: 1.4-1.9), 1.8 times higher for AAPI men (95% CI: 1.5-2.3), and 2.1 times higher for White men (95% CI: 1.9-2.2). Conclusions: The incidence of late-stage prostate cancer has increased significantly in all US male despite race and ethnicity. However, reginal-stage prostate cancer increased the most over time in AAPI and Black men, while newly-diagnosed distant prostate cancer increased the most over time in White men.

Preclinical drug screens identified disulfiram as a potent in vitro inhibitor of prostate cancer (PCa) cell growth. Although many mechanisms for its anticancer activity have been proposed, tumor suppressor gene re-expression through promoter demethylation emerged as one of the more plausible.We conducted an open-label, dose escalation trial of disulfiram in men with non-metastatic recurrent PCa after local therapy. Dose escalation occurred if a demethylating 'response' (that is, 10% decrease in peripheral blood mononuclear cell (PBMC) global 5-methyl cytosine (5(me)C) content) was observed in3 patients in cohort 1. Cohorts 1 and 2 received disulfiram 250 mg and 500 mg daily, respectively. The primary end point was the proportion of subjects with a demethylation response. Secondary end points included the rate of PSA progression at 6 months, changes in PSA doubling time and safety/tolerability.Changes in global 5(me)C content were observed in two of nine patients (22.2%) in cohort 1 and 3 of 10 (30.0%) in cohort 2. Only five subjects were on trial for 6 months, all were in cohort 1 and all had PSA progression by 6 months. No changes in PSA kinetics were observed in either cohort. Disulfiram was poorly tolerated with six patients experiencing grade 3 adverse events (three per cohort). Three of the responders displayed pretreatment instability in their 5(me)C content.A minority of patients had transient global PBMC demethylation changes. Instability in 5(me)C may limit the reproducibility of these findings, limiting our ability to confirm our hypothesis. Given the toxicities and no clinical benefits, further development of disulfiram should not be pursued in this population.

Epigenetic aberrations contribute to prostate cancer carcinogenesis and disease progression. Efforts have been made to target DNA methyltransferase and histone deacetylases (HDACs) in prostate cancer and other solid tumors but have not had the success that was seen in the hematologic malignancies. Oral, less toxic, and more specific agents are being developed in solid tumors including prostate cancer. Combinations of epigenetic agents alone or with a targeted agent such as androgen receptor signaling inhibitors are promising approaches and will be discussed further.

Background Pembrolizumab is a monoclonal antibody that is designed against programmed cell death protein 1 (PD-1). Pembrolizumab and other immunocheckpoint-blocking monoclonal antibodies work by modulating a patient’s own immune system to increase anti-tumor activity. While immunocheckpoint blockade has shown promising results, only 20–40 % of patients experience objective clinical benefit. Differences in individual tumor biology and the presence multiple immune checkpoints present a challenge for treatment. Because radiotherapy has immunomodulatory effects on the tumor microenvironment, it has the potential to synergize with immunotherapy and augment tumor response. NCT02318771 is a phase 1 clinical trial designed to investigate the immunomodulatory effects of radiation therapy in combination with pembrolizumab. Case presentation The patient is a 64-year-old male with metastatic clear cell renal cell carcinoma, Fuhrman grade 4, pathologically staged as T3 N0. Metastatic disease was well controlled for several years with sunitinib. Following disease progression, he was switched to axitinib. When disease progression continued, the patient was enrolled in NCT02318771, a phase 1 clinical trial combining radiotherapy and pembrolizumab. The patient experienced unusually rapid disease progression during treatment, which was confirmed by repeated CT scans to rule out pseudoprogression. Tissue biopsies and peripheral blood draws were obtained before, during, and after treatment. Samples were analyzed to provide plausible rationale for rapid treatment failure. Conclusions Biomarker analysis demonstrated an absence of TILs, which may be a cause of treatment failure as pembrolizumab works through T cell-dependent mechanisms. Furthermore, the presence of other non-redundant immune checkpoints in the periphery and tumor microenvironment presents a treatment challenge. Additionally, the radiation dose and fractionation schedule may have played a role in treatment failure as these factors play a role in the effect radiotherapy on the tumor microenvironment as well as the potential for synergy with immunotherapy. Trial registration An Exploratory Study to Investigate the Immunomodulatory Activity of Radiation Therapy (RT) in Combination With MK-3475 in Patients With Recurrent/Metastatic Head and Neck, Renal Cell Cancer, Melanoma and Lung Cancer, NCT02318771 .

Lessons Learned Patients with metastatic castration-resistant prostate cancer did not tolerate the combination of alisertib with abiraterone and prednisone. There was no clear signal indicating that adding alisertib might be beneficial for those patients progressing on abiraterone. Background. We hypothesized that Aurora A kinase (AK) contributes to castrate resistance in prostate cancer (PCa) and that inhibiting AK with alisertib can resensitize PCa cells to androgen receptor (AR) inhibitor abiraterone. Methods. This was a phase I/II trial to determine the safety and efficacy of alisertib when given in combination with abiraterone plus prednisone (AP). Metastatic castration-resistant prostate cancer (mCRPC) patients were treated with dose escalation (alisertib at 30, 40, and 50 mg orally b.i.d., days 1–7 every 21 days) per standard 3+3 design. Results. Nine of 43 planned subjects were enrolled. The maximum tolerated dose (MTD) was not reached, and the dose-limiting toxicities (DLTs) included neutropenic fever (1 of 9), neutropenia (1 of 9), fatigue with memory impairment (1 of 9), and diarrhea/mucositis (1 of 9). No prostate-specific antigen (PSA) decrease or circulating tumor cell (CTC) changes were observed during the study. Pharmacodynamically, adding alisertib did not affect total testosterone or dehydroepiandrosterone (DHEA) levels. There was some change in neuroendocrine markers after therapy. Mean duration on study was 2.5 months. The trial was terminated early. Conclusion. A tolerable dose of alisertib in combination with AP in mCRPC was not established in this study. There was no clear signal indicating that alisertib might be beneficial for patients with mCRPC progressing on abiraterone.

BACKGROUND: Several phase II trials in men with noncastrate PSA-recurrent prostate cancer have assessed the impact of novel nonhormonal agents on PSA kinetics. However, it is unknown whether changes in PSA kinetics influence metastasis-free survival (MFS). METHODS: We performed a retrospective post hoc analysis of 146 men treated in 4 phase II trials examining the investigational agents marimastat (a matrix metalloproteinase inhibitor; n = 39), imatinib (a tyrosine kinase inhibitor; n = 25), ATN-224 (a copper/zinc-superoxide dismutase inhibitor; n = 22), and lenalidomide (an antiangiogenic/immunomodulatory drug; n = 60). We investigated factors influencing MFS, including within-subject changes in PSA kinetics (PSA slope, doubling time, and velocity) before and after treatment initiation. RESULTS: After a median follow-up of 16.8 months, 70 patients (47.9%) developed metastases. In multivariable Cox regression models, factors that were independently predictive of MFS after adjusting for age and other clinical prognostic variables were baseline PSA doubling time (PSADT) (P = .05), baseline PSA slope (P = .01), on-study change in PSADT (P = .02), and on-study change in PSA slope (P = .03). In a landmark Kaplan-Meier analysis, median MFS was 63.5 months (95% confidence interval [CI], 34.6-not reached) and 28.9 months (95% CI, 13.5-68.0) for men with or without any decrease in PSA slope by 6 months after treatment, respectively. CONCLUSIONS: This hypothesis-generating analysis suggests that within-subject changes in PSADT and PSA slope after initiation of experimental therapy may correlate with MFS in men with biochemically recurrent prostate cancer. If validated in prospective trials, changes in PSA kinetics may represent a reasonable intermediate end point for screening new agents in these patients. Cancer 2011;. © 2011 American Cancer Society.

Remarkable clinical efficacy and durable responses to antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway have been observed in patients with multiple cancers, including classical Hodgkin lymphomas (cHL). However, the responses in the majority of Non-Hodgkin lymphoma patients, including mantle cell lymphoma (MCL), treated with anti- PD1/PDL1 antibodies have been modest to date. It has been postulated that the immune suppressive nature of the tumor microenvironment (TME) may play a role in limiting the efficacy of checkpoint blockade strategies. As such, identification of critical molecules in TME required for driving response and resistance is key to improve lymphoma immunotherapy. We have therefore generated in vivo and ex-vivo MCL lymphoma-stroma co-culture models and capitalize this model with primary human MCL cells as well. First, we found that co-injection of murine Fc-muMCL1 cells with stromal cells significantly promote lymphoma growthas compared to Fc-muMCL1 cells injected alone. This aggressive growth was associated with less tumor infiltrating cytotoxic T-cells in the TME. Second, to identify the tolerogenic mechanism(s) that drive immunosuppression in TME, we co-cultured MCL cells with stroma cells ex-vivo and found an increased translation and transcription of PD-L1 via upregulation of c-Myc. Furthermore, co-culture of patient primary lymphoma cells with stromal cell dramatically increases PD-L1 expression in both stromal cells and lymphoma cells. Tumor infiltrating T cells also induce PD-L1 expression in stromal cells. Of note, just by knocking down c-Myc in stromal cell we were able to block co-culture-induced PD-L1 expression, highlighting a critical role for c-Myc in driving this tolerogenic process in the TME. In lieu of the above findings, next we treated murine MCL in vitro with a bromodomain inhibitor (JQ1) and observed a significant decrease in c-Myc/PD-L1 expression which was associated with increased immunogenicity of malignant B-cells leading to a better T-cell activation. More importantly, treatment of MCL-bearing mice with a combination of a bromodomain inhibitor with anti-PD1 antibody resulted in enhanced inhibition of MCL growth, increased effector memory T cells and improved function of tumor infiltrating T cells in vivo. No such effects were observed in MCL-bearing mice treated with either agent alone. Taken together, we have identified the c-Myc/PD-L1 axis in stromal cells that by creating a tolerogenic/immunosuppressive TME imposes a significant barrier to the efficacy of checkpoint blockade therapy in lymphomas. This barrier seems not to be unsurmountable since the addition of a bromodomain inhibitor augmented the efficacy of checkpoint blockade by inducing a more immunogenic TME in MCL. Disclosures No relevant conflicts of interest to declare.

Prostate cancer possesses its unique feature of low proliferation rate and slow growth. Ca(2+)-induced apoptosis is not dependent on cell cycle progression and targeting this pathway could circumvent the problems encountered using current cytotoxic chemotherapies for prostate cancer. Hypoxia-inducible factor 1alpha (HIF-1alpha) is another novel cancer drug target and inhibitors of hypoxia-response pathway are being developed. Digoxin and other cardiac glycosides, known inhibitors of the alpha-subunit of sarcolemmal Na(+)K(+)-ATPase, were recently found to block tumor growth via the inhibition of HIF-1alpha synthesis. Thus, cardiac glycosides disrupt two important cellular pathways and, therefore, may be useful as an anticancer therapy. This review will focus on HIF-1alpha and calcium signaling as novel cancer drug targets in prostate cancer. The possible application of digoxin and other cardiac glycosides in cancer therapeutics especially in prostate cancer is discussed.

Purpose: This was a phase I trial to determine the minimal effective dose and optimal dose schedule for 5-azacytidine (5-AC) in combination with sodium phenylbutyrate in patients with refractory solid tumors. The pharmacokinetics, pharmacodynamics, and antineoplastic effects were also studied. Experimental Design: Three dosing regimens were studied in 27 patients with advanced solid tumors, and toxicity was recorded. The pharmacokinetics of the combination of drugs was evaluated. Repeat tumor biopsies and peripheral blood mononuclear cells (PBMC) were analyzed to evaluate epigenetic changes in response to therapy. EBV titers were evaluated as a surrogate measure for gene re-expression of epigenetic modulation in PBMC. Results: The three dose regimens of 5-AC and phenylbutyrate were generally well tolerated and safe. A total of 48 cycles was administrated to 27 patients. The most common toxicities were bone marrow suppression–related neutropenia and anemia, which were minor. The clinical response rate was disappointing for the combination of agents. One patient showed stable disease for 5 months whereas 26 patients showed progressive disease as the best tumor response. The administration of phenylbutyrate and 5-AC did not seem to alter the pharmacokinetics of either drug. Although there were individual cases of targeted DNA methyltransferase activity and histone H3/4 acetylation changes from paired biopsy or PBMC, no conclusive statement can be made based on these limited correlative studies. Conclusion: The combination of 5-AC and phenylbutyrate across three dose schedules was generally well tolerated and safe, yet lacked any real evidence for clinical benefit. (Clin Cancer Res 2009;15(19):6241–9)

Meeting abstracts Pembrolizumab is an antibody designed against programmed cell-death protein-1 (PD-1), which is expressed on the surface of activated T cells. Tumor cells can upregulate PD-L1, which binds to PD-1 and mediates T cell anergy. By antagonizing the PD-1/PD-L1 binding, pembrolizumab can

inTroDucTion Tumor lysis syndrome (TLS) is a metabolic disturbance caused by the destruction of rapidly dividing cancer cells following administration of cytotoxic chemotherapy. The subsequent release of intracellular material results in hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia. The clinical presentation of TLS, including acute kidney injury, results from these electrolyte abnormalities and can be life-threatening. Here we present the second reported case of TLS in a woman with endometrial cancer.

BACKGROUND Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized primarily in the stromal compartment of the human prostate and may regulate stromal as well as epithelial cell growth and survival. The primary cognate HB-EGF receptor, ErbB1, has been shown recently to be transactivated by G-protein coupled receptors (GPCRs) through regulated proteolytic cleavage of the membrane-bound, precursor form of HB-EGF. Previous studies have demonstrated that human prostate tissue, especially tissue from benign prostatic hyperplasia (BPH), has high angiotensin converting enzyme activity, and a high density of angiotensin (Ang) receptors in periurethral stromal cells. Because the pressor peptide Ang II signals through GPCRs, we tested the possibility that Ang II could transactivate ErbB1/ErbB2 in human prostate stromal (hPS) cells. METHODS Primary and early passage hPS cells were used as an in vitro model. Cells were stimulated by recombinant HB-EGF or Ang II and total cell lysates were harvested for immunoprecipitation and Western blot. Cell growth was measured by [3H]thymidine incorporation assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT) assay. RESULTS Ang II receptors AT1 and AT2 were expressed in hPS cells. ErbB1 and ErbB2 receptors were activated by HB-EGF. Furthermore, Ang II was able to transactivate both ErbB1 and ErbB2, and this transactivation activity could be abolished by pretreatment with [Glu-52]-diphtheria toxin/CRM197, a specific inhibitor of HB-EGF bioactivity. Consistent with its transactivation activity, Ang II modestly promoted hPS cell growth and this effect was abolished by pretreatment with the ErbB1 antagonist AG1478. CONCLUSION These experiments suggest a regulatory role for Ang II in the prostate stroma and implicate the endogenous stromal growth factor HB-EGF as a mediator of Ang II signaling in the prostate. Prostate 54: 1–7, 2003. © 2002 Wiley-Liss, Inc.

Peptide growth factors have been implicated in progression of prostate cancer (PCa) to the androgen-independent state; however, much of the evidence linking diffusible mitogens and survival factors to this process remains circumstantial. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a prostate stroma-derived factor, promotes survival, proliferation, and neuroendocrine differentiation of androgen-dependent LNCaP PCa cells in vitro. To test whether sustained exposure to HB-EGF can confer an androgen-independent phenotype, we generated stable populations of LNCaP cells that express constitutively a secreted form of HB-EGF (LNCaP/sHB). LNCaP/sHB cells proliferated more rapidly under androgen-depleted conditions in vitro and formed larger tumors with higher frequency in intact and castrated severe combined immunodeficient mice, in comparison to control cells. LNCaP/sHB tumors also expressed higher levels of the neuroendocrine marker, neuron-specific enolase, compared with control tumors. In castrates, increased neuron-specific enolase expression in LNCaP/sHB tumors was associated with reduced androgen receptor (AR) levels. In vitro, AR protein levels were reduced in LNCaP/sHB cells, and in transient transfection assays using an androgen-responsive promoter (mouse mammary tumor virus-long terminal repeat), LNCaP/sHB cells showed reduced sensitivity to dihydrotestosterone compared with controls. This is the first demonstration that continuous exposure of AR-positive PCa cells to a single growth factor can promote an androgen-independent phenotype in vivo. These findings also emphasize the potential role of pathways other than the AR axis in acquisition of androgen independence.

e16058 Background: Preclinical data suggest there are synergistic effects of radiation therapy (RT) and check point inhibitors in anticancer immunity. The primary objective of this study was to explore the immunomodulatory activity of RT alone or in combination with Pembrolizumab (pembro) in solid tumors including renal cell cancer (RCC) patients (pts). Methods: RCC pts who progressed after at least one front-line therapy were eligible. Pts were treated with either RT (8Gy x 1 or 4Gy x 5) followed by (f/b)pembro or 1 dose pembro f/b RT f/b pembro. Pre- and post RT tumor biopsy was obtained to evaluate PD-L1 expression (assay by QualTeck). Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. Treatment response was measured based on modified RECIST criteria. Results: Twelve RCC patients were enrolled including 2 with non-clear cell subtype. One pt was not evaluable since pt quickly deteriorated and was taken off study. As of January 13, 2017, 2 pts are still on active treatment. Two pts had partial response (18%) and were on study for at 54 and 63 weeks. One responder was treated with 8 Gy f/b pembro while 1 was treated with 1 dose pembro f/b RT f/b pembro. Five pts had stable disease of 18 to 45 weeks and 4 pts (36%, non-responders) had progression in 9 weeks. For adverse events, 1 pt developed grade 3 pneumonitis after 10 cycles of pembro (RT to adrenal mets). Grade 3 AEs include Fatigue, Nausea, Hyperglycemia, Lymphopenia, thrombocytopenia and AST elevation (post RT for liver mets). PDL1 expression and tumor infiltrating lymphocytes presence after RT showed various patterns. Preliminary flow cytometry showed persistent higher numbers of monocytes in non-responders comparing with responders. CD4+, CD8+ and NK cells and other markers are under analyzed and the results will be presented. Conclusions: The combination of RT (8Gy or 20Gy) with pembro is feasible and tolerated, and demonstrates clinical activity. The AE profile is similar to single agent pembro. Monocytes, T and NK cell kinetics are being examined. (ClinicalTrials.gov ID: NCT02318771) Clinical trial information: NCT02318771.

e593 Background: The use of radium-223 in patients with metastatic castration-resistant prostate cancer (mCRPC) improves overall survival (OS) and quality of life. Combination of radium-223 with second-generation anti-androgens has further improved OS; however, the optimal length of radium-223 treatment for maximal effect remains unknown. Methods: We reviewed 35 consecutive patients with mCRPC who received radium-223 from December 2012 to August 2015 at Thomas Jefferson University. Patients were divided into two groups: those who received full treatment of 6 injections (n = 18) versus those who received less than 6 injections (n = 17). Kaplan-Meier analysis of OS were tested for difference by treatment group using Log Rank test. Univariable association with survival outcomes was calculated with univariable Cox regression and Log Rank tests. Results: Mean age was 73 ± 10 years and Karnofsky performance status (KPS) ranged from 50-90 (median, 80). Median follow-up was 13.9 months. Eighteen patients were receiving concurrent second generation anti-androgens at the start of treatment. Median OS was 12 months for patients who received 6 injections and 6.48 months for patients who received less than 6 injections (p = 0.0045). The results of univariate Cox regression analysis revealed full treatment was associated with increased OS (p = 0.0013). On multivariate analysis accounting for KPS, full treatment was significantly associated with improved OS (p = 0.0028). Conclusions: In this retrospective, single-institution analysis, we demonstrated that full course completion of radium-223 was associated with improved OS in patients with mCRPC. These patients should be optimally supported during treatment to allow for therapy completion.

80 Background: Introducing amino acid sequence changes in highly expressed self-antigens for androgen sensitive prostate cancer pts might be sufficient to break tolerance, thus a DNA vaccine was developed using SynCon PSA and PSMA (INO-5150) that share 96.8 and 91.6% sequence identities to these native antigens, respectively. Administration of these antigens to prostate cancer pts along with plasmid encoded adjuvant IL-12 (INO-9012) via electroporation (EP) using the CELLECTRA5P device is postulated to break tolerance, resulting in an antigen-specific immune response which could lead to stabilization of disease progression. Methods: This Phase I, open-label, multicenter study included prostate cancer pts post-definitive therapy with a rising PSA ≥ 1.0 ng/ml after surgery, or ≥ 2.0 ng/ml above nadir after RT and PSA doubling time > 3 months, testosterone > 150 ng/dL and no evidence of metastasis within 12 months. INO-5150 with or without INO-9012 was administered IM followed by EP in 4 arms: low (2 mg) or high dose (8.5 mg) INO-5150 alone or with 1 mg INO-9012 on Day 0 and at week 3, 12, and 24 in 60 planned pts (15 pts/arm). DLT assessments were performed after dosing of the first 3 pts of each arm at Week 4. Results: Enrollment is complete in all 4 arms and at data cut-off (10Oct16), 62 enrolled pts received at least one, 60 pts received 3 and about half, 28 pts (10 in arm A, 8 in B, 7 in C, and 3 in D) received all 4 vaccinations. Safety: there were no DLTs noted. Four pts had five Grade 3 SAEs noted as pre-syncope, cardiac disorder, hospitalization for fall, ALT and AST elevation. No Grade 4-5 AEs were noted. Grade 1-3 treatment-emergent AEs occurred in 50 (81%) pts: 12 (75%) in arm A, 13 (87%) B, 13 (87%) C, and 12 (75%) in D. The most common AEs were injection site pain (24/39%), erythema (13/21%), swelling (12/19%), bruising (10/16%), hyperglycemia (8/13%) and fatigue (6/10%), all Grade 1-2. Assessments of immunological response, PSA kinetics and correlation with clinical outcome are ongoing and will be presented. Conclusions: INO-5150 (+) or (-) INO-9012 is generally safe and well-tolerable at all 4 dose levels in a biochemically relapsed prostate cancer patient population. Clinical trial information: NCT02514213.

Purpose Neoadjuvant cisplatin-based chemotherapy is standard of care for muscle-invasive bladder cancer (MIBC); however, it is infrequently adopted in practice because of concerns regarding toxicity and delay to cystectomy. We hypothesized that three cycles of neoadjuvant accelerated methotrexate, vinblastine, doxorubicin, and cisplatin (AMVAC) would be safe, shorten the time to surgery, and yield similar pathologic complete response (pT0) rates compared with historical controls. Patients and Methods Patients with cT2-T4a and N0-N1 MIBC were eligible and received three cycles of AMVAC with pegfilgrastim followed by radical cystectomy with lymph node dissection. The primary end point was pT0 rate. Telomere length (TL) and p53 mutation status were correlated with response and toxicity. Results Forty-four patients were accrued; 60% had stage III to IV disease; median age was 64 years. Forty patients were evaluable for response, with 15 (38%; 95% CI, 23% to 53%) showing pT0 at cystectomy, meeting the primary end point of the study. Another six patients (14%) were downstaged to non–muscle invasive disease. Most (82%) experienced only grade 1 to 2 treatment-related toxicities. There were no grade 3 or 4 renal toxicities and no treatment-related deaths. One patient developed metastases and thus did not undergo cystectomy; all others (n = 43) proceeded to cystectomy within 8 weeks after last chemotherapy administration. Median time from start of chemotherapy to cystectomy was 9.7 weeks. TL and p53 mutation did not predict response or toxicity. Conclusion AMVAC is well tolerated and results in similar pT0 rates with 6 weeks of treatment compared with standard 12-week regimens. Further analysis is ongoing to ascertain whether molecular alterations in tumor samples can predict response to chemotherapy.

The physiological mechanisms by which soluble mediators of cell proliferation and survival alter expansion of the prostatic stroma in benign prostatic hyperplasia are poorly understood. We recently identified heparin-binding epidermal growth factor like growth factor (HB-EGF) as a product predominantly of the smooth muscle cell compartments of the adult human prostate. We assess the potential role of this growth factor as a stromal cell regulator.Primary cultures of desmin and alpha-actin positive human prostate stromal cells were shown to express several cell associated HB-EGF isoforms as well as the primary cognate HB-EGF receptor, ErbB1/HER1, suggesting the existence of an autocrine or juxtacrine regulatory loop. The related receptor tyrosine kinase, ErbB2/HER2, was also expressed as assessed by reverse transcriptase (RT) polymerase chain reaction (PCR). HB-EGF messenger RNA levels in human prostate stromal cells increased modestly (70%) in response to a repetitive mechanical stimulus, a lower response than has been reported for neonatal rat bladder smooth muscle cells, in which HB-EGF was originally identified as a mechanically responsive gene.HB-EGF, epidermal growth factor and basic fibroblast growth factor stimulated human prostate stromal cell growth, while a specific antagonist of HB-EGF, [Glu52]-diphtheria toxin/CRM197, inhibited human prostate stromal growth in serum-free medium by a mechanism that did not involve increased apoptosis. A function blocking antibody against CD9/DRAP27/MRP-1, a cell surface binding partner of the membrane form of HB-EGF, also stimulated human prostate stromal cell proliferation.HB-EGF is an endogenously produced human prostate stromal cell growth factor and, thus, may have a role as a physiologically relevant autocrine or juxtacrine mediator of stromal expansion in benign prostatic hyperplasia.

311 Background: Aurora A kinase (AK) phosphorylates and interacts with androgen receptor (AR) and enhances AR DNA binding activity. We hypothesized that AK contributes to castration resistance in PCa, and targeting AK can inhibit AR function and re-sensitize PCa cells to AR signaling inhibitors such as abiraterone. Methods: Aphase I/II, open label, single institution trial to determine the safety and efficacy of AK inhibitor alisertib when given in combination with abiraterone plus prednisone (AP) was planned. Eligible subjects had mCRPC with disease progression on abiraterone. Androgen deprivation plus abiraterone and prednisone were maintained through the trial. Subjects were treated with dose escalation per standard 3+3 design. Planned 3 dose cohorts of alisertib were 30, 40, 50 mg PO BID D 1-7 every 21 D. Correlative studies included kinetics of neuroendocrine tumor markers, enumeration and AK gene amplification of CTCs. Results: Nine of 43 planned subjects were enrolled, 3 in cohort 1 without DLT. Two DLTs developed in cohort 2, thus 3 more patients were enrolled into cohort 1, and 2 developed DLT (total 2/6) in this cohort. The grade 3/4 toxicities (4/9) include neutropenic fever (1/9), neutropenia (1/9), fatigue with memory impairment (1/9), and mucositis (diarrhea and mouth sores, 1/9). Pharmacodynamically, adding alisertib did not affect circulating total testosterone and DHEA levels. Only one patient had decreased Chromogranin A level lasted for 4 months when compared to baseline. Four (4/9) patients had ≥ 5 CTCs at baseline but no conversion ( to < 5) was observed. No PSA decrease was observed during the study. Mean duration of study was 2.5 months. The toxicity / efficacy ratio did not warrant further dose expansion and the trial was terminated early after discussion with sponsor. Conclusions: A tolerable dose of alisertib in combination with AP in mCRPC was not established in this study. There was no clear signal indicating that alisertib might be beneficial for patients with mCRPC progressing on abiraterone. Clinical trial information: NCT01848067.

BACKGROUND The clinical success of the nucleoside analogs 5-aza-cytidine (5-azaC) and 5-aza-2′deoxycytidine (5-aza-dC) as DNA methyltransferase (DNMT) inhibitors has spurred interest in the development of non-nucleoside inhibitors with improved pharmacologic and safety profiles. Because DNMT catalysis features attack of cytosine bases by an enzyme thiol group, we tested whether disulfiram (DSF), a thiol-reactive compound with known clinical safety, demonstrated DNMT inhibitory activity. METHODS Inhibition of DNMT1 activity by DSF was assessed using methyltransferase activity assays with recombinant DNMT1. Next, prostate cancer cell lines were exposed to DSF and assessed for: i) reduction of global 5-methyl cytosine (5meC) content using liquid chromatography/tandem mass spectrometry (LC-MS/MS); ii) gene-specific promoter demethylation by methylation-specific PCR (MSP); and iii) gene-reactivation by real-time RT-PCR. DSF was also tested for growth inhibition using prostate cancer cell lines propagated in vitro in cell culture and in vivo as xenografts in nude mice. RESULTS Disulfiram showed a dose-dependent inhibition of DNMT1 activity on a hemimethylated DNA substrate. In prostate cancer cells in culture, DSF exposure led to reduction of global genomic 5meC content, increase in unmethylated APC and RARB gene promoters, and associated re-expression of these genes, but did not significantly alter prostate-specific antigen (PSA) expression. DSF significantly inhibited growth and clonogenic survival of prostate cancer cell lines in culture and showed a trend for reduced growth of prostate cancer xenografts. CONCLUSIONS Disulfiram is a non-nucleoside DNMT1 inhibitor that can reduce global 5meC content, reactivate epigenetically silenced genes, and significantly inhibit growth in prostate cancer cell lines. Prostate 77:333–343, 2011. © 2010 Wiley-Liss, Inc.

26 Background: Cabazitaxel (C) improves overall survival and has been approved in second line chemotherapy for patients (pts) with metastatic castrate resistant PCa. We hypothesized that C is a radiosensitizer and performed a phase I trial of weekly C with standard IMRT and ADT for pts with high risk localized PCa. Methods: Conventional “3 + 3” design. Pts with newly diagnosed high risk PCa defined as either Gleason score (GS) 8 – 10, or T3/T4 with GS 7, or PSA > 20 with GS 7 are eligible. Dose escalation occurred only after all the cohort pts completed weekly C ( 4 cohorts at the dose of 4, 6, 8 and 10 mg/m2) and the total planned dose IMRT. ADT was started two months prior to C and IMRT then continued on for a total of 24 months. Daily bicalutamide started within 1 day to 2 weeks prior to LHRH agonist therapy and stopped on the last day of IMRT for approximately 4 month duration. IMRT is delivered to a total dose of 75.6 Gy at 1.8 Gy daily fraction for 8.5 – 9 wks. The primary objective of this study was to determine the safe dose of the combination of weekly C with IMRT and ADT . Results: At present, 14 pts of mean age 62 yrs (range 53 – 82), T2 - T4; mean PSA 89 (range 4 – 253); and median GS 9 (GS 7 – 10) were enrolled and 10 pts completed the chemo-radiation therapy. Grade 3 dose-limiting toxicites in the first two pts at the 8 mg/m2 C dose level were diarrhea and elevated transaminase, thus the maximum tolerable dose (MTD) of C with IMRT was determined to be 6 mg/m2. An expansion cohort is ongoing. In this dose level, there were ≤ grade 2 toxicities only (diarrhea, hypophosphatemia and leukopenia). No long term toxicities are observed. Standardized measurement of health status and quality of life will be presented. Conclusions: Concurrent weekly C with current standard IMRT is feasible with no unexpected toxicity. In combination with IMRT and ADT, the MTD of weekly C is 6 mg/m2 in pts with newly diagnosed high risk PCa. Clinical trial information: NCT01420250.

e15519 Background: There is no standard second line therapy for metastatic urothelial cancer (mUC) following platinum chemotherapy (CT). Taxanes have shown clinical activity in this setting. Cabazi...

4513 Background: Neoadjuvant cisplatin (cis) based chemotherapy prior to radical cystectomy (RC) is standard of care for localized MIBC. Combination gemcitabine (gem) and cis is frequently used, bu...

179 Background: The first described roles for PARP-1 were in the repair of DNA damage and genomic maintenance, however, recent studies have identified PARP-1 as harboring critical context-dependent transcriptional regulatory functions. Our group recently discovered that PARP-1 enzymatic activity is a critical effector of androgen receptor (AR) function in models of prostatic adenocarcinoma (PCa), and is recruited to regulatory sites of select AR target genes. Pharmacological inhibition of PARP-1 enzymatic activity results in diminished AR and PARP-1 residency at AR target gene regulatory loci, reduced AR target gene expression (more than 50%), and reduced AR-driven, PCa-associated phenotypes, including castrate-resistant PCa (CRPC) AR function, tumor cell growth, and transition to CRPC. These data, in part, served as critical rationale for current clinical trials combining PARP inhibition and abiraterone for patients with metastatic CRPC NCT01576172 . Given the clinical importance of targeting AR function in PCa, preclinical studies were performed to assess the impact of leveraging the dual roles of PARP-1 as a means to improve therapy for advanced disease. Methods: In vitro and in vivo model systems were utilized to assess impact on AR and tumor growth. Multiple PARP inhibitors were used for additional studies. The impact of PARP-1 on gene expression was also assessed using unbiased analyses. PARP-1 mutants were also utilized to segregate the DNA damage and transcriptional regulatory roles of PARP-1. Results: Critically, PARP-1 inhibitors cooperated with castration to elicit an enhanced therapeutic response, and PARP-1 inhibitors were effective at suppressing CRPC growth in vivo. Multiple PARP inhibitors diminished AR chromatin occupancy, altered AR transcriptional output and reduction in PCa and CRPC cell growth. Conclusions: These data identify PARP-1 as a feasible therapeutic target for advanced prostate cancer. Multiple PARP inhibitors show pre-clinical efficacy in models of PCa and CRPC and merit consideration for clinical trial, and a concept will be presented.