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The vitamin D receptor (VDR) binds the secosteroid hormone 1,25(OH)2D3 with high affinity and regulates gene programs that control a serum calcium levels, as well as cell proliferation and differentiation. A significant focus has been to exploit the VDR in cancer settings. Although preclinical studies have been strongly encouraging, to date clinical trials have delivered equivocal findings that have paused the clinical translation of these compounds. However, it is entirely possible that mining of genomic data will help to refine precisely what are the key anticancer actions of vitamin D compounds and where these can be used most effectively.

Folate impacts the genome and epigenome by feeding into one-carbon metabolism to produce critical metabolites, deoxythymidine monophosphate and s-adenosylmethionine. The impact of folate exposure and intervention timing on cancer progression remains controversial. Due to polyamine metabolism’s extraordinary biosynthetic flux in prostate cancer (CaP) we demonstrated androgen stimulated CaP is susceptible to dietary folate deficiency. We hypothesized dietary folate levels may also affect castration recurrent CaP. We used the CWR22 human xenograft model which recurs following androgen withdrawal. Engrafted mice were fed a folate depleted or supplemented diet beginning at androgen withdrawal, or prior to xenograft implantation. Both folate depletion and supplementation at the time of withdrawal significantly decreased recurrence incidence. Folate supplementation prior to xenograft implantation increased time to recurrence, suggesting a protective role. By contrast, folate depleted recurrent tumors exhibited transcriptional adaptive responses that maintained high polyamine levels at the expense of increased DNA damage and DNA methylation alterations. Mining of publically available data demonstrated folate related pathways are exceptionally dysregulated in human CaP, which correlated with decreased time to biochemical recurrence. These findings highlight the potential for novel therapeutic interventions that target these metabolic pathways in CaP and provide a rationale to apply such strategies alongside androgen withdrawal.

Recently, we undertook a pan-cancer analyses of the nuclear hormone receptor (NR) superfamily in The Cancer Genome Atlas (TCGA), and revealed that the vitamin D receptor (NR1I1/VDR) was commonly and significantly down-regulated specifically in colon adenocarcinoma cohort (COAD). To examine the consequence of down-regulated VDR expression we re-analyzed VDR chromatin immunoprecipitation sequencing (ChIP-Seq) data from LS180 colon cancer cells (GSE31939). This analysis identified 1809 loci that displayed significant (p.adj

No approved therapy exists for cancer‐associated cachexia. The colon‐26 mouse model of cancer cachexia mimics recent late‐stage clinical failures of anabolic anti‐cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx‐024. The histone deacetylase inhibitor (HDACi) AR‐42 exhibited anti‐cachectic activity in this model. We explored combined SARM/AR‐42 therapy as an improved anti‐cachectic treatment paradigm. A reduced dose of AR‐42 provided limited anti‐cachectic benefits, but, in combination with GTx‐024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR‐42 suppressed the IL‐6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx‐024‐mediated β‐catenin target gene regulation was apparent in cachectic mice only when combined with AR‐42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx‐024 therapy and a blockade of GTx‐024‐mediated anabolic signaling. AR‐42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx‐024. Combining GTx‐024, a clinically established anabolic therapy, with AR‐42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients., Cancer cachexia increases morbidity and mortality of cancer patients. Given the multifactorial pathogenesis of cachexia, effective treatment remains elusive. Here, a novel therapeutic strategy combining anti‐catabolic and anabolic activities is established in a murine model of cancer cachexia.

The 3D organization of the genome within the cell nucleus has come into sharp focus over the last decade. This has largely arisen because of the application of genomic approaches that have revealed numerous levels of genomic and chromatin interactions, including topologically associated domains (TADs). The current review examines how these domains were identified, are organized, how their boundaries arise and are regulated, and how genes within TADs are coordinately regulated. There are many examples of the disruption to TAD structure in cancer and the altered regulation, structure and function of TADs are discussed in the context of hormone responsive cancers, including breast, prostate and ovarian cancer. Finally, some aspects of the statistical insight and computational skills required to interrogate TAD organization are considered and future directions discussed.

Background Virtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA. Methods Rapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3’UTR of the lymphocyte-specific protein tyrosine kinase (LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth. Results In this study, a previously unannotated lncRNA lying within exon six and 3’UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA “HULLK” for Hormone-Upregulated lncRNA within LCK. Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation. Conclusions HULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa. Electronic supplementary material The online version of this article (10.1186/s12943-019-1039-6) contains supplementary material, which is available to authorized users.

Radioiodine treatment fails ≥25% of patients with thyroid cancer and has been proposed as a potential treatment for breast cancer. Cellular iodide uptake is governed by the sodium iodide symporter (NIS), which is frequently mislocalized in thyroid and breast tumours. However, the trafficking of NIS to the plasma membrane (PM) is ill-defined. Through mass spectrometry, co-immunoprecipitation, cell surface biotinylation and proximity ligation assays we identify two proteins which control NIS subcellular trafficking: ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP). HiLo microscopy revealed ARF4 enhanced NIS trafficking in co-incident PM vesicles, governed by a C-terminal VXPX motif, whilst papillary thyroid cancers (PTC) demonstrate repressed ARF4 expression. In contrast, VCP, the central protein in ER-associated degradation, specifically bound NIS and decreased its PM localization. Five chemically distinct allosteric VCP inhibitors all overcame VCP-mediated repression of NIS function. In mice, two re-purposed FDA-approved VCP inhibitors significantly enhanced radioiodine uptake into thyrocytes, whilst human primary thyrocytes showed similar increases. Critically, PTC patients with high tumoural VCP expression who received radioiodine had strikingly worse disease-free survival. These studies now delineate the mechanisms of NIS trafficking, and for the first time open the therapeutic possibility of systemically enhancing radioiodine uptake in patients via FDA-approved drugs.One Sentence SummaryNovel NIS interactors ARF4 and VCP alter NIS trafficking in vitro, and FDA-approved VCP inhibitors can significantly enhance radioiodine uptake.

Bioinformatics applies unbiased approaches to develop statistically robust insight into health and disease. At the global, or "20,000 ft" view bioinformatic analyses of NR signaling can measure how the NRs are implicated in human health and disease through the impact of genome-wide significant genetic variation, family-wide NR expression patterns or considering where NRs are significantly identified in other high-dimensional data analyses. With a more NR-centric, or "2000 ft" view, bioinformatic approaches can interrogate events downstream of a given NR. Integrative approaches aim to combine multiple NR-centric high-dimensional data both derived in cell models and primary human tissue to reveal how NR-transcriptional networks relate to human health and disease. Bioinformatic approaches to such high-dimensional data are central and require specialist statistical insight and computational skills, coupled with a dexterous understanding of the biological question. A current challenge is determining the optimal mechanism to share such bioinformatic approaches through the biological research community.

Background Long thought “relics” of evolution, not until recently have pseudogenes been of medical interest regarding regulation in cancer. Often, these regulatory roles are a direct by-product of their close sequence homology to protein-coding genes. Novel pseudogene-gene (PGG) functional associations can be identified through the integration of biomedical data, such as sequence homology, functional pathways, gene expression, pseudogene expression, and microRNA expression. However, not all of the information has been integrated, and almost all previous pseudogene studies relied on 1:1 pseudogene–parent gene relationships without leveraging other homologous genes/pseudogenes. Results We produce PGG families that expand beyond the current 1:1 paradigm. First, we construct expansive PGG databases by (i) CUDAlign graphics processing unit (GPU) accelerated local alignment of all pseudogenes to gene families (totaling 1.6 billion individual local alignments and >40,000 GPU hours) and (ii) BLAST-based assignment of pseudogenes to gene families. Second, we create an open-source web application (PseudoFuN [Pseudogene Functional Networks]) to search for integrative functional relationships of sequence homology, microRNA expression, gene expression, pseudogene expression, and gene ontology. We produce four “flavors” of CUDAlign-based databases (>462,000,000 PGG pairwise alignments and 133,770 PGG families) that can be queried and downloaded using PseudoFuN. These databases are consistent with previous 1:1 PGG annotation and also are much more powerful including millions of de novo PGG associations. For example, we find multiple known (e.g., miR-20a-PTEN-PTENP1) and novel (e.g., miR-375-SOX15-PPP4R1L) microRNA-gene-pseudogene associations in prostate cancer. PseudoFuN provides a “one stop shop” for identifying and visualizing thousands of potential regulatory relationships related to pseudogenes in The Cancer Genome Atlas cancers. Conclusions Thousands of new PGG associations can be explored in the context of microRNA-gene-pseudogene co-expression and differential expression with a simple-to-use online tool by bioinformaticians and oncologists alike.

Retinoblastoma is a rare pediatric tumor of the retina, caused by the homozygous loss of the Retinoblastoma 1 (RB1) tumor suppressor gene. Previous microarray studies have identified changes in the expression profiles of coding genes; however, our understanding of how non-coding genes change in this tumor is absent. This is an important area of research, as in many adult malignancies, non-coding genes including LNC-RNAs are used as biomarkers to predict outcome and/or relapse. To establish a complete and in-depth RNA profile, of both coding and non-coding genes, in Retinoblastoma tumors, we conducted RNA-seq from a cohort of tumors and normal retina controls. This analysis identified widespread transcriptional changes in the levels of both coding and non-coding genes. Unexpectedly, we also found rare RNA fusion products resulting from genomic alterations, specific to Retinoblastoma tumor samples. We then determined whether these gene expression changes, of both coding and non-coding genes, were also found in a completely independent Retinoblastoma cohort. Using our dataset, we then profiled the potential effects of deregulated LNC-RNAs on the expression of neighboring genes, the entire genome, and on mRNAs that contain a putative area of homology. This analysis showed that most deregulated LNC-RNAs do not act locally to change the transcriptional environment, but potentially function to modulate genes at distant sites. From this analysis, we selected a strongly down-regulated LNC-RNA in Retinoblastoma, DRAIC, and found that restoring DRAIC RNA levels significantly slowed the growth of the Y79 Retinoblastoma cell line. Collectively, our work has generated the first non-coding RNA profile of Retinoblastoma tumors and has found that these tumors show widespread transcriptional deregulation.

Introduction: Vitamin D in its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) binds to the vitamin D receptor (VDR) to regulate genes, for example, in skeletal mineralization. However, it also has other potent biological functions in regulating apoptosis, proliferation, and inflammation. In carcinogenesis, 1,25(OH)2D3 may be exploited to regulate these crucial pathways. It is also clear that a frequent cancer disparity exists amongst African American (AA) men with prostate cancer compared to European Americans (EA). AA men show a higher incidence rate and two to three times increased risk of mortality than EA counterparts. Various groups have suggested that 1,25(OH)2D3 levels and/or VDR functions are risk factors linked with increased prostate cancer incidence in AA men. Incidentally, reports showed that VD plays a crucial role in regulating circadian rhythm (CR). There is, therefore, a need to understand and evaluate 1,25(OH)2D3-dependent CR regulation and the association with racial disparity in prostate cancer. This study aimed to determine if there are differentially expressed VD metabolic enzymes in AA and EA and evaluate if the differential expression correlates with CR genes. Methods: The Cancer Genome Atlas Research Network (TCGA), 2015 database was queried for expression of VD metabolizing enzymes and CR genes. The search was carried out on prostate adenocarcinoma expressions of AA and EA. VD metabolizing enzymes queried are CYP2R1, CYP24A1, CYP27B1, CYP27A1, while CR genes queried include ARNTL, CLOCK, CRY1, CRY2, CSNK1E, NPAS2, PER1, PER2, PER3, and TIMELESS. Prostate adenocarcinoma racial differential expressions of AA and EA were evaluated, and a correlation study was done using the Pearson correlation. Results: VD metabolic enzyme, CYP2R1, and CR gene, ARNTL were significantly upregulated in AA compared to EA counterparts. Although CYPR1 correlates negatively with CLOCK, CRY2, and PER3 in both races, CYPR1 specifically showed a positive correlation with CR gene CRY1 in EA and negative correlations with CR genes NPAS2 and CSNK1E in AA. However, a significant correlation between CYP2R1 and ARNTL in EA and AA was not observed. Conclusion: The data suggest a relationship between racial influence and prostate cancer associated with VD metabolism and CR regulation. Hence, it is crucial to elucidate CYP2R1 regulation in prostate cancer related to VD levels and CR regulation, especially with a focus on racial disparities. Citation Format: Isaacson Bababode Adelani, Solomon Oladapo Rotimi, Clayton Yates, Moray Campbell. Specific racial CYP2R1 correlation with circadian rhythm genes in prostate adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2529.

African American (AA) men have a 2 to 3 times higher prostate cancer mortality rates than European American (EA) men. Studies suggested that tumor biology may influence racial/ethnic survival and account for 24% of the disparities in PCa that remains even when controlled for access to care and stage at presentation. Additionally, men of African ancestry from the Caribbean and South America experience incidence and mortality rates similar to AA men, suggesting a possible ancestral basis for some of these expected outcomes. No study has currently assessed whether African descent men are affected by a systemic inflammatory process with changes in the immune system that increases the risk of lethal PCa. To assess the hypothesis that African ancestry drives aggressive prostate cancer and leads to genetic alterations with upregulation of unique immune-inflammatory signatures in men of African descent, we performed a genome-wide RNA sequencing analysis. We analyzed RNA isolated from FFPE tumor tissue obtained from 15 patients who self-reported as AA and 13 patients who self-reported as EA (n=28). To verify self-reported race, we used ADMIXTURE to generate a quantitative estimate of each individual ancestral composition. Notably, 14 patients who self-reported as AA also have a predominant African ancestry, particularly from African Caribbean's subpopulation, and one patient who self-reported as AA was actually Ad Mixed American. Furthermore, we conducted a descriptive statistical analysis of the study population; patients were stratified by race and pathology stage. Our results show that AA men are diagnosed with PCa at a younger age and higher pathology stage (≥ T2), contributing to a lower survival rate in AA. Gene-level expression was measured from STAR counts using Ensembl gene annotation. The resulting datasets were analyzed for differential gene expression and enriched pathways based on the patient's ancestry, race, and Gleason Score. Our analyses reveal that interferon-inducible genes (ISG15, IFT1, STAT1) are positively enriched (p-value 0.05), while neutrophil degranulation and Interleukins genes (IL8, CXCL8, KRTs, IL6, CXCL6) are negatively enriched (p-value 0.05) in AA men. These enriched gene sets may indicate that immune/inflammatory signatures play an important role in driving aggressive prostate cancer in AA. Additionally, we run GSEA-based on the immunological signature mode and used gene ontology function in EdgeR package; both showed that immune-related signaling pathways are enriched in AA men. These findings were confirmed in other RNA-Seq data sets attained from FFPE tissues. Similar immune/inflammatory patterns were observed in AA PCa cell lines (RC). Our study provides new insight into understanding how genetic ancestry and upregulation of unique immune-inflammatory signatures may contribute to PCa racial disparities in AA men cohorts from African ancestry. Citation Format: Isra A. Elhussin, Jason A. White, Tamaro S. Hudson, Moray J. Campbell, Chanita Hughes-Halbert, Stefan Ambs, Clayton Yates. Prostate cancer: Immune-inflammation signature in men of African ancestry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2196.

The current study aimed to define the genomic functions of the vitamin D receptor (VDR) in African American (AA) prostate cancer (PCa) compared to European American (EA) counterparts. VDR-dependent ChIP-Seq and RNA-Seq gene was undertaken in EA (non-malignant HPr1AR and malignant LNCaP) and AA (non-malignant RC43N and malignant RC43T) prostate models, combined with analyses of three PCa cohorts. In AA prostate models the VDR is highly expressed, binds more frequently, and is enriched in active and poised enhancers. Motif analyses revealed selective enrichment, including ZBTB33/KAISO in AA cells and ERG family members in EA cells and, similarly, GIGGLE analyses revealed AA VDR cistromes were significantly overlapped with core circadian rhythm transcription factors (e.g. NONO). Combining VDR-dependent ChIP-Seq and RNA-Seq established that AA cells displayed a significantly stronger transcriptional response, compared to EA cells, and was most responsive in non-malignant RC43N. For example, RC43N transcriptional responses were enriched for circadian rhythm (NES 2.7) and inflammation, whereas in RC43T the same gene networks were repressed. To reveal how VDR/1,25(OH)2D3 signaling is corrupted in AA PCa, we mined TCGA data and revealed that the BAZ1A/SMARCA5 chromatin remodeling complex was uniquely altered in TMPRSS2:ERG fusion negative AA PCa. We are currently examining the impact of BAZ1A on the 1,25(OH)2D3 responsiveness. We also identified miRNA associated with progression from HGPIN to PCa in AA men, and that ~30% were bound by VDR and regulated by 1,25(OH)2D3, although ~5% of EA progression miRNA were VDR-responsive. For example, VDR binds to miR-199b, is uniquely 1,25(OH)2D3 up-regulated in RC43N but repressed in RC43T, and associates with AA progression from HGPIN to PCa. MiR-199b regulates expression of NPAS2, a core circadian transcription factor. Finally, leveraging a previously analyzed cohort of 1,25(OH)2D3-treated PCa patients revealed that AA tumors were intrinsically more 1,25(OH)2D3-responsive than EA counterparts, reflecting the cell line analyses. 1,25(OH)2D3 regulated circadian transcriptional regulators (e.g. NOCT and MYBBP1A) and inflammatory signals. Together, these data suggest VDR transcriptional control in AA men is more dynamic than in EA men, and is primed to govern inflammatory and circadian rhythm pathways. This is frequently disrupted, including by altered BAZ1A/SMARCA5 expression and/or reduced environmental-regulated serum vitamin D3 levels, and leads to altered regulation of circadian rhythm process, and inflammatory signals. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes that contributes to PCa health disparities. Citation Format: Manjunath Siddappa, Sajad D. Wani, Jaimie S. Gray, Mark D. Long, Hedieh Jafari, Hsuchang Wu, Honhe Wang, Rebecca Morgan, Gary Hardiman, James Marshall, Chanita Hughes-Halbert, Lara E. Sucheston-Campbell, Clayton L. Yates, Moray J. Campbell. Epigenetic disruption of vitamin D receptor signaling in African American prostate cancer alters circadian signaling networks [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR05.

Bioinformatics applies unbiased approaches to develop statistically robust insight into health and disease. At the global, or "20,000 ft" view bioinformatic analyses of NR signaling can measure how the NRs are implicated in human health and disease through the impact of genome-wide significant genetic variation, family-wide NR expression patterns or considering where NRs are significantly identified in other high-dimensional data analyses. With a more NR-centric, or "2000 ft" view, bioinformatic approaches can interrogate events downstream of a given NR. Integrative approaches aim to combine multiple NR-centric high-dimensional data both derived in cell models and primary human tissue to reveal how NR-transcriptional networks relate to human health and disease. Bioinformatic approaches to such high-dimensional data are central and require specialist statistical insight and computational skills, coupled with a dexterous understanding of the biological question. A current challenge is determining the optimal mechanism to share such bioinformatic approaches through the biological research community.

Castration-resistant prostate cancer (CRPC) is defined by tumor microenvironment heterogeneity affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), and aberrant activation of transcription factors including androgen receptor (AR) and c-Myc. Using in vitro, in vivo, and animal models, we find a direct correlation between miR-644a downregulation and dysregulation of essential cellular processes. MiR-644a downregulated expression of diverse tumor microenvironment drivers including c-Myc, AR coregulators, and antiapoptosis factors Bcl-xl and Bcl2. Moreover, miR-644a modulates epithelial–mesenchymal transition (EMT) by directly targeting EMT-promoting factors ZEB1, cdk6, and Snail. Finally, miR-644a expression suppresses the Warburg effect by direct targeting of c-Myc, Akt, IGF1R, and GAPDH expression. RNA sequencing analysis revealed an analogous downregulation of these factors in animal tumor xenografts. These data demonstrate miR-644a mediated fine-tuning of oncogenesis, stimulating pathways and resultant potentiation of enzalutamide therapy in CRPC patients. Significance: This study demonstrates that miR-644a therapeutically influences the CRPC tumor microenvironment by suppressing androgen signaling and additional genes involved in metabolism, proliferation, Warburg effect, and EMT, to potentiate the enzalutamide therapy.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and poses a significant health burden due to its rising incidence. Although the proto-oncogene pituitary tumor–transforming gene 1 (PTTG) predicts poor patient outcome, its mechanisms of action are incompletely understood. We show here that the protein PBF modulates PTTG function, is overexpressed in HNSCC tumors, and correlates with significantly reduced survival. Lentiviral shRNA attenuation of PTTG or PBF expression in HNSCC cells with either wild-type or mutant p53, and with and without HPV infection, led to dysregulated expression of p53 target genes involved in DNA repair and apoptosis. Mechanistically, PTTG and PBF affected each other's interaction with p53 and cooperated to reduce p53 protein stability in HNSCC cells independently of HPV. Depletion of either PTTG or PBF significantly repressed cellular migration and invasion and impaired colony formation in HNSCC cells, implicating both proto-oncogenes in basic mechanisms of tumorigenesis. Patients with HNSCC with high tumoral PBF and PTTG had the poorest overall survival, which reflects a marked impairment of p53-dependent signaling. Significance: These findings reveal a complex and novel interrelationship between the expression and function of PTTG, PBF, and p53 in human HNSCC that significantly influences patient outcome. Cancer Res; 78(20); 5863–76. ©2018 AACR.

Although limited by severe side effects and development of resistance, platinum-based therapies still represent the most common first-line treatment for non-small cell lung cancer (NSCLC). However, a crucial need in the clinical management of NSCLC is represented by the identification of cases sensitive to DNA damage response (DDR)-targeting drugs, such as cisplatin or PARP inhibitors. Here, we provide a molecular rationale for the stratification of NSCLC patients potentially benefitting from platinum compounds based on the expression levels of RANBP9, a recently identified player of the cellular DDR. RANBP9 was found overexpressed by immunohistochemistry (IHC) in NSCLC compared to normal adjacent tissues (NATs) (n = 147). Moreover, a retrospective analysis of 132 platinum-treated patients from the multi-centric TAILOR trial showed that RANBP9 overexpression levels are associated with clinical response to platinum compounds [Progression Free Survival Hazard Ratio ((RANBP9 high vs low)) 1.73, 95% CI 1.15–2.59, p = 0.0084; Overall Survival HR ((RANBP9 high vs low)) 1.99, 95% CI 1.27–3.11, p = 0.003]. Accordingly, RANBP9 KO cells showed higher sensitivity to cisplatin in comparison with WT controls both in vitro and in vivo models. NSCLC RANBP9 KO cells were also more sensitive than control cells to the PARP inhibitor olaparib alone and in combination with cisplatin, due to defective ATM-dependent and hyper-activated PARP-dependent DDR. The current investigation paves the way to prospective studies to assess the clinical value of RANBP9 protein levels as prognostic and predictive biomarker of response to DDR-targeting drugs, leading to the development of new tools for the management of NSCLC patients.

Background The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. Methods In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. Results These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. Conclusions These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer.

// Vineet K. Dhiman 1 , Kristopher Attwood 2 , Moray J. Campbell 3 and Dominic J. Smiraglia 1 1 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Department of Biostatistics and Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA Correspondence to: Dominic J. Smiraglia, email: // Keywords : DNA methylation, androgen receptor, gene transcription, nuclear receptor, androgen regulated target genes, Chromosome Section Received : November 23, 2015 Accepted : November 24, 2015 Published : December 04, 2015 Abstract DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1 . Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1 . These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation.

Nuclear receptors (NR) act as an integrated conduit for environmental and hormonal signals to govern genomic responses, which relate to cell fate decisions. We review how their integrated actions with each other, shared co-factors and other transcription factors are disrupted in cancer. Steroid hormone nuclear receptors are oncogenic drivers in breast and prostate cancer and blockade of signaling is a major therapeutic goal. By contrast to blockade of receptors, in other cancers enhanced receptor function is attractive, as illustrated initially with targeting of retinoic acid receptors in leukemia. In the post-genomic era large consortia, such as The Cancer Genome Atlas, have developed a remarkable volume of genomic data with which to examine multiple aspects of nuclear receptor status in a pan-cancer manner. Therefore to extend the review of NR function we have also undertaken bioinformatics analyses of NR expression in over 3000 tumors, spread across six different tumor types (bladder, breast, colon, head and neck, liver and prostate). Specifically, to ask how the NR expression was distorted (altered expression, mutation and CNV) we have applied bootstrapping approaches to simulate data for comparison, and also compared these NR findings to 12 other transcription factor families. Nuclear receptors were uniquely and uniformly downregulated across all six tumor types, more than predicted by chance. These approaches also revealed that each tumor type had a specific NR expression profile but these were most similar between breast and prostate cancer. Some NRs were down-regulated in at least five tumor types (e.g., NR3C2/MR and NR5A2/LRH-1)) whereas others were uniquely down-regulated in one tumor (e.g., NR1B3/RARG). The downregulation was not driven by copy number variation or mutation and epigenetic mechanisms maybe responsible for the altered nuclear receptor expression.

To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVID's biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.