Elien Smeets

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

Treatment-induced mutations in the ligand-binding domain of the androgen receptor (AR) are known to change antagonists into agonists. Recently, the F877L mutation has been described to convert enzalutamide into an agonist. This mutation was seen to co-occur in the endogenous AR allele of LNCaP cells, next to the T878A mutation. Here, we studied the effects of enzalutamide on the F877L and T878A mutants, as well as the double-mutant AR (F877L/T878A). Molecular modeling revealed favorable structural changes in the double-mutant AR that lead to a decrease in steric clashes for enzalutamide. Ligand-binding assays confirmed that the F877L mutation leads to an increase in relative binding affinity for enzalutamide, but only the combination with the T878A mutation resulted in a strong agonistic activity. This correlated with changes in coregulator recruitment and chromatin interactions. Our data show that enzalutamide is only a very weak partial agonist of the AR F877L, and a strong partial agonist of the double-mutant AR. Mol Cancer Ther; 15(7); 1702–12. ©2016 AACR.

Genomic and epigenomic analysis of high-risk prostate cancer reveals changes in hydroxymethylation and TET1

The clinical heterogeneity of prostate cancer (PCa) makes it difficult to identify those patients that could benefit from more aggressive treatments. As a contribution to a better understanding of the genomic changes in the primary tumor that are associated with the development of high-risk disease, we performed exome sequencing and copy number determination of a clinically homogeneous cohort of 47 high-risk PCas. We confirmed recurrent mutations in SPOP, PTEN and TP53 among the 850 point mutations we detected. In seven cases, we discovered genomic aberrations in the TET1 (Ten-Eleven Translocation 1) gene which encodes a DNA hydroxymethylase than can modify methylated cytosines in genomic DNA and thus is linked with gene expression changes. TET1 protein levels were reduced in tumor versus non-tumor prostate tissue in 39 of 40 cases. The clinical relevance of changes in TET1 levels was demonstrated in an independent PCa cohort, in which low TET1 mRNA levels were significantly associated with worse metastases-free survival. We also demonstrate a strong reduction in hydroxymethylated DNA in tumor tissue in 27 of 41 cases. Furthermore, we report the first exploratory (h)MeDIP-Seq analyses of eight high-risk PCa samples. This reveals a large heterogeneity in hydroxymethylation changes in tumor versus non-tumor genomes which can be linked with cell polarity.

The role of TET-mediated DNA hydroxymethylation in prostate cancer

Ten-eleven translocation (TET) proteins are recently characterized dioxygenases that regulate demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further derivatives. The recent finding that 5hmC is also a stable and independent epigenetic modification indicates that these proteins play an important role in diverse physiological and pathological processes such as neural and tumor development. Both the genomic distribution of (hydroxy)methylation and the expression and activity of TET proteins are dysregulated in a wide range of cancers including prostate cancer. Up to now it is still unknown how changes in TET and 5(h)mC profiles are related to the pathogenesis of prostate cancer. In this review, we explore recent advances in the current understanding of how TET expression and function are regulated in development and cancer. Furthermore, we look at the impact on 5hmC in prostate cancer and the potential underlying mechanisms. Finally, we tried to summarize the latest techniques for detecting and quantifying global and locus-specific 5hmC levels of genomic DNA.

Treatment-induced changes in the androgen receptor axis: Liquid biopsies as diagnostic/prognostic tools for prostate cancer

Prostate cancer progression and treatment relapse is associated with changes in the androgen receptor axis, and analysis of alternations of androgen receptor signaling is valuable for prognostics and treatment optimization. The profile of androgen receptor axis is currently obtained from biopsy specimens, which are not always easy to obtain. Moreover, the information acquired only provides a snapshot of the tumor biology, with strict spatial and temporal limitations. On the other hand, circulation is easily accessible source of both circulating tumor cells and circulating tumor DNA, which can be sampled at numerous time points. This Review will explore the potential use of androgen receptor axis alternations detectable in the blood in therapeutic decision-making and precision medicine for advancing metastatic castration-resistant prostate cancer.

Abstract B075: The role of TET1 and hydroxymethylation in high-risk prostate cancer

The Ten-eleven translocation (TET) proteins are dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) as part of the DNA demethylation pathway. Gene silencing through cytosine methylation contributes to cancer formation, but how hydroxymethylation affects gene expression is largely unknown. In this work, TET1 and genomic hydroxymethylation were investigated in a high-risk prostate cancer cohort for which exome sequencing data are available (1). Immunohistochemical analysis of tumor versus nontumor prostate biopsies from high-risk prostate cancer showed a strong reduction of genome-wide 5hmC and milder but still significant reduction of 5mC levels in prostate cancer tissue compared to control tissue. Copy number analysis of high-risk prostate cancer showed a loss of the TET1 genomic region in 6 out of 39 samples of our cohort. As expected, the mRNA levels of TET1 were decreased in these tumor samples. Recently, an alternative promoter of TET1 was discovered. This promoter generates a novel isoform lacking the CXXC-domain, which is called TET1ALT (2). We therefore performed functional analyses of the two TET1 promoters in prostate cancer cells. Interestingly, the promoter controlling expression of the endogenous full-length TET1 is less active in prostate cancer cell line PC-3, while the alternative promoter generating TET1ALT is more active in these cells. This is shown by luciferase constructs driven by the two promoter regions. In conclusion, our findings indicate that there is a drop in (hydroxy)methylation levels in high-risk prostate cancer that correlates with decreased TET1 expression levels and in some cases by a copy number loss of TET1. We are currently working on a more detailed analysis of TET1 promoter usage and control. This will unravel the regulation of hydroxymethylation in prostate cancer, which is crucial for the understanding of prostate cancer development and progression. References: 1. Spans L, Van den Broeck T, Smeets E, et al. Genomic and epigenomic analysis of high-risk prostate cancer reveals changes in hydroxymethylation and TET1. Oncotarget 2016;7(17):24326-38. 2. Good CR, Madzo J, Patel B, et al. A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer. Nucleic Acids Res 2017;45(14):8269-81. Citation Format: Elien Smeets, Lien Spans, Stefan Prekovic, Thomas Van den Broeck, Lisa Moris, Thomas Gevaert, Christine Helsen, Steven Joniau, Frank Claessens. The role of TET1 and hydroxymethylation in high-risk prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B075.

Abstract B065: Genomic analysis of localized prostate cancer identifies AZIN1 as driver of metastatic progression

High-risk PCa (HRPCa) remains very heterogeneous with an unacceptable variation in patient outcome after radical prostatectomy, with cancer-specific mortality rates ranging from 4.6% to 20.3% at 10 years’ follow-up. Clearly, we need a better understanding of the tumor biology to enhance the subclassification, enable the identification of lethal PCa, and eventually allow a more precise decision-making regarding treatment. We created a matched case-control study of two clinically identical HRPCa patient groups, both treated with radical prostatectomy but where one developed metastatic recurrence (n=19) and the other did not (n=25), despite very-long-term follow-up. The integrated analysis of copy number aberrations and transcriptome analysis highlighted a focal amplification of 8q22.3 in the metastatic group, associated with a higher expression of antizyme inhibitor 1 (AZIN1) in our cohort. This association of high AZIN1 levels with the metastatic HRPCa phenotype suggests a role for AZIN1 as possible predictor and/or target for metastatic HRPCa. AZIN1 is one of the regulators of the polyamine synthesis, which play a central role in many cellular processes. Our in vitro analyses confirmed that modulation of AZIN1 expression determines both growth and migratory potential of prostate cancer cells. Future experiments are planned to confirm these data in the in vivo setting. RNA sequencing after knockdown of AZIN1 in PCa cells revealed several transcriptional programs that are activated/deactivated upon AZIN1 knockdown. This showed among others a significant upregulation of genes involved in extracellular matrix composition, including genes encoding for subunits of the collagen IV, which is an integral part of the basement membrane. Ongoing experiments focus on identifying the mechanism by which AZIN1 regulates collagen IV expression. Citation Format: Thomas Van den Broeck, Lisa Moris, Thomas Gevaert, Elien Smeets, Stefan Prekovic, Christine Helsen, Hendrik van Poppel, Wouter Everaerts, Christine Buerki, Elai Davicioni, Steven Joniau, Frank Claessens. Genomic analysis of localized prostate cancer identifies AZIN1 as driver of metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B065.

The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3

The nuclear vitamin D receptor (VDR) is generally recognized as a ligand-dependent transcription factor that mediates the actions of its natural ligand, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on multiple target genes involved in mineral homeostasis, bone development, as well as immune reactivity. As the VDR is widely distributed in nearly all cells of the body, it implies that the vitamin D endocrine system may regulate many cell types and functions. Experiments in VDR null mice established that the VDR has intrinsically critical roles in skin and keratinocyte biology but not in immune responses. Oppositely, absence of the VDR ligand is linked to susceptibility to autoimmunity, illustrating a potential role for the unliganded VDR in the immune system. This discrepancy stimulated us to further investigate the impact of the VDR on the phenotype and function of myeloid dendritic cells (DCs) generated ex vivo from bone marrow precursors of VDR null (with a truncated VDR) and VDR ΔAF2 mice (with a mutated C-terminal activation factor 2 domain thus rendering ligand-induced gene transcription impossible). Absent or unliganded VDR did not affect bone marrow-derived myeloid DC generation. DCs obtained from VDR null and VDR ΔAF2 bone marrow cells had comparable MHC-II, and costimulatory molecule CD86, CD80 and CD40 expression than DCs from wild-type bone marrow cells. Additionally, an unliganded VDR did not affect the cytokine production nor the antigen-specific T cell stimulatory capacity of bone marrow-derived DCs. In conclusion, we showed that although clear effects of 1α,25-dihydroxyvitamin D3 are described on DC generation, absence of VDR or presence of an unliganded VDR does not affect the profile and function of ex vivo generated bone marrow-derived DCs.