ONECUT2 regulates RANKL-dependent enterocyte and microfold cell differentiation in the small intestine; a multi-omics study.

Microfold (M) cells reside in the intestinal epithelium of Peyer's patches (PP). Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an enterocyte and M cell precursor population. We used our newly developed computational tool SCEPIA to link precursor cell-specific gene expression to transcription factor motif activity in cis-regulatory elements, uncovering high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling axis to support enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating cell fate balance in the PP, and it provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium.

Delivery of antisense oligonucleotides for splice-correction of androgen receptor pre-mRNA in castration-resistant prostate cancer models using cell-penetrating peptides.

BACKGROUND: Cell-penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre-mRNA, thereby preventing the generation of the splice variant AR-V7 mRNA. AON-mediated knockdown of AR-V7 resulted in inhibition of androgen-independent cell proliferation. In this study, we evaluated the CPP-mediated delivery of this AON into castration-resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). METHODS: Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter-Glo cell viability assay. The effectivity of CPP-mediated delivery of the splice-correcting AON-intronic splicing enhancer (ISE) targeting the ISE in the castration-resistant prostate cancer (CRPC)-derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR-V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR-V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. RESULTS: The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine-rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier-associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 µM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON-ISE directed against AR pre-mRNA achieved significant downregulation of AR-V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen-independent cell proliferation of DuCaP and VCaP cells. CONCLUSIONS: This proof-of-principle study constitutes the basis for further development of CPP-mediated delivery of AONs for targeted therapy in prostate cancer.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Androgen receptor signalling confers clonogenic and migratory advantages in urothelial cell carcinoma of the bladder.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR-positive UCC-derived cell line model, UM-UC-3-AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell-like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen-stimulated UM-UC-3-AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

Suppression of prostate tumor cell survival by antisense oligonucleotide-mediated inhibition of AR-V7 mRNA synthesis.

One of the mechanisms by which advanced prostate cancer develops resistance to androgen deprivation therapy is the elevated expression of C-terminally truncated androgen receptor (AR) variants. These variants, such as AR-V7, originate from aberrant splicing of the AR pre-mRNA and the inclusion of a cryptic exon containing a premature stop codon in the mRNA. The resulting loss of the ligand-binding domain allows AR-V7 to act as a constitutively active transcription factor. Here, we designed two antisense oligonucleotides (AONs) directed against cryptic splicing signals within the AR pre-mRNA. These two AONs, AON-ISE and AON-ESE, demonstrated high efficiency in silencing AR-V7 splicing without affecting full-length AR expression. The subsequent downregulation of AR-V7-target gene UBE2C was accompanied by inhibition of androgen-independent cell proliferation and induction of apoptosis in castration-resistant prostate cancer (CRPC)-derived cell line models 22Rv1, DuCaP, and VCaP. Our results show that splicing-directed AONs can efficiently prevent expression of AR-V7, providing an attractive new therapeutic option for the treatment of CRPC.

Integrative multi-omics analysis of intestinal organoid differentiation.

Intestinal organoids accurately recapitulate epithelial homeostasis in vivo, thereby representing a powerful in vitro system to investigate lineage specification and cellular differentiation. Here, we applied a multi-omics framework on stem cell-enriched and stem cell-depleted mouse intestinal organoids to obtain a holistic view of the molecular mechanisms that drive differential gene expression during adult intestinal stem cell differentiation. Our data revealed a global rewiring of the transcriptome and proteome between intestinal stem cells and enterocytes, with the majority of dynamic protein expression being transcription-driven. Integrating absolute mRNA and protein copy numbers revealed post-transcriptional regulation of gene expression. Probing the epigenetic landscape identified a large number of cell-type-specific regulatory elements, which revealed Hnf4g as a major driver of enterocyte differentiation. In summary, by applying an integrative systems biology approach, we uncovered multiple layers of gene expression regulation, which contribute to lineage specification and plasticity of the mouse small intestinal epithelium.

Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting.

Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.