An RNA-dependent and phase-separated active subnuclear compartment safeguards repressive chromatin domains.

The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.

BAZ2A-mediated repression via H3K14ac-marked enhancers promotes prostate cancer stem cells.

Prostate cancer (PCa) is one of the most prevalent cancers in men. Cancer stem cells are thought to be associated with PCa relapse. Here, we show that BAZ2A is required for PCa cells with a cancer stem-like state. BAZ2A genomic occupancy in PCa cells coincides with H3K14ac-enriched chromatin regions. This association is mediated by BAZ2A-bromodomain (BAZ2A-BRD) that specifically binds H3K14ac. BAZ2A associates with inactive enhancers marked by H3K14ac and repressing transcription of genes frequently silenced in aggressive and poorly differentiated PCa. BAZ2A-mediated repression is also linked to EP300 that acetylates H3K14ac. BAZ2A-BRD mutations or treatment with inhibitors abrogating BAZ2A-BRD/H3K14ac interaction impair PCa stem cells. Furthermore, pharmacological inactivation of BAZ2A-BRD impairs Pten-loss oncogenic transformation of prostate organoids. Our findings indicate a role of BAZ2A-BRD in PCa stem cell features and suggest potential epigenetic-reader therapeutic strategies to target BAZ2A in aggressive PCa.

Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4.

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.

Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Graphene Oxide Functional Nanohybrids with Magnetic Nanoparticles for Improved Vectorization of Doxorubicin to Neuroblastoma Cells.

With the aim to obtain a site-specific doxorubicin (DOX) delivery in neuroblastoma SH-SY5Y cells, we designed an hybrid nanocarrier combining graphene oxide (GO) and magnetic iron oxide nanoparticles (MNPs), acting as core elements, and a curcumin⁻human serum albumin conjugate as functional coating. The nanohybrid, synthesized by redox reaction between the MNPs@GO system and albumin bioconjugate, consisted of MNPs@GO nanosheets homogeneously coated by the bioconjugate as verified by SEM investigations. Drug release experiments showed a pH-responsive behavior with higher release amounts in acidic (45% at pH 5.0) vs. neutral (28% at pH 7.4) environments. Cell internalization studies proved the presence of nanohybrid inside SH-SY5Y cytoplasm. The improved efficacy obtained in viability assays is given by the synergy of functional coating and MNPs constituting the nanohybrids: while curcumin moieties were able to keep low DOX cytotoxicity levels (at concentrations of 0.44⁻0.88 µM), the presence of MNPs allowed remote actuation on the nanohybrid by a magnetic field, increasing the dose delivered at the target site.

In vivo [64Cu]CuCl2 PET imaging reveals activity of Dextran-Catechin on tumor copper homeostasis.

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels in vivo is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [64Cu]CuCl2 PET imaging protocol to monitor its effect on copper homeostasis in tumors. Methods: Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin in vivo and its effect on tumor copper homeostasis. PET imaging with [64Cu]CuCl2 was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment. Results: CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. In vivo studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [64Cu]CuCl2 retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. Ex vivo analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity. Conclusion: Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments.