The BET degrader ZBC260 suppresses stemness and tumorigenesis and promotes differentiation in triple-negative breast cancer by disrupting inflammatory signaling.

BACKGROUND: Breast cancer stem cells (BCSCs) are resistant to standard therapies, facilitate tumor dissemination, and contribute to relapse and progression. Super-enhancers are regulators of stemness, and BET proteins, which are critical for super-enhancer function, are a potential therapeutic target. Here, we investigated the effects of BET proteins on the regulation of breast cancer stemness using the pan-BET degrader ZBC260. METHODS: We evaluated the effect of ZBC260 on CSCs in TNBC cell lines. We assessed the effect of ZBC260 on cellular viability and tumor growth and measured its effects on cancer stemness. We used RNA sequencing and stemness index to determine the global transcriptomic changes in CSCs and bulk cells and further validated our findings by qPCR, western blot, and ELISA. RESULTS: ZBC260 potently inhibited TNBC growth both in vitro and in vivo. ZBC260 reduced stemness as measured by cell surface marker expression, ALDH activity, tumorsphere number, and stemness index while increasing differentiated cells. GSEA analysis indicated preferential downregulation of stemness-associated and inflammatory genes by ZBC260 in ALDH+ CSCs. CONCLUSIONS: The BET degrader ZBC260 is an efficient degrader of BET proteins that suppresses tumor progression and decreases CSCs through the downregulation of inflammatory genes and pathways. Our findings support the further development of BET degraders alone and in combination with other therapeutics as CSC targeting agents.

LncRNA XIST regulates breast cancer stem cells by activating proinflammatory IL-6/STAT3 signaling.

Aberrant expression of XIST, a long noncoding RNA (lncRNA) initiating X chromosome inactivation (XCI) in early embryogenesis, is a common feature of breast cancer (BC). However, the roles of post-XCI XIST in breast carcinogenesis remain elusive. Here we identify XIST as a key regulator of breast cancer stem cells (CSCs), which exhibit aldehyde dehydrogenase positive (ALDH+) epithelial- (E) and CD24loCD44hi mesenchymal-like (M) phenotypes. XIST is variably expressed across the spectrum of BC subtypes, and doxycycline (DOX)-inducible knockdown (KD) of XIST markedly inhibits spheroid/colony forming capacity, tumor growth and tumor-initiating potential. This phenotype is attributed to impaired E-CSC in luminal and E- and M-CSC activities in triple-negative (TN) BC. Gene expression profiling unveils that XIST KD most significantly affects cytokine-cytokine receptor interactions, leading to markedly suppressed expression of proinflammatory cytokines IL-6 and IL-8 in ALDH- bulk BC cells. Exogenous IL-6, but not IL-8, rescues the reduced sphere-forming capacity and proportion of ALDH+ E-CSCs in luminal and TN BC upon XIST KD. XIST functions as a nuclear sponge for microRNA let-7a-2-3p to activate IL-6 production from ALDH- bulk BC cells, which acts in a paracrine fashion on ALDH+ E-CSCs that display elevated cell surface IL-6 receptor (IL6R) expression. This promotes CSC self-renewal via STAT3 activation and expression of key CSC factors including c-MYC, KLF4 and SOX9. Together, this study supports a novel role of XIST by derepressing let-7 controlled paracrine IL-6 proinflammatory signaling to promote CSC self-renewal.

Targeting Breast Cancer Stem Cell State Equilibrium through Modulation of Redox Signaling.

Although breast cancer stem cells (BCSCs) display plasticity transitioning between quiescent mesenchymal-like (M) and proliferative epithelial-like (E) states, how this plasticity is regulated by metabolic or oxidative stress remains poorly understood. Here, we show that M- and E-BCSCs rely on distinct metabolic pathways and display markedly different sensitivities to inhibitors of glycolysis and redox metabolism. Metabolic or oxidative stress generated by 2DG, H2O2, or hypoxia promotes the transition of ROSlo M-BCSCs to a ROShi E-state. This transition is reversed by N-acetylcysteine and mediated by activation of the AMPK-HIF1α axis. Moreover, E-BCSCs exhibit robust NRF2-mediated antioxidant responses, rendering them vulnerable to ROS-induced differentiation and cytotoxicity following suppression of NRF2 or downstream thioredoxin (TXN) and glutathione (GSH) antioxidant pathways. Co-inhibition of glycolysis and TXN and GSH pathways suppresses tumor growth, tumor-initiating potential, and metastasis by eliminating both M- and E-BCSCs. Exploiting metabolic vulnerabilities of distinct BCSC states provides a novel therapeutic approach targeting this critical tumor cell population.

RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA.

Ribonucleic acid-mediated transcriptional gene silencing (known as RNA-directed DNA methylation, or RdDM, in Arabidopsis thaliana) is important for influencing gene expression and the inhibition of transposons by the deposition of repressive chromatin marks such as histone modifications and DNA methylation. A key event in de novo methylation of DNA by RdDM is the production of long non-coding RNA (lncRNA) by RNA polymerase V (Pol V). Little is known about the events that connect Pol V transcription to the establishment of repressive chromatin modifications. Using RNA immunoprecipitation, we elucidated the order of events downstream of lncRNA production and discovered interdependency between lncRNA-associated proteins. We found that the effector protein ARGONAUTE4 (AGO4) binds lncRNA independent of the RNA-binding protein INVOLVED IN DE NOVO2 (IDN2). In contrast, IDN2 binds lncRNA in an AGO4-dependent manner. We further found that the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) also associates with lncRNA produced by Pol V and that this event depends on AGO4 and IDN2. We propose a model where the silencing proteins AGO4, IDN2 and DRM2 bind to lncRNA in a stepwise manner, resulting in DNA methylation of RdDM target loci.

Biological and clinical significance of cancer stem cell plasticity.

In the past decade, the traditional view of cancers as a homogeneous collection of malignant cells is being replaced by a model of ever increasing complexity suggesting that cancers are complex tissues composed of multiple cell types. This complex model of tumorigenesis has been well supported by a growing body of evidence indicating that most cancers including those derived from blood and solid tissues display a hierarchical organization of tumor cells with phenotypic and functional heterogeneity and at the apex of this hierarchy are cells capable of self-renewal. These "tumor imitating cells" or "cancer stem cells" drive tumorigenesis and contribute to metastasis, treatment resistance and tumor relapse. Although tumor stem cells themselves may display both genetic and phenotypic heterogeneity, recent studies have demonstrated that cancer stem cells maintain plasticity to transition between mesenchymal-like (EMT) and epithelial-like (MET) states, which may be regulated by the tumor microenvironment. These stem cell state transitions may play a fundamental role in tumor progression and treatment resistance. In this review, we discuss the emerging knowledge regarding the plasticity of cancer stem cells with an emphasis on the signaling pathways and noncoding RNAs including microRNAs (miRNA) and long non-coding RNAs (lncRNAs) in regulation of this plasticity during tumor growth and metastasis. Lastly, we point out the importance of targeting both the EMT and MET states of CSCs in order to eliminate these lethal seeds of cancers.

A SWI/SNF chromatin-remodeling complex acts in noncoding RNA-mediated transcriptional silencing.

RNA-mediated transcriptional silencing prevents deleterious effects of transposon activity and controls the expression of protein-coding genes. It involves long noncoding RNAs (lncRNAs). In Arabidopsis thaliana, some of those lncRNAs are produced by a specialized RNA Polymerase V (Pol V). The mechanism by which lncRNAs affect chromatin structure and mRNA production remains mostly unknown. Here we identify the SWI/SNF ATP-dependent nucleosome-remodeling complex as a component of the RNA-mediated transcriptional silencing pathway. We found that SWI3B, an essential subunit of the SWI/SNF complex, physically interacts with a lncRNA-binding protein, IDN2. SWI/SNF subunits contribute to lncRNA-mediated transcriptional silencing. Moreover, Pol V mediates stabilization of nucleosomes on silenced regions. We propose that Pol V-produced lncRNAs mediate transcriptional silencing by guiding the SWI/SNF complex and establishing positioned nucleosomes on specific genomic loci. We further propose that guiding ATP-dependent chromatin-remodeling complexes may be a more general function of lncRNAs.

A direct docking mechanism for a plant GSK3-like kinase to phosphorylate its substrates.

Glycogen synthase kinase 3 (GSK3) is a highly conserved serine/threonine protein kinase that plays important roles in a variety of physiological and developmental processes in animals. It is well known that the GSK3 kinase-catalyzed protein phosphorylation often requires a stable kinase-substrate docking interaction, which is achieved mainly by two mechanisms as follows: priming phosphorylation of a substrate by a distinct kinase to create a docking phosphate group and scaffold protein-mediated protein complex formation. Brassinosteroid-INsensitive 2 (BIN2) is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling by phosphorylating BES1 (bri1 EMS suppressor 1) and BZR1 (brassinazole-resistant 1), two highly similar transcription factors critical for bringing about characteristic BR responses. However, little is known about the biochemical mechanism by which BIN2 phosphorylates its substrates. Here, we show that BIN2 interacts directly with BZR1 through a 12-amino acid BIN2-docking motif adjacent to the C terminus of BZR1. Interestingly, this 12-amino acid motif is sufficient to allow a Drosophila GSK3 substrate Armadillo to be phosphorylated by BIN2 in vitro. Deletion of this motif inhibits the phosphorylation and subsequent degradation of BZR1 in vivo, resulting in phenotypic suppression of a hypermorphic bin2 mutation and enhanced resistance to a BR biosynthesis inhibitor. We thus concluded that BIN2 utilizes a direct kinase-substrate docking mechanism to phosphorylate BZR1 and regulate its protein stability.

Regulation of Arabidopsis brassinosteroid signaling by atypical basic helix-loop-helix proteins.

Basic helix-loop-helix (bHLH) proteins are highly conserved transcription factors critical for cell proliferation and differentiation. Recent studies have implicated bHLH proteins in many plant signaling processes, including brassinosteroid (BR) signaling. Here, we report identification of two families of atypical bHLH proteins capable of modulating BR signaling. We found that activation-tagged bri1 suppressor 1-Dominant (atbs1-D), previously identified as a dominant suppressor of a weak BR receptor mutant bri1-301, was caused by overexpression of a 93-amino acid atypical bHLH protein lacking amino acids critical for DNA binding. Interestingly, atbs1-D only suppresses weak BR mutants, while overexpression of a truncated ATBS1 lacking the basic motif also rescues bri1-301, suggesting that ATBS1 likely stimulates BR signaling by sequestering negative BR signaling components. A yeast two-hybrid screen using ATBS1 as bait discovered four ATBS1-Interacting Factors (AIFs) that are members of another atypical bHLH protein subfamily. AIF1 exhibits an overlapping expression pattern with ATBS1 and its homologs and interacts with ATBS1 in vitro and in vivo. AIF1 overexpression nullifies the suppressive effect of atbs1-D on bri1-301 and results in dwarf transgenic plants resembling BR mutants. By contrast, silencing of AIF1 partially suppressed the bri1-301 phenotype. Our results suggested that plants use these atypical bHLH proteins to regulate BR signaling.

PALB2 regulates recombinational repair through chromatin association and oligomerization.

Maintenance of genomic stability ensures faithful transmission of genetic information and helps suppress neoplastic transformation and tumorigenesis. Although recent progress has advanced our understanding of DNA damage checkpoint regulations, little is known as to how DNA repair, especially the RAD51-dependent homologous recombination repair pathway, is executed in vivo. Here, we reveal novel properties of the BRCA2-associated protein PALB2 in the assembly of the recombinational DNA repair machinery at DNA damage sites. Although the chromatin association of PALB2 is a prerequisite for subsequent BRCA2 and RAD51 loading, the focal accumulation of the PALB2 x BRCA2 x RAD51 complex at DSBs occurs independently of known DNA damage checkpoint and repair proteins. We provide evidence to support that PALB2 exists as homo-oligomers and that PALB2 oligomerization is essential for its focal accumulation at DNA breaks in vivo. We propose that both PALB2 chromatin association and its oligomerization serve to secure the BRCA2 x RAD51 repair machinery at the sites of DNA damage. These attributes of PALB2 are likely instrumental for proficient homologous recombination DNA repair in the cell.

Regulation of the Arabidopsis GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 through proteasome-mediated protein degradation.

Glycogen synthase kinase 3 (GSK3) is a unique serine/threonine kinase that is implicated in a variety of cellular processes and is regulated by phosphorylation or protein-protein interaction in animal cells. BIN2 is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling. Genetic studies suggested that BIN2 is inhibited in response to BR perception at the cell surface to relieve its inhibitory effects on downstream targets; however, little is known about biochemical mechanisms of its inhibition. Here, we show that BIN2 is regulated by proteasome-mediated protein degradation. Exogenous application of a BR biosynthesis inhibitor and an active BR increased and decreased the amount of BIN2 proteins, respectively. Interestingly, the gain-of-function bin2-1 mutation significantly stabilizes BIN2, making it unresponsive to BR-induced BIN2 depletion. Exogenous application of different plant growth hormones revealed that BIN2 depletion is specifically induced by BR through a functional BR receptor, while treatment of a proteasome inhibitor, MG132, not only prevented the BR-induced BIN2 depletion but also nullified the inhibitory effect of BR on the BIN2 kinase activity. Taken together, our results strongly suggest that proteasome-mediated protein degradation constitutes an important regulatory mechanism for restricting the BIN2 activity.

Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice.

The rice Eui (ELONGATED UPPERMOST INTERNODE) gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins (GAs). In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmy1A and GAmyb were highly upregulated in eui aleurone cells in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 (GIBBERELLIN INSENSITIVE DWARF 1), GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA20ox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.

ELONGATED UPPERMOST INTERNODE encodes a cytochrome P450 monooxygenase that epoxidizes gibberellins in a novel deactivation reaction in rice.

The recessive tall rice (Oryza sativa) mutant elongated uppermost internode (eui) is morphologically normal until its final internode elongates drastically at the heading stage. The stage-specific developmental effect of the eui mutation has been used in the breeding of hybrid rice to improve the performance of heading in male sterile cultivars. We found that the eui mutant accumulated exceptionally large amounts of biologically active gibberellins (GAs) in the uppermost internode. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase, CYP714D1. Using heterologous expression in yeast, we found that EUI catalyzed 16alpha,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall and dwarfed phenotypes of the eui mutant and Eui-overexpressing transgenic plants, respectively, 16alpha,17-epoxidation reduced the biological activity of GA(4) in rice, demonstrating that EUI functions as a GA-deactivating enzyme. Expression of Eui appeared tightly regulated during plant development, in agreement with the stage-specific eui phenotypes. These results indicate the existence of an unrecognized pathway for GA deactivation by EUI during the growth of wild-type internodes. The identification of Eui as a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops.