Transcription Complex Formation Research Articles

YAP represses the TEAD-NF-κB complex and inhibits the growth of clear cell renal cell carcinoma.

The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.

ACSL3 regulates breast cancer progression via lipid metabolism reprogramming and the YES1/YAP axis.

Mitochondrial fatty acid oxidation is a metabolic pathway whose dysregulation is recognized as a critical factor in various cancers, because it sustains cancer cell survival, proliferation, and metastasis. The acyl-CoA synthetase long-chain (ACSL) family is known to activate long-chain fatty acids, yet the specific role of ACSL3 in breast cancer has not been determined. We assessed the prognostic value of ACSL3 in breast cancer by using data from tumor samples. Gain-of-function and loss-of-function assays were also conducted to determine the roles and downstream regulatory mechanisms of ACSL3 in vitro and in vivo. ACSL3 expression was notably downregulated in breast cancer tissues compared with normal tissues, and this phenotype correlated with improved survival outcomes. Functional experiments revealed that ACSL3 knockdown in breast cancer cells promoted cell proliferation, migration, and epithelial-mesenchymal transition. Mechanistically, ACSL3 was found to inhibit β-oxidation and the formation of associated byproducts, thereby suppressing malignant behavior in breast cancer. Importantly, ACSL3 was found to interact with YES proto-oncogene 1, a member of the Src family of tyrosine kinases, and to suppress its activation through phosphorylation at Tyr419. The decrease in activated YES1 consequently inhibited YAP1 nuclear colocalization and transcriptional complex formation, and the expression of its downstream genes in breast cancer cell nuclei. ACSL3 suppresses breast cancer progression by impeding lipid metabolism reprogramming, and inhibiting malignant behaviors through phospho-YES1 mediated inhibition of YAP1 and its downstream pathways. These findings suggest that ACSL3 may serve as a potential biomarker and target for comprehensive therapeutic strategies for breast cancer.

The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/β-catenin Signaling via Epigenetic Dysregulation that Enhances Sp1-β catenin-p300 Interactions in Hepatocellular Carcinoma.

The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3) is an epigenetic player that modulates chromatin accessibility to activate p53 during liver injury. Nonetheless, a precise tumor suppressive and epigenetic role of NR2E3 in hepatocellular carcinoma (HCC) development remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice consistently exhibits accelerated liver tumor formation accompanied by enhanced activation of Wnt/β-catenin signaling pathway and inactivation of p53 signaling. At cellular level, the loss of NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates key genes in the WNT/β-catenin pathway with increased chromatin accessibility. This event is mediated through increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. These findings demonstrate that the loss of NR2E3 activates Wnt/β-catenin signaling at cellular and organism levels and this dysregulation is associated with aggressive HCC development and poor clinical outcomes. In summary, NR2E3 is a novel tumor suppressor with a significant prognostic value, maintaining epigenetic homeostasis to suppress the Wnt/β-catenin signaling pathway that promotes HCC development.

Abstract 3011: The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) stands as the fourth leading cause of global cancer-related mortality due to late-stage detection, tumor heterogeneity, and drug resistance. The pivotal role of epigenetic factors in HCC development and progression necessitates the identification of critical epigenetic regulators. The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3), primarily associated with retinal development, has emerged as a potential tumor suppressor. Earlier studies showed that the loss of NR2E3 leads to epigenetic repression of the estrogen receptor α (ER) through the involvement of Lysine Specific histone Demethylase 1 (LSD1). NR2E3 also forms a transcriptional complex with Sp1, governing the aryl hydrocarbon receptor (AHR) expression, a crucial factor in liver xenobiotic metabolism. In vivo, NR2E3 ablation results in p53 inactivation and severe liver injuries when exposed to liver toxicants, highlighting its role in activating p53. Nonetheless, the precise tumor suppressive and epigenetic role of NR2E3 in HCC remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice show accelerated liver tumor formation and progression accompanied by enhanced activation of the Wnt/β-catenin signaling pathway and inactivation of the p53 signaling pathway. At a cellular level, losing NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates vital genes in the Wnt/β-catenin pathway with enhanced chromatin accessibility. This event is mediated through the increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. Correspondingly, treatment of p300 inhibitor C646 decreased the expression of critical genes in the Wnt/β-catenin signaling pathway. These findings demonstrate that the loss of NR2E3 promotes Wnt/β-catenin signaling activation at cellular, organismal, and clinical levels. In summary, NR2E3 is a novel tumor suppressor that maintains epigenetic homeostasis, thereby preventing activation of Wnt/β-catenin signaling that promotes HCC formation. Citation Format: Yuet-Kin Leung, Sung-Gwon Lee, Jiang Wang, Shuk-Mei Ho, Nancy Rusch, Chungoo Park, Kyounghyun Kim. The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3011.

Tubercidin inhibits PRRSV replication via RIG-I/NF-κB pathways and interrupting viral nsp2 synthesis.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases, which causes huge economic loss worldwide. However, there is no effective therapeutic method for PRRS prevention and control. Here, we found that tubercidin, a naturally extracted adenosine analog, exhibited strong anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Mechanically, tubercidin inhibited viral binding, replication, and release. Tubercidin suppressed PRRSV non-structural protein 2 expression, which is important for the formation of replication and transcription complex, leading to the block of viral RNA synthesis and PRRSV replication. Moreover, tubercidin could activate retinoic acid-inducible gene I/nuclear factor kappa-light-chain-enhancer of activated B cell innate immune signaling pathway and increased the expression of interferons and proinflammatory cytokines, which was the other way to inhibit PRRSV replication. Our work evaluated the potential value of tubercidin as an antiviral agent on PRRSV replication and provided a new way to prevent PRRSV replication in vitro.

SUMOylation Fine-Tunes Endothelial HEY1 in the Regulation of Angiogenesis.

Angiogenesis, which plays a critical role in embryonic development and tissue repair, is controlled by a set of angiogenic signaling pathways. As a TF (transcription factor) belonging to the basic helix-loop-helix family, HEY (hairy/enhancer of split related with YRPW motif)-1 (YRPW motif, abbreviation of 4 highly conserved amino acids in the motif) has been identified as a key player in developmental angiogenesis. However, the precise mechanisms underlying HEY1's actions in angiogenesis remain largely unknown. Our previous studies have suggested a potential role for posttranslational SUMOylation in the dynamic regulation of vascular development and organization. Immunoprecipitation, mass spectrometry, and bioinformatics analysis were used to determine the biochemical characteristics of HEY1 SUMOylation. The promoter-binding capability of HEY1 was determined by chromatin immunoprecipitation, dual luciferase, and electrophoretic mobility shift assays. The dimerization pattern of HEY1 was determined by coimmunoprecipitation. The angiogenic capabilities of endothelial cells were assessed by CCK-8 (cell counting kit-8), 5-ethynyl-2-deoxyuridine staining, wound healing, transwell, and sprouting assays. Embryonic and postnatal vascular growth in mouse tissues, matrigel plug assay, cutaneous wound healing model, oxygen-induced retinopathy model, and tumor angiogenesis model were used to investigate the angiogenesis in vivo. We identified intrinsic endothelial HEY1 SUMOylation at conserved lysines by TRIM28 (tripartite motif containing 28) as the unique E3 ligase. Functionally, SUMOylation facilitated HEY1-mediated suppression of angiogenic RTK (receptor tyrosine kinase) signaling and angiogenesis in primary human endothelial cells and mice with endothelial cell-specific expression of wild-type HEY1 or a SUMOylation-deficient HEY1 mutant. Mechanistically, SUMOylation facilitates HEY1 homodimer formation, which in turn preserves HEY1's DNA-binding capability via recognition of E-box promoter elements. Therefore, SUMOylation maintains HEY1's function as a repressive TF controlling numerous angiogenic genes, including RTKs and Notch pathway components. Proangiogenic stimuli induce HEY1 deSUMOylation, leading to heterodimerization of HEY1 with HES (hairy and enhancer of split)-1, which results in ineffective DNA binding and loss of HEY1's angiogenesis-suppressive activity. Our findings demonstrate that reversible HEY1 SUMOylation is a molecular mechanism that coordinates endothelial angiogenic signaling and angiogenesis, both in physiological and pathological milieus, by fine-tuning the transcriptional activity of HEY1. Specifically, SUMOylation facilitates the formation of the HEY1 transcriptional complex and enhances its DNA-binding capability in endothelial cells.

MiR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

Metadherin orchestrates PKA and PKM2 to activate β-catenin signaling in podocytes during proteinuric chronic kidney disease

Podocyte damage is the major cause of glomerular injury and proteinuria in multiple chronic kidney diseases. Metadherin (MTDH) is involved in podocyte apoptosis and promotes renal tubular injury in mouse models of diabetic nephropathy and renal fibrosis; however, its role in podocyte injury and proteinuria needs further exploration. Here, we show that MTDH was induced in the glomerular podocytes of patients with proteinuric chronic kidney disease and correlated with proteinuria. Podocyte-specific knockout of MTDH in mice reversed proteinuria, attenuated podocyte injury, and prevented glomerulosclerosis after advanced oxidation protein products challenge or adriamycin injury. Furthermore, specific knockout of MTDH in podocytes repressed β-catenin phosphorylation at the Ser675 site and inhibited its downstream target gene transcription. Mechanistically, on the one hand, MTDH increased cAMP and then activated protein kinase A (PKA) to induce β-catenin phosphorylation at the Ser675 site, facilitating the nuclear translocation of MTDH and β-catenin; on the other hand, MTDH induced the deaggregation of pyruvate kinase M2 (PKM2) tetramers and promoted PKM2 monomers to enter the nucleus. This cascade of events leads to the formation of the MTDH/PKM2/β-catenin/CBP/TCF4 transcription complex, thus triggering TCF4-dependent gene transcription. Inhibition of PKA activity by H-89 or blockade of PKM2 deaggregation by TEPP-46 abolished this cascade of events and disrupted transcription complex formation. These results suggest that MTDH induces podocyte injury and proteinuria by assembling the β-catenin-mediated transcription complex by regulating PKA and PKM2 function.

Gut Microbiota Regulates Epigenetic Remodelling in the Amygdala: A Role in Repeated Mild Traumatic Brain Injury (rMTBI)-Induced Anxiety.

Gut microbiota serves in the development and maintenance of phenotype. However, the underlying mechanisms are still in its infancy. The current study shows epigenetic remodelling in the brain as a causal mechanism in the gut microbiota-brain axis. Like in trauma patients, gut dysbiosis and anxiety were comorbid in adult male Wistar rats subjected to repeated mild traumatic brain injuries (rMTBI). rMTBI caused epigenetic dysregulation of brain-derived neurotrophic factor (Bdnf) expression in the amygdala, owing to the formation of transcriptional co-repressor complex due to dynamic interaction between histone deacetylase and DNA methylation modification at the Bdnf gene promoter. The probiosis after faecal microbiota transplantation (FMT) from healthy naïve rats or by administration of single strain probiotic (SSP), Lactobacillus rhamnosus GG (LGG), recuperated rMTBI-induced anxiety. Concurrently, LGG infusion or naïve FMT also dislodged rMTBI-induced co-repressor complex resulting in the normalization of Bdnf expression and neuronal plasticity as measured by Golgi-Cox staining. Furthermore, sodium butyrate, a short-chain fatty acid, produced neurobehavioural effects similar to naïve FMT or LGG administration. Interestingly, the gut microbiota from rMTBI-exposed rats per se was able to provoke anxiety in naïve rats in parallel with BDNF deficits. Therefore, gut microbiota seems to be causally linked with the chromatin remodelling necessary for neuroadaptations via neuronal plasticity which drives experience-dependent behavioural manifestations.

Dimerization choice and alternative functions of ZBTB transcription factors.

Zinc Finger DNA-binding domain-containing proteins are the most populous family among eukaryotic transcription factors. Among these, members of the BTB domain-containing ZBTB sub-family are mostly known for their transcriptional repressive functions. In this Viewpoint article, we explore molecular mechanisms that potentially diversify the function of ZBTB proteins based on their homo and heterodimerization, alternative splicing and post-translational modifications. We describe how the BTB domain is as much a scaffold for the assembly of co-repressors, as a domain that regulates protein stability. We highlight another mechanism that regulates ZBTB protein stability: phosphorylation in the zinc finger domain. We explore the non-transcriptional, structural roles of ZBTB proteins and highlight novel findings that describe the ability of ZBTB proteins to associate with poly adenosine ribose in the nucleus during the DNA damage response. Herein, we discuss the contribution of BTB domain scaffolds to the formation of transcriptional repressive complexes, to chromosome compartmentalization and their non-transcriptional, purely structural functions in the nucleus.

Bergamottin Inhibits PRRSV Replication by Blocking Viral Non-Structural Proteins Expression and Viral RNA Synthesis.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes economic losses in the swine industry worldwide. However, current vaccines cannot provide effective protection against PRRSV, and PRRSV-specific treatments for infected herds are still unavailable. In this study, we found that bergamottin showed strong inhibitory effects against PRRSV replication. Bergamottin inhibited PRRSV at the stage of the replication cycle. Mechanically, bergamottin promoted the activation of IRF3 and NF-κB signaling, leading to the increased expression of proinflammatory cytokines and interferon, which inhibited viral replication to some extent. In addition, bergamottion could reduce the expression of the non-structural proteins (Nsps), leading to the interruption of replication and transcription complex (RTC) formation and viral dsRNA synthesis, ultimately restraining PRRSV replication. Our study identified that bergamottin possesses potential value as an antiviral agent against PRRSV in vitro.

RNA activation in ticks

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3′- untranslated region (3’-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3ʹ-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

Ligustrazine alleviates pulmonary arterial hypertension in rats by promoting the formation of myocardin transcription complex in the nucleus of pulmonary artery smooth muscle cells.

Pulmonary arterial hypertension (PAH) is a pathophysiological state of abnormally elevated pulmonary arterial pressure caused by drugs, inflammation, toxins, viruses, hypoxia, and other risk factors. We studied the therapeutic effect and target of tetramethylpyrazine (tetramethylpyrazine [TMP]; ligustrazine) in the treatment of PAH and we speculated that dramatic changes in myocardin levels can significantly affect the progression of PAH. In vivo, the results showed that administration of TMP significantly prolonged the survival of PAH rats by reducing the proliferative lesions, right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), and the Fulton index in the heart and lung of PAH rats. In vitro, TMP can regulate the levels of smooth muscle protein 22-alpha (SM22-α), and myocardin as well as intracellular cytokines such as NO, transforming growth factor beta (TGF-β), and connective tissue growth factor (CTGF) in a dose-dependent manner (25, 50, or 100 μM). Transfection of myocardin small interfering RNA (siRNA) aggravated the proliferation of pulmonary artery smooth muscle cells (PSMCs), and the regulatory effect of TMP on α-smooth muscle actin (α-SMA) and osteopontin (OPN) disappeared. The application of 10 nM estrogen receptor alpha (ERα) inhibitor MPP promoted the proliferation of PSMCs, but it does not affect the inhibition of TMP on PSMCs proliferation. Finally, we found that TMP promoted the nucleation of myocardin-related transcription factor-A (MRTF-A) and combined it with myocardin. In conclusion, TMP can inhibit the transformation of PSMCs from the contractile phenotype to the proliferative phenotype by promoting the formation of the nuclear (MRTF-A/myocardin) transcription complex to treat PAH.

Identification of mechanism of cancer-cell-specific reactivation of hTERT offers therapeutic opportunities for blocking telomerase specifically in human colorectal cancer.

Transcriptional reactivation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter present in 19% of cancers are recognized as key drivers of hTERT reactivation, mechanisms by which wildtype hTERT (WT-hTERT) promoter is reactivated, in majority of human cancers, remain unknown. Using primary colorectal cancers (CRC) we identified Tert INTeracting region 2 (T-INT2), the critical chromatin region essential for reactivating WT-hTERT promoter in CRCs. Elevated β-catenin and JunD level in CRC facilitates chromatin interaction between hTERT promoter and T-INT2 that is necessary to turn on hTERTexpression. Pharmacological screens uncovered salinomycin, which inhibits JunD mediated hTERT-T-INT2 interaction that is required for the formation of a stable transcription complex on the hTERT promoter. Our results showed for the first time how known CRC alterations, such as APC, lead to WT-hTERT promoter reactivation during stepwise-tumorigenesis and provide a new perspective for developing cancer-specific drugs.

Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL.

Cereblon (CRBN), a substrate receptor of the E3 ubiquitin ligase complex CRL4CRBN, is the target of the small molecules lenalidomide and avadomide. Upon binding of the drugs, Aiolos and Ikaros are recruited to the E3 ligase, ubiquitylated, and subsequently degraded. In diffuse large B-cell lymphoma (DLBCL) cells, Aiolos and Ikaros are direct transcriptional repressors of interferon-stimulated genes (ISG) and degradation of these substrates results in increased ISG protein levels resulting in decreased proliferation and apoptosis. Herein, we aimed to uncover the mechanism(s) Aiolos and Ikaros use to repress ISG transcription and provide a mechanistic rationale for a combination strategy to enhance cell autonomous activities of CRBN modulators (CELMoD). We conducted paired RNA sequencing with histone modification and Aiolos/Ikaros chromatin immunoprecipitation sequencing to identify genes regulated by these transcription factors and to elucidate correlations to drug sensitivity. We confirmed Aiolos/Ikaros mediated transcriptional complex formation in DLBCL patient samples including those treated with avadomide. In DLBCL, the repression of ISG transcription is accomplished in part through recruitment of large transcriptional complexes such as the nucleosome remodeling and deacetylase, which modify the chromatin landscape of these promoters. A rational combination approach of avadomide with a specific histone deacetylase inhibitor leads to a significant increase in ISG transcription compared with either single agent, and synergistic antiproliferative activity in DLBCL cell lines. Our results provide a novel role for lineage factors Aiolos and Ikaros in DLBCL as well as further insight into the mechanism(s) of Aiolos and Ikaros-mediated transcriptional repression and unique therapeutic combination strategies.

YB-1 is a positive regulator of KLF5 transcription factor in basal-like breast cancer.

Y-box binding protein 1 (YB-1) is a well-known oncogene highly expressed in various cancers, including basal-like breast cancer (BLBC). Beyond its role as a transcription factor, YB-1 is newly defined as an epigenetic regulator involving RNA 5-methylcytosine. However, its specific targets and pro-cancer functions are poorly defined. Here, based on clinical database, we demonstrate a positive correlation between Kruppel-like factor 5 (KLF5) and YB-1 expression in breast cancer patients, but a negative correlation with that of Dachshund homolog 1 (DACH1). Mechanistically, YB-1 enhances KLF5 expression not only through transcriptional activation that can be inhibited by DACH1, but also by stabilizing KLF5 mRNA in a RNA 5-methylcytosine modification-dependent manner. Additionally, ribosomal S6 kinase 2 (RSK2) mediated YB-1 phosphorylation at Ser102 promotes YB-1/KLF5 transcriptional complex formation, which co-regulates the expression of BLBC specific genes, Keratin 16 (KRT16) and lymphocyte antigen 6 family member D (Ly6D), to promote cancer cell proliferation. The RSK inhibitor, LJH685, suppressed BLBC cell tumourigenesis in vivo by disturbing YB-1-KLF5 axis. Our data suggest that YB-1 positively regulates KLF5 at multiple levels to promote BLBC progression. The novel RSK2-YB-1-KLF5-KRT16/Ly6D axis provides candidate diagnostic markers and therapeutic targets for BLBC.

LSM12 facilitates the progression of colorectal cancer by activating the WNT/CTNNB1 signaling pathway.

Aberrant activation of the WNT signaling pathway is a joint event in colorectal cancer (CRC), but the molecular mechanism is still unclear. Recently, RNA-splicing factor LSM12 (like-Sm protein 12) is highly expressed in CRC tissues. This study aimed to verify whether LSM12 is involved in regulating CRC progression via regulating the WNT signaling pathway. Here, we found that LSM12 is highly expressed in CRC patient-derived tissues and cells. LSM12 is involved in the proliferation, invasion, and apoptosis of CRC cells, similar to the function of WNT signaling in CRC. Furthermore, protein interaction simulation and biochemical experiments proved that LSM12 directly binds to CTNNB1 (also known as β-Catenin) and regulates its protein stability to affect the CTTNB1-LEF1-TCF1 transcriptional complex formation and the associated WNT downstream signaling pathway. LSM12 depletion in CRC cells decreased the in vivo tumor growth through repression of cancer cell growth and acceleration of cancer cell apoptosis. Taken together, we suggest that the high expression of LSM12 is a novel factor leading to aberrant WNT signaling activation, and that strategies targeting this molecular mechanism may contribute to developing a new therapeutic method for CRC.

Lactate secreted by PKM2 upregulation promotes Galectin-9-mediated immunosuppression via inhibiting NF-\u03baB pathway in HNSCC

Pyruvate kinase M2 as a key rate-limiting enzyme in glycolysis, it plays a critical role in metabolic reprogramming and carcinogenesis. However, whether PKM2 can promote immunosuppressive microenvironment formation remains unknown in head and neck squamous cell carcinoma (HNSCC). PKM2 expression was detected using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of Galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. The mechanism of lactate regulating Galectin-9 expression through NF-κB signaling was explored in vitro. Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. The glycolysis and glycolytic capacity are significantly decreased after PKM2 silencing. Lactate secretion induced by PKM2 significantly promotes migration and invasion capacity. Furthermore, a positive correlation between PKM2 and Galectin-9 expression is observed in HNSCC tissues. The induction of Galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor. Mechanically, lactate impeded the suppressive transcriptional complex formation of NF-κB and histone deacetylase 3 (HDAC3), which released the transcription of Galectin-9 mediated by NF-κB signaling. Our findings demonstrate that lactate produced by PKM2 upregulation promotes tumor progression and Galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-Galectin-9 axis might be a potential therapeutic target in HNSCC.

A novel transcriptional complex on the VE-cadherin promoter regulated the downregulation of VE-cadherin in the Down Syndrome Candidate Region 1 isoform 1L-mediated angiogenesis

Angiogenesis is critical for many diseases. Previously, we reported that Down Syndrome Candidate Region 1 isoform 1L (DSCR1-1L) was one of the most up-regulated genes in endothelial cells induced by VEGF and histamine, and regulated endothelial cell proliferation and Matrigel angiogenesis in mice. However, it was not known whether DSCR1-1L regulated angiogenesis in vivo and what was the molecular mechanism underlying it. In this study, gene knockdown and overexpression models were established to study the role of DSCR1-1L in angiogenesis in vivo. Further, the downstream regulatory target of DSCR1-1L was explored with molecular biological methods in vascular endothelial cells. We found that DSCR1-1L shRNAs significantly inhibited angiogenesis induced by VEGF in mice (p < 0.0001). In the gain-of-function assay, overexpression of DSCR1-1L cDNA in mouse endothelium of EC-FH-DSCR1-1L transgenic mice was sufficient to induce angiogenesis significantly (p < 0.01). DSCR1-1L regulated angiogenesis in the early stage by down-regulation of the VE-cadherin expression through targeting its transcription, but not mRNA stability. Three DSCR1-1L-targeted DNA elements in the VE-cadherin promoter were identified by promoter reporter assays, among which, a novel specific transcriptional complex was found. The DNA sequence (CTTCTG) in the VE-cadherin promoter was identified to directly interact with proteins by Electrophoresis Mobility Shift Assays and DNase I footprint assay. Hence, DSCR1-1L is an excellent therapeutic target for angiogenic diseases through down-regulating the formation of a novel transcriptional complex on the VE-cadherin promoter. DSCR1-1L shRNAs and cDNA have the potential to be developed for clinical application. Our results also contribute significantly to the field of mechanistic studies.

Development of Small Molecule Inhibitors Targeting PBX1 Transcription Signaling as a Novel Cancer Therapeutic Strategy

PBX1 (pre-B cell leukemia transcription factor 1) is a transcription factor involved in diverse cellular functions including organ development, stem cell renewal, and tumorigenesis. PBX1 is localized at chr1q23.3, a frequently amplified chromosomal region, and it is overexpressed in many human malignancies including breast, lung, melanoma, and ovarian carcinomas. Cancer cells with elevated PBX1 signaling are particularly vulnerable to PBX1-withdrawal. We designed a series of small molecule compounds capable of docking to the interface between PBX1 and its cognate DNA target sequence and identified a lead compound, T417, which efficiently hindered the formation of the PBX1 transcriptional complex and affected the transcription of PBX1 target genes. In cell-based assays, T417 significantly suppressed long-term self-renewal and proliferation of cancer cells expressing high levels of PBX1 but not of those expressing low levels of PBX1. T417 also re-sensitized platinum-resistant ovarian tumor cells to carboplatin and produced synergistic anti-tumorigenic effects in vivo in combination with carboplatin. Normal tissues were spared, likely due to the lower PBX1 expression levels. Since PBX1 functions as a molecular hub in developing cancer recurrence and treatment resistance, our data highlight the potential of targeting the PBX-DNA interface as a therapeutic strategy for patients whose tumors rely on PBX1 activation for survival.