Nuclear Kinase Research Articles

NUCKS1 promotes breast cancer cell proliferation and metastasis via PI3K/ AKT pathway

Purpose: To investigate the role of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in breast cancer. 
 Methods: Breast cancer cells were maintained in RPMI-1640 medium containing 10 % fetal bovine serum in a incubator at 37 °C. Cell proliferation was determined by CCK8 and colony formation assays. Flow cytometry and Transwell assays were used to determine cell cycle and metastasis, respectively. 
 Results: Expression of NUCKS1 was significantly elevated in breast cancer (p < 0.01). Overexpression of NUCKS1 significantly increased cell viability (p < 0.01), and promoted proliferation of breast cancer cells. Knockdown of NUCKS1 inhibited cell proliferation, and induced cell cycle arrest at G1 phase. However, overexpression of NUCKS1 promoted cell cycle progression via down-regulation of p21 and up-regulation of cyclin D1 and CDK1. Cell migration and invasion were induced by overexpression of NUCKS1, and suppressed by silencing of NUCKS1. Overexpression of NUCKS1 enhanced p-AKT and p-PI3K expression, while knockdown of NUCKS1 reduced the expression of p-AKT and p-PI3K in breast cancer cells. 
 Conclusion: NUCKS1 promotes breast cancer cell proliferation and metastasis via activation of PI3K/AKT signaling. The silencing of NUCKS1 can be used as a strategy to develop therapies for the management of breast cancer.

Huoxiang Zhengqi alleviates azoxymethane/dextran sulfate sodium-induced colitis-associated cancer by regulating Nrf2/NF-κB/NLRP3 signaling.

Colitis-associated cancer (CAC) is a subtype of inflammatory bowel disease (IBD)-associated colorectal cancer. Huoxiang Zhengqi (HXZQ) is a classical Chinese herbal medicine and has been used to treat intestinal disorders, however, anti-CAC effects and underlying mechanisms of HXZQ have not been reported. An azoxymethane/dextran sulfate sodium-induced CAC mice model was used to investigate the anti-CAC effect of HXZQ. HXZQ significantly reduced colonic inflammation, suppressed the size and number of tumors, and reduced the levels of pro-inflammatory cytokines (interleukin [IL]-1α, IL-1β, IL-6, IL-17A, IL-21, IL-23, granulocyte macrophage-colony stimulating factor, and tumor necrosis factor-α) and oxidative stress markers (reactive oxygen species and malondialdehyde), and increased the levels of anti-inflammatory cytokines (IL-10 and IL-27) in CAC mice. Intestinal microbiota and serum metabolomics analyses indicated that HXZQ altered the gut microbial composition and the abundance of 29 serum metabolites in CAC mice. Additionally, HXZQ activated the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signaling pathway and increased the levels of antioxidants such as catalase (CAT), heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductases-1 (NQO-1), and superoxide dismutase-1 (SOD-1). HXZQ inhibited the activation of the nuclear factor kappa-B (NF-κB) signaling pathway and decreased the expression of NLR family pyrin domain containing 3 (NLRP3) by inhibiting the phosphorylation of inhibitor of nuclear factor kappa-B (IκB), inhibitor of nuclear factor kappa-B kinase (IKK), and NF-κB. In conclusion, HXZQ alleviated CAC in mice by modulating the intestinal microbiota and metabolism, activating Nrf2-mediated antioxidant response, and inhibiting NF-κB-mediated NLRP3 inflammasome activation against inflammation. The present data provide a reference for the use of HXZQ as a therapeutic or combination agent for clinical CAC treatment.

VRK1 as a synthetic lethal target in VRK2 promoter-methylated cancers of the nervous system.

Collateral lethality occurs when loss of a gene/protein renders cancer cells dependent on its remaining paralog. Combining genome-scale CRISPR/Cas9 loss-of-function screens with RNA sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase vaccinia-related kinase 1 (VRK1) for their survival in vivo. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout sensitized cells to VRK1 loss, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 loss. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced barrier-to-autointegration factor phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas.

Cryptotanshinone inhibits ovarian tumor growth and metastasis by degrading c-Myc and attenuating the FAK signaling pathway.

Cryptotanshinone (CT), a natural compound derived from Salvia miltiorrhiza Bunge that is also known as the traditional Chinese medicine Danshen, exhibits antitumor activity in various cancers. However, it remains unclear whether CT has a potential therapeutic benefit against ovarian cancers. The aim of this study was to test the efficacy of CT in ovarian cancer cells in vitro and using a xenograft model in NSG mice orthotopically implanted with HEY A8 human ovarian cancer cells and to explore the molecular mechanism(s) underlying CT’s antitumor effects. We found that CT inhibited the proliferation, migration, and invasion of OVCAR3 and HEY A8 cells, while sensitizing the cell responses to the chemotherapy drugs paclitaxel and cisplatin. CT also suppressed ovarian tumor growth and metastasis in immunocompromised mice orthotopically inoculated with HEY A8 cells. Mechanistically, CT degraded the protein encoded by the oncogene c-Myc by promoting its ubiquitination and disrupting the interaction with its partner protein Max. CT also attenuated signaling via the nuclear focal adhesion kinase (FAK) pathway and degraded FAK protein in both cell lines. Knockdown of c-Myc using lentiviral CRISPR/Cas9 nickase resulted in reduction of FAK expression, which phenocopies the effects of CT and the c-Myc/Max inhibitor 10058-F4. Taken together, our studies demonstrate that CT inhibits primary ovarian tumor growth and metastasis by degrading c-Myc and FAK and attenuating the FAK signaling pathway.

MiR-92b-5p negatively regulates IKK through targeting its ORF region in the innate immune responses of amphioxus (Branchiostoma belcheri)

Cephalochordate (Amphioxus), situated at a key phylogenetic position in the phylum Chordata, serves as a model organism for studying the origin and evolution of the vertebrate innate immune. In this study, five members of precursor miR-92 family (miR-92a-1, miR-92a-2, miR-92b, miR-92c and miR-92d) are identified in Branchiostoma belcheri, and their evolutionary conservation and potential molecular functions in innate immunity are analyzed. Among them, miR-92b-5p was validated in HEK293 cells to target the coding region but not classic 3′UTR of IKK (inhibitor of nuclear factor kappa-B kinase) mRNA, one integral component of MAPK and TLR4 immune signaling. Furthermore, the spatiotemporal expression patterns of miR-92b-5p and IKK were examined in different tissues or different time points (2 h, 4 h, 8 h, 12 h, 24 h and 48 h) post LPS stimulation at RNA and protein level in vivo. The seemingly inverse expression pattern between miR-92b-5p and IKK supports the involvement of miR-92b-5p in Branchiostoma belcheri innate immune response. In conclusion, our work not only illustrates the evolutionary pattern of Branchiostoma belcheri miR-92 family across chordates, but also reveals that miR-92b-5p could target IKK expression to regulate innate immune response.

Antioxidant and Anti-Inflammatory Actions of Polyphenols from Red and White Grape Pomace in Ischemic Heart Diseases.

Grape pomace (GP) represents a very reliable source of polyphenols because it could be found globally as a remnant of the wine industry. During the winemaking process, two types of GP are generated: red GP and white GP, according to the produced wine, red or white. Grape pomace represents a viable source of polyphenols, mainly flavanols, procyanidins anthocyanins, and resveratrol which possess antioxidant and anti-inflammatory activities. Multiple differences were observed between red and white GP in terms of their antioxidant and anti-inflammatory activity in both in vitro and in vivo studies. Although most studies are focused on the antioxidant and anti-inflammatory effect of red grape pomace, there are still many variables that need to be taken into consideration, as well as extensive study of the white GP. It was observed that in both in vitro and in vivo studies, the GP polyphenols have a direct antioxidant activity by acting as a free radical scavenger or donating a hydrogen atom. It also possesses an indirect antioxidant and anti-inflammatory activity by reducing mitochondrial reactive oxygen species (ROS) generation, malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κβ), and inhibitor of nuclear factor kappa-B kinase subunit beta (Iκκβ) levels or nitrate oxide-4 (NOX4) expression and by increasing the levels of antioxidants enzymes like superoxide dismutase (SOD), catalase (CAT) glutathione reductase (GRx) and glutathione peroxidase(GPx). Besides these activities, many beneficial effects in ischemic heart diseases were also observed, such as the maintenance of the ventricular function as close as possible to normal, and the prevention of infarcted area extension. In this context, this review intends to present the actual knowledge of grape pomace’s potential antioxidant and anti-inflammatory activity in ischemic heart disease, knowledge gathered from existing in vitro and in vivo studies focused on this.

Regulation of Atherosclerosis by Toll-Like Receptor 4 Induced by Serum Amyloid 1: A Systematic In Vitro Study.

The objective of this study was to investigate the effects of serum amyloid 1 (SAA1) on activation of endothelial cells, formation of foam cells, platelet aggregation, and monocyte/platelet adhesion to endothelial cells. The effect of SAA1 on the inflammatory activation of endothelial cells was investigated by detecting the expression of inflammatory factors and adhesion molecules. The role of SAA1 in formation of foam cells was verified by detecting lipid deposition and expression of molecules related to the formation of foam cells. After platelets were stimulated by SAA1, the aggregation rate was evaluated to determine the effect of SAA1 on platelet aggregation. Monocytes/platelets were cocultured with human umbilical vein endothelial cells (HUVECs) pretreated with or without SAA1 to determine whether SAA1 affected monocyte/platelet adhesion to endothelial cells. By inhibiting toll-like receptor 4 (TLR4) function, we further identified the role of TLR4 signaling in SAA1-mediated endothelial inflammatory activation, foam-cell formation, and monocyte/platelet adhesion to HUVECs. SAA1 significantly increased the expression of adhesion molecules and inflammatory factors in HUVECs. Moreover, SAA1 also promoted lipid deposition and the expression of inflammatory factors and low-density lipoprotein receptor-1 (LOX-1) in THP-1-derived macrophages. In addition, SAA1 induced platelet aggregation and enhanced monocyte/platelet adhesion to HUVECs. However, the TLR4 antagonist significantly inhibited SAA1-induced endothelial cell activation, foam-cell formation, and monocyte/platelet adhesion to HUVECs and downregulated the expression of myeloid differentiation factor 88 (MyD88), phosphor-inhibitor of nuclear factor κB kinase subunit α/β (P-IKKα/β), phospho-inhibitor of nuclear factor κB subunit α (P-IKBα), and phosphorylation of nuclear transcription factor-κB p65 (P-p65) in SAA1-induced HUVECs and THP-1 cells. Conclusively, it is speculated that SAA1 promotes atherosclerosis through enhancing endothelial cell activation, platelet aggregation, foam-cell formation, and monocyte/platelet adhesion to endothelial cells. These biological functions of SAA1 may depend on the activation of TLR4-related nuclear factor-kappa B (NF-κB) signaling pathway.

HM-Chromanone, a Major Homoisoflavonoid in Portulaca oleracea L., Improves Palmitate-Induced Insulin Resistance by Regulating Phosphorylation of IRS-1 Residues in L6 Skeletal Muscle Cells.

This study investigated the effect of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) on palmitate-induced insulin resistance and elucidated the underlying mechanism in L6 skeletal muscle cells. Glucose uptake was markedly decreased due to palmitate-induced insulin resistance in these cells; however, 10, 25, and 50 µM HM-chromanone remarkably improved glucose uptake in a concentration-dependent manner. HM-chromanone treatment downregulated protein tyrosine phosphatase 1B (PTP1B) and phosphorylation of c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), which increased because of palmitate mediating the insulin-resistance status in cells. HM-chromanone promoted insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and suppressed palmitate-induced phosphorylation of IRS-1 serine. This activated phosphoinositide 3-kinase (PI3K) and stimulated protein kinase B (AKT) phosphorylation. Phosphorylated AKT promoted the translocation of Glucose transporter type 4 to the plasma membrane and significantly enhanced glucose uptake into muscle cells. Additionally, HM-chromanone increased glycogen synthesis through phosphorylating glycogen synthase kinase 3 alpha/beta (GSK3 α/β) via AKT. Consequently, HM-chromanone may improve insulin resistance by downregulating the phosphorylation of IRS-1 serine through inhibition of negative regulators of insulin signaling and inflammation-activated protein kinases in L6 skeletal muscle cells.

PAD4-dependent citrullination of nuclear translocation of GSK3β promotes colorectal cancer progression via the degradation of nuclear CDKN1A

Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3β (GSK3β) in CRC cells, and GSK3β Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3β in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3β from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3β/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC.

A Haspin promoter element induces tissue-specific methylation of a transcription region and the regulation of gene expression in mouse ova.

HASPIN is a nuclear serine-threonine kinase originally identified in the mouse testis. Its 193 bp DNA promoter element (hereafter, 193PE) regulates bidirectional, synchronous gene expression in the germ cells of male mice. Recent studies have shown that Haspin is also expressed in trace amounts in somatic cells; HASPIN also functions in oocytes. Haspin expression is regulated by the tissue-specific methylation of Haspin genomic DNA regions, including somatic cells. This study investigated relationship between 193PE and DNA methylation by examining methylation status of transgenic mice carrying 193PE and a reporter gene. In somatic (liver) cells carrying the reporter gene, 193PE induced methylation as well as trace expression of the reporter gene. In the testis, 193PE induced hypomethylation and intense reporter gene expression. Expression of HASPIN in an egg was assessed using human chorionic gonadotrophin to induce ovulation in female transgenic mice. The results showed that 193PE induced tissue-specific methylation, which resulted in reporter gene expression in a mouse egg.

Enhancing SIRT1 Gene Expression Using Small Activating RNAs: A Novel Approach for Reversing Metabolic Syndrome.

Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.

Abstract 1570: Macrophage mediated cell-kill evasion by human prostate cancer cells

Abstract Macrophages can adopt a cytotoxic M1 phenotype which can eliminate cancer cells. However, despite the presence of macrophages in the prostate, prostate cancer (PCa) is a prevalent cancer in men. This suggests that PCa cells can evade M1 macrophage mediated cell-kill. We have shown that human THP-1 derived M1-like macrophages readily kill human prostate cancer LNCap cells, while in testosterone proficient conditions (Fraction Growth over Mono-culture (FGM): 0.1). However, in testosterone deprived conditions LNCap cells survive (FGM: 1.1). In addition, when exposed to M1-like macrophage conditioned medium, LNCap cells are killed in testosterone proficient conditions and not in depleted conditions (FGM: 0.2 and 0.7, respectively). This suggests that M1 macrophage mediated PCa cell-kill depends on Androgen Receptor (AR) signaling and the release of soluble factors by macrophages. This is supported by cell-kill of castration-resistant LNCap-abl cells with an AR amplification (FGM: 0.1) in testosterone deprived conditions and no cell-kill in both testosterone deprived and proficient conditions of castration resistant PC3 cells without AR expression when co-cultured with M1-like macrophages (FGM: 0.7 and 0.7. respectively). To dissect the mechanism of M1 macrophage mediated cell-kill evasion, a genome wide CRISPR knock-out screen was performed in LNCap cells in co-culture with M1-like macrophages. LNCap cells were transfected with the Brunello library and cultured in testosterone proficient conditions. After 72 hours of co-culture, cells were harvested and DNA was isolated and submitted for sequencing. After quality check analysis, five gene knock-outs with a significant (log2FcMedian >1.5) survival advantage were identified, AR, PRKCD (Protein Kinase C Delta), IKBKA (Inhibitor of nuclear factor kappa-B kinase subunit α:), IKBKB (Inhibitor of nuclear factor kappa-B kinase subunit β) and IKBKG (Inhibitor of nuclear factor kappa-B kinase subunit γ). The latter three genes are components of the IkB kinase (IKK) complex, which is essential for activation of members of the nuclear factor-kB (NFkB) family of transcription factors. The hits as identified in the CRISPR knock-out screen, were validated in single guide knock-out LNCap cells in co-culture with M1-like macrophages. Additionally, we have shown that AR signaling enhances PRKCD expression, both at the RNA and protein level. Moreover, expression of PRKCD is significantly lower in prostatectomy specimen of 56 patients treated for 3 months with the AR signalling inhibitor enzalutamide prior to surgery, than untreated matched controls. Furthermore, LNCap cells lacking PRKCD, IKBKB and IKBKG were significantly less affected in their growth by reactive oxygen species (H2O2) than non-targeting LNCap cells. Ongoing studies are aimed to dissect how these critical factors are interconnected in LNCap cells, M1 macrophage mediated cell-kill evasion as a result. Citation Format: Anniek Zaalberg, Emma Minnee, Wilbert Zwart, Andries M. Bergman. Macrophage mediated cell-kill evasion by human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1570.

Abstract 823: ABL1 kinase regulates leukemia phenotype and response to therapy

Abstract The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase. Cytoplasmic ABL1 stimulates, whereas nuclear ABL1 inhibits cell growth. Analysis of the CGAP Mitelman database detected loss of ABL1 allele in hematologic malignancies. Among the top genetic aberrations detected in acute myeloid leukemias were t(8;21) generating fusion protein AML1-ETO and NUP98 translocations, e.g., t(1;11) encoding NUP98-PMX1.Expression of AML1-ETO and NUP98-PMX1 in Lin-cKit+ stem cell enriched bone marrow cells from Abl1-/-;Vav-Cre and Abl1+/+;Vav-Cre mice revealed that oncogene-expressing Abl1-/- cells gained tremendous proliferation advantage. Conversely, overexpression of ABL1 and DPH (ABL1 kinase agonist)-mediated activation of ABL1 kinase resulted in reduced proliferation of AML1-ETO and NUP98-PMX1 cells. Since the role of ABL1 in DNA damage response (DDR) is well established, we applied the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi) to identify potential therapeutic vulnerabilities. AML1-ETO;Abl1-/- cells were more sensitive to all three inhibitors when compared to AML1-ETO;Abl1+/+ cells. Remarkably, NUP98-PMX1;Abl1-/- cells were exceptionally sensitive to ATRi, but not to ATMi and DNA-PKi when compared to NUP98-PMX1;Abl+/+ cells. ABL1 did not affect the sensitivity of transformed cells to DNA damaging agent doxorubicin. To pinpoint mechanisms collaborating with Abl1 deletion in transformation of hematopoietic cells, Abl1 was knockout in 32Dcl3 cells by CRISPR/Cas9 to obtain 32Dcl3-Abl1ko cells. 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells were challenged to induce transformation by starving them from growth factors (IL3). Only 32Dcl3-Abl1ko cell populations generated growth factor-independent cells. This indicates that the absence of Abl1 may facilitate malignant transformation of hematopoietic cells. RNA-seq was performed to compare gene expression in 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells in the presence or absence of IL-3. Pathway analysis of FPKM reads (top 30 significant canonical pathways) and Venn diagram analysis followed by KEGG pathway analysis (291 genes uniquely expressed in growth factor-independent 32Dcl3-Abl1ko cells) implicated PI3K-AKT pathway signaling including mTOR and sirtuins (SITR) in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib, mTOR inhibitor rapamycin and SIRT2 inhibitor thiomyristoyl exerted selective activity against Lin-cKit+ AML1-ETO;Abl1-/- and/or NUP98-PMX1;Abl1-/- cells when compared to Abl1+/+ counterparts. Moreover, inhibition of ABL1 kinase by imatinib in Lin-cKit+ AML1-ETO;Abl1+/+ and/or NUP98-PMX1;Abl+/+ cells induced the sensitivity to PI3K-ATK pathway inhibitors. In conclusion, we showed that normal ABL1 kinase plays a tumor suppressor role and is a potential therapeutic target in hematological malignancies induced by AML1-ETO and NUP98-PMX1. The latter statement is being currently tested in animal models. Citation Format: Konstantin Golovine, Zhaorui Lian, Kumaraswamy Naidu Chitrala, Gleb Ablakov, Margaret Nieborowska-Skorska, Jian Huang, Tomasz Skorski. ABL1 kinase regulates leukemia phenotype and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 823.

Hsa_circ_0001550 facilitates colorectal cancer progression through mediating microRNA-4262/nuclear casein kinase and cyclin-dependent kinase substrate 1 cascade.

BackgroundCircular RNAs (circRNAs) play important roles in various malignancies, such as colorectal cancer (CRC). However, the function of hsa_circ_0001550 in CRC remains to be elucidated.MethodsThe expression levels of hsa_circ_0001550, microRNA (miR)‐4262, and nuclear casein kinase and cyclin‐dependent kinase substrate 1 (NUCKS1) were determined by real‐time qPCR. Cell biological behaviors were evaluated via colony formation assay, transwell assay, flow cytometry, and sphere formation assays. The target relationship was validated via dual‐luciferase reporter and RNA pull‐down assays. Protein expression was analyzed by western blot. Xenograft tumor model was adopted to evaluate hsa_circ_0001550 function in vivo.ResultsHsa_circ_0001550 enrichment was enhanced in CRC tissue specimens and cell lines. Hsa_circ_0001550 absence hindered CRC cell proliferation, metastasis, stemness, and caused apoptosis. Hsa_circ_0001550 targeted miR‐4262, and hsa_circ_0001550 absence‐caused impacts were diminished by anti‐miR‐4262. MiR‐4262 targeted NUCKS1. Hsa_circ_0001550 had positive regulation on NUCKS1 expression. NUCKS1 overexpression overturned the influences of hsa_circ_0001550 silencingon CRC cell progression. Hsa_circ_0001550 interference notably blocked in vivo xenograft tumor growth.ConclusionHsa_circ_0001550 facilitated CRC progression by binding to miR‐4262 to positively regulate NUCKS1 abundance.

MiR-203a-3p, ABL1 and TP63 gene expression is altered in the endometrium of women with endometriosis

Objective Many genes and miRNAs have been shown to be associated with the pathogenesis of endometriosis. TP63 (p63) is implicated in lineage specification, proliferative potential, differentiation, cell death and survival. The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase implicated in cell differentiation, cell division, and cell adhesion. Moreover, hsa-miR-203a-3p was reported to play pivotal roles in tumor progression by targeting multiple genes, including ABL1 and TP63. The aim of this study was to investigate the expression of ABL1, TP63, and miR-203a-3p in endometriosis. Methods This study included 30 women with endometriosis (stage III: n = 12 and stage IV: n = 18) and 30 age-matched controls. Total RNA extraction and cDNA synthesis were performed, and a quantitative polymerase chain reaction technique was used to determine the expression of miR-203a-3p, TP63, and ABL1. Results TP63 and ABL1 were significantly overexpressed in stages III and IV endometriosis as compared to controls (p < .0001). Moreover, overexpression of ABL1 and TP63 was observed in stage IV compared to stage III (p = .0006 and p = .0002, respectively). Furthermore, significant increase miR-203a-3p expression has been seen in stage IV endometriosis compared to controls (p = .006). The expression of miR-203a-3p in stage III was not significant compared to stage IV and control (p = .33 and p = .43, respectively). Conclusion It is concluded that miR-203a-3p, ABL1 and TP63 gene expression is altered in the endometrium of patients with endometriosis. It is also suggested that miR-203a-3p, ABL1, and TP63 might be candidate factors for the pathogenesis of endometriosis and suggesting its therapeutic potential in endometriosis.

Role of IKKε in the Metabolic Diseases: Physiology, Pathophysiology, and Pharmacology.

IKKε (inhibitor of nuclear factor kappa-B kinase ε) is a member of the noncanonical NF-κB pathway. It participates in the inflammatory response and innate immunity against bacteria. In recent decades, IKKε has been closely associated with metabolic regulation. Inhibition of the IKKε pathway can improve fat deposition in the liver, reduce subcutaneous fat inflammation, and improve liver gluconeogenesis in obesity. IKKε is expected to be a new therapeutic target for metabolic diseases such as nonalcoholic fatty liver disease, diabetes, and obesity. Herein, we summarize the structural characterization, physiological function, and pathological role of IKKε in metabolic diseases and small molecule inhibitors of IKKε.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor–induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

Small-molecule IKKβ activation modulator (IKAM) targets MAP3K1 and inhibits pancreatic tumor growth

Activation of inhibitor of nuclear factor NF-κB kinase subunit-β (IKKβ), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKβ knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKβ and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKβ is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKβ in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKβ inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKβ but inhibited the IKKβ kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKβ phosphorylation was inhibited by 39-100, thus we termed it IKKβ activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKβ levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKβ activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

Retraction: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

Retraction: Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

IL-3-Induced Immediate Expression of c-fos and c-jun Is Modulated by the IKK2-JNK Axis.

Interleukin (IL)-3 is a pleiotropic cytokine that regulates the survival, proliferation, and differentiation of hematopoietic cells. The binding of IL-3 to its receptor activates intracellular signaling, inducing transcription of immediate early genes (IEGs) such as c-fos, c-jun, and c-myc; however, transcriptional regulation under IL-3 signaling is not fully understood. This study assessed the role of the inhibitor of nuclear factor-κB kinases (IKKs) in inducing IL-3-mediated expression of IEGs. We show that IKK1 and IKK2 are required for the IL-3-induced immediate expression of c-fos and c-jun in murine hematopoietic Ba/F3 cells. Although IKK2 is well-known for its pivotal role as a regulator of the canonical nuclear factor-κB (NF-κB) pathway, activation of IKKs did not induce the nuclear translocation of the NF-κB transcription factor. We further revealed the important role of IKK2 in the activation of c-Jun N-terminal kinase (JNK), which mediates the IL-3-induced expression of c-fos and c-jun. These findings indicate that the IKK2-JNK axis modulates the IL-3-induced expression of IEGs in a canonical NF-κB-independent manner.