Expression Of DYRK2 Research Articles

CircCOX6A1 suppresses osteogenic differentiation and aggravates osteoporosis via miR-512-3p/DYRK2 axis.

Osteoporosis (OP), characterized by compromised bone integrity and increased fracture risk, poses a significant health challenge. Circular RNAs (circRNAs) have emerged as crucial regulators in various pathophysiological processes, prompting investigation into their role in osteoporosis. This study aimed to elucidate the involvement of circCOX6A1 in OP progression and understand its underlying molecular mechanisms. The primary objective was to explore the impact of circCOX6A1 on bone marrow-derived mesenchymal stem cells (BMSCs) and its potential interactions with miR-512-3p and DYRK2. GSE161361 microarray analysis was employed to assess circCOX6A1 expression in OP patients. We utilized in vitro and in vivo models, including BMSC cultures, osteogenic differentiation assays, and an OVX-induced mouse model of OP. Molecular techniques such as quantitative RT-PCR, western blotting, and functional assays like alizarin red staining (ARS) were employed to evaluate circCOX6A1 effects on BMSC proliferation, apoptosis, and osteogenic differentiation. The interaction between circCOX6A1, miR-512-3p, and DYRK2 was investigated through dual luciferase reporter assays, RNA immunoprecipitation, and RNA pull-down assays. CircCOX6A1 was found to be upregulated in osteoporosis patients, and its expression inversely correlated with osteogenic differentiation of BMSCs. CircCOX6A1 knockdown enhanced osteogenic differentiation, as evidenced by increased mineralized nodule formation and upregulation of osteogenic markers. In vivo, circCOX6A1 knockdown ameliorated osteoporosis progression in OVX mice. Mechanistically, circCOX6A1 acted as a sponge for miR-512-3p, subsequently regulating DYRK2 expression. This study provides compelling evidence for the role of circCOX6A1 in osteoporosis pathogenesis. CircCOX6A1 negatively regulates BMSC osteogenic differentiation through the miR-512-3p/DYRK2 axis, suggesting its potential as a therapeutic target for mitigating OP progression.

Dyrk2 gene transfer suppresses hepatocarcinogenesis by promoting the degradation of Myc and Hras

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and has a poor prognosis. However, the molecular mechanisms underlying hepatocarcinogenesis and progression remain unknown. Invitro gain- and loss-of-function analyses in cell lines and xenografts revealed that dual-specificity tyrosine-regulated kinase 2 (DYRK2) influences tumour growth in HCC. To investigate the role of Dyrk2 during hepatocarcinogenesis, we developed liver-specific Dyrk2 conditional knockout mice and an invivo gene delivery system with a hydrodynamic tail vein injection and the Sleeping Beauty transposon. The antitumour effects of Dyrk2 gene transfer were investigated in a murine autologous carcinogenesis model. Dyrk2 expression was reduced in tumours, and that its downregulation was induced before hepatocarcinogenesis. Dyrk2 gene transfer significantly suppressed carcinogenesis. It also suppresses Myc-induced de-differentiation and metabolic reprogramming, which favours proliferative, and malignant potential by altering gene profiles. Dyrk2 overexpression caused Myc and Hras degradation at the protein level rather than at the mRNA level, and this degradation mechanism was regulated by the proteasome. Immunohistochemical analyses revealed a negative correlation between DYRK2 expression and MYC and longer survival in patients with HCC with high-DYRK2 and low-MYC expressions. Dyrk2 protects the liver from carcinogenesis by promoting Myc and Hras degradation. Our findings would pave the way for a novel therapeutic approach using DYRK2 gene transfer. Hepatocellular carcinoma (HCC) is one of the most common cancers, with a poor prognosis. Hence, identifying molecules that can become promising targets for therapies is essential to improve mortality. No studies have clarified the association between DYRK2 and carcinogenesis, although DYRK2 is involved in tumour growth in various cancer cells. This is the first study to show that Dyrk2 expression decreases during hepatocarcinogenesis and that Dyrk2 gene transfer is an attractive approach with tumour suppressive activity against HCC by suppressing Myc-mediated de-differentiation and metabolic reprogramming that favours proliferative and malignant potential via Myc and Hras degradation.

A pan-cancer analysis of the oncogenic role of dual-specificity tyrosine (Y)-phosphorylation- regulated kinase 2 (DYRK2) in human tumors

Although there have been studies correlating DYRK2 with a number of human cancers, there has been no pan-cancer analysis. Therefore, through the TCGA database, we conducted a related study on the expression of DYRK2 in cancers.The expression of DYRK2 is obviously increased in some cancers, while the opposite is true in others, and there is a clear association between its expression and the prognosis of cancer patients.The mutation of DYRK2 is also significantly correlated with patients’ prognosis in certain human tumors. In addition, phosphorylation and methylation levels of DYRK2 are different between tumor tissues and adjacent normal tissues in various tumors. In the tumour microenvironment, the expression of DYRK2 correlates with cancer-associated fibroblast infiltration, such as BLCA or HNSC. In order to fully understand the role of DYRK2 in different tumors, we conducted a pan-cancer analysis.

Combination of DYRK2 and TERT Expression Is a Powerful Predictive Marker for Early-stage Breast Cancer Recurrence.

DYRK2 is a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase induces degradation of telomerase reverse transcriptase (TERT). The expression of both proteins in breast cancer were investigated as predictors of recurrence. Two hundred and twenty-one patients with early breast cancer treated at our institute between 2000 and 2009, were included. We used immunohistochemical analyses to measure the expression of DYRK2 and TERT and correlated it with clinicopathological factors and survival. DYRK2 and TERT were positive in 58 (26%) and 86 (39%) of 221 patients, respectively. There was no correlation between DYRK2 and TERT expression and clinicopathological factors. Better disease-free survival was observed in the DYRK2-positive group (p=0.032), and poorer disease-free survival was noted in the TERT-positive group (p=0.023). The DYRK2-positive TERT-negative group exhibited significantly better disease-free survival than the other groups (p=0.006). The combination of DYRK2 and TERT may be a powerful tool to stratify breast cancer patients.

Vitamin K3 (menadione) Induces Cytotoxicity in Chronic Myeloid Leukemia Stem Cells By Upregulating DYRK2 through the Inhibition of SIAH2 Ubiquitin Ligase

Leukemic stem cells (LSCs) generated by the transformation of normal hematopoietic stem cells (HSCs) by BCR-ABL are elusive targets that can initiate and sustain leukemia owing to their unique capacity to regenerate themselves during cell division (self-renewal) and differentiate into a bulk of leukemia cells. Although chronic myeloid leukemia (CML) can be successfully managed using tyrosine kinase inhibitors (TKIs), drug discontinuation trials have shown that 30-60% of patients, depending on the drug used for first- and second-line treatment, can remain in treatment-free remission. Thus, discontinuation is still considered experimental and there are not safety guidelines to stop treatment. A better understanding of self-renewal mechanisms in LSCs will support the development of LSC-specific drugs to induce treatment-free remission in chronic-phase patients by eradicating BCR-ABL-positive LSCs. We recently discovered in mouse models that deletion of the Klf4 gene encoding for the transcription factor KLF4 severely abrogated progression of BCR-ABL(p210)-induced CML by impairing survival and self-renewal of LSCs but not normal hematopoietic stem cells (HSCs). Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated DYRK2 protein levels, which was associated with p53-mediated apoptosis and inhibition of self-renewal via depletion of c-Myc protein in LSCs and not in normal HSCs. Here, we report that this mechanism is active in bone marrow cells from chronic-phase CML patients and that stabilization of DYRK2 protein by inhibition of the ubiquitin E3 ligase with vitamin K3 (VK3) has the potential therapeutic approach. Treatment of CML cell lines and bone marrow cells from CML patients with VK3 induced dose-dependent cytotoxicity and apoptosis. This was correlated with increased DYRK2 expression, depletion of c-Myc, and activation of p53-mediated apoptosis. Most importantly, VK3 induced DYRK2 expression and abrogated capacity of purified CD34+ CML stem cells, but not normal CD34+ cells, to generate colonies in methylcellulose assays. Cytotoxicity of VK3 largely depended on the expression of DYRK2, the reduced form menadiol inhibited cell viability similarly to menadione (VK3), and the presence of p53 enhanced cytotoxicity by inducing apoptosis in addition to inhibition of self-renewal. VK3 augmented cytotoxicity of Imatinib in CML cell lines and inhibited cell survival in Imatinib-resistant CML cells. The ability to target CML LSCs was confirmed by in vivo administration of VK3 in BCR-ABL induced mouse model followed by transplantation of leukemic bone marrow in secondary recipient mice. Altogether, our results suggest that stabilization of DYRK2 by inhibition of SIAH2 can induce apoptosis in bulk leukemia cells and LSCs by activating p53 and depleting c-Myc. DisclosuresNo relevant conflicts of interest to declare.

MiR-187 modulates cardiomyocyte apoptosis and oxidative stress in myocardial infarction mice via negatively regulating DYRK2

Myocardial infarction is a serious representation of cardiovescular disease, MicroRNAs play a role in modifying I/R injury and myocardial infarct remodeling. The present study therefore examined the potential role of miR-187 in cardiac I/R injury and its underlying mechanisms. miR-187 was inhibited or overexpressed in cardiomyocytes H/R models by pretreatment with miR-187 mimic or inhibitor to confirm the function of miR-187 in H/R. DYRK2 was inhibited or overexpressed in cardiomyocytes H/R models by pretreatment with DYRK2 inhibitor. A myocardium I/R mouse model was established. Circulating levels of miR-187 or DYRK2 was detected by quantitative realtime PCR and protein expression was detected by western blotting. The cell viability in all groups was determined by MTT assay and the apoptosis ratio was detected by flow cytometry after staining with Annexin V-FITC. The effect of miR-187 on cellular ROS generation was examined by DCFH-DA. The level of lipid peroxidation and SOD expression were determined by MDA and SOD assay. The findings indicated that miR-187 may be a possible regulator in the protective effect of H/R-induced cardiomyocyte apoptosis, cellular oxidative stress and leaded to DYRK2 suppression at a posttranscriptional level. Moreover, the improvement of miR-187 on H/R-induced cardiomyocyte injury contributed to the obstruction of DYRK2 expression. In addition, these results identified DYRK2 as the functional downstream target of miR-187 regulated myocardial infarction and oxidative stress.These present work provided the first insight into the function of miR-187 in successfully protect cardiomyocyte both in vivo and in vitro, and such a protective effect were mediated through the regulation of DYRK2 expression.

Frequent DYRK2 gene amplification in micropapillary element of lung adenocarcinoma - an implication in progression in EGFR-mutated lung adenocarcinoma.

The present study aimed to discern the molecular alterations involved in the progression of EGFR-mutated lung adenocarcinoma (LADC). We previously demonstrated that the micropapillary (mPAP) element is the most important histological factor for assessing malignant grades in LADCs. Therefore, mPAP and other elements were separately collected from three cases of EGFR-mutated LADC using laser capture microdissection and subjected to a comprehensive mRNA expression analysis. We focused on DYRK2 in this study because its level showed a substantial increase in EGFR-mutated LADCs with mPAP. We also immunohistochemically examined 130 tumors for the expression of DYRK2. The results confirmed a strong expression of DYRK2 in EGFR-mutated LADC with mPAP. Fluorescent in situ hybridization (FISH) analyses targeting the DYRK2 locus revealed frequent gene amplification in EGFR-mutated LADC, specifically occurring in the high-grade components, like mPAP. In summary, the results of this study suggest that DYRK2 overexpression through gene amplification is one of the molecular mechanisms responsible for promoting the progression of EGFR-mutated LADC.

Abstract P3-10-10: DYRK2 is a novel therapeutic target in ER negative breast cancer

Abstract Background Dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) belongs to a family of CMGC kinases that function as modulators of different downstream pathways that allow cells to cope with hypoxia, DNA damage and various stress signals. Additionally, DYRK2 has been implicated in various human cancers with both pro- and anti-tumour roles, which are probably cancer type- and cell type-dependent. Furthermore, studies show that DYRK2 is involved in epithelial-mesenchymal transition, hence suggesting a role in tumour metastasis. The current study investigates the prognostic role of DYRK2 in breast cancer and investigates its potential as a novel therapeutic target. Methods Immunohistochemistry was employed to investigate if nuclear expression of DYRK2 was associated with clinical outcome measures in a cohort of 715 patients. Expression was determined using the weighted histoscore method. Antibody specificity was confirmed in paraffin embedded cell pellets +/- DYRK2 silencing. Cell counts in parental and CRISPR-mediated DYRK2 knocked-out MDA-MB-468 and MDA-MB-231 cells (ER, PR, HER2, AR negative) were measured using Alamar Blue; NSGTMmice (n=8) were injected subcutaneously with MDA-MDB-231 with or without DYRK2 depletion to assess tumour growth in vivo. Results In a cohort of 715 patients, median follow-up was 160 months with 155 breast cancer deaths and 135 deaths due to other causes. The majority of patients were over 50 years of age (71%), had ductal carcinoma (88%), tumours <20mm in size (56%) and node negative disease (57%). 489 patients had ER positive disease, 226 had ER negative disease and of these 148 had TN (triple-negative) disease. DYRK2 expression was observed in the cell cytoplasm and nucleus and ranged from 3 to 200 weighted histoscore units (WHS) and ROC analysis was used to determine cut-offs, tumours with a cytoplasmic and nuclear WHS <145 were classified as low expression and tumours with a cytoplasmic and nuclear WHS >145 were classified as high expression. In the full cohort (p=0.087) and ER negative (p=0.066) cohort DYRK2 was not associated with cancer specific survival. However in TN disease high DYRK2 expression was associated with cancer specific survival (p=0.012, mean survival 145 months versus 107 months). This was potentiated in patients with ER, PR, HER2, AR negative disease (p=0.005, mean survival 166 months versus 100 months) and independent in multivariate analysis with age, histological tumour type, tumour size tumour grad, nodal status, ki67 index, chemotherapy, radiotherapy and recurrence (p=0.13, HR 3.920). Following this observation, patients with ER, AR negative disease were investigated and again high DYRK2 expression was associated with cancer specific survival (p=0.0003, mean survival 163 months versus 86 months) and was independent when combined in multivariate analysis (p=0.001, HR 4.154). To investigate if DYRK2 was a potential target in TN breast cancer, the effect of silencing DYRK2 was investigated. CRISPR-mediated DYRK2 depletion impeded cell proliferation in TN cell-lines and markedly reduced tumour burden in mouse MDA-MDB-231 xenografts (p<0.0001). Conclusions Our studies indicate that DYRK2 is indeed a potential therapeutic target for patients with TN breast cancer or ER, AR negative breast cancer. Citation Format: Edwards J, Baillie G, Quinn J, Monreno R, Banerjee S, Tomkinson N, MacKay S, De La Vega L. DYRK2 is a novel therapeutic target in ER negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-10.

Abstract 145: Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells

Abstract Leukemia stem cells (LSCs) are a rare population able to sustain and recapitulate leukemia through a poorly understood mechanism of self-renewal. Because more than half of patients relapse after the cessation of TKI therapy, it is clear that a cure is not possible with TKIs alone, and LSC-specific drugs are urgently needed to simultaneously eliminate bulk leukemia with TKIs and LSCs. Here we report that KLF4 promotes disease progression in chronic myeloid leukemia (CML) by repressing an inhibitory mechanism in LSCs that can be activated with small molecules. Deletion of the Klf4 gene severely abrogated maintenance of BCR-ABL(p210)-induced CML by impairing survival and self-renewal in LSCs whereas increased self-renewal was observed in hematopoietic stem cells during serial transplantation. Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated levels of the DYRK2 kinase in LSCs, which was associated with p53-mediated apoptosis and inhibition of self-renewal through depletion of c-Myc protein. Supporting this model, stabilization of DYRK2 protein, by inhibiting the ubiquitin E3 ligase SIAH2 with vitamin K3, promoted apoptosis in a panel of CML cell lines (K562, KU-812, and KCL-22) by inducing DYRK2 expression. Knocking out the DYRK2 gene in K562 cells by Cas9/CRISPR abrogated cytotoxicity induced by vitamin K3 and the presence of p53 significantly lowered IC50. In vivo treatment of CML mice diminished the number of LSCs evaluated in secondary transplants. In humans, vitamin K3 induced apoptosis in bone marrow cells from CML patients but not in healthy individuals by inducing DYRK2, p53 phosphorylation, and c-Myc depletion; furthermore, vitamin K3 abrogated capacity of CD34+ cells to generate colonies in methylcellulose only in CML. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling both p53 and c-Myc mediated regulation of survival and self-renewal in CML LSCs that can be activated pharmacologically. Citation Format: Chun Shik Park, Ye Shen, Andrew Lewis, Koramit Suppipat, Monica Puppi, Julie Tomolonis, Taylor Chen, Paul Pang, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Rachel Rau, Daniel Lacorazza. Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 145.

Dyrk2 involved in regulating LPS-induced neuronal apoptosis

The activation of relevant signaling pathways plays a very important role in LPS-induced neuronal damage. Dual-specificity tyrosine-phosphorylation-regulated kinase 2(Dyrk2), as a phosphokinase that can directly or indirectly phosphorylate signal molecules, was recently reported to down-regulate Type I Interferon(TIF) by promoting ser527 phosphorylation of TBK1. To further investigate the role of Dyrk2 in neuroinflammation, we for the first time focused on its function in LPS-induced neuronal damage. We found LPS stimulation increased the expression of Dyrk2 in the nucleus and cytoplasm of neurons. In addition, overexpression of Dyrk2 not only reduced the level of TNF-α induction, but also obviously inhibited LPS-induced neuronal apoptosis. We further found that Dyrk2 promoted the induction of phospho-Akt, phospho-p65 and phospho-p38MAPK (p38 mitogen-activated protein kinase), but immunoprecipitation showed Dyrk2 interacted with and Akt, p38MAPK and IκBα (IkappaB-alpha), except NF-κB subunit p65. These findings suggest Dyrk2 can inhibit LPS-induced neuronal apoptosis and plays key roles in LPS-indcued signaling pathways by its phosphokinase function. These data provide a novel viewpoint that Dyrks family may have an important role in neuroinflammation, and provide a potential molecular target for improving neuronal apoptosis.

Abstract 3334: KLF4 promotes self-renewal by repressing DYRK2-mediated degradation of c-Myc in leukemic stem cells: development of targeted therapy

Abstract Chronic myeloid leukemia (CML) is the first blood cancer known to originate from a single hematopoietic stem cell (HSC) by expression of BCR-ABL, a product of the chromosomal translocation t(9;22), that slowly progress to a lethal fast-growing leukemia caused by malignant reprogramming of progenitor cells (blast crisis). Although CML can be successfully managed with targeted therapy by suppressing BCR-ABL kinase activity with tyrosine kinase inhibitors (TKI), patients remain in remission as long as they adhere to a lifelong treatment. The leukemic stem cell (LSC) population emerges as a key ‘CML reservoir’ that escapes TKI therapy by developing BCR-ABL-independent mechanisms of self-renewal and survival. LSC still remains an elusive target because of our poor understanding of specific self-renewal mechanisms and inability to selectively eliminate LSC without damaging normal hematopoiesis. Thus, there is a need for alternative drugs for relapse patients to prevent reactivation of BCR-ABL-positive LSC after stopping chemotherapy or emergence of chemoresistance and as frontline therapy to achieve treatment-free remission. We found that somatic deletion of the transcriptional factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. This data suggest that KLF4 promotes self-renewal of LSCs whereas serial transplantation indicates that KLF4 restricts stress self-renewal of normal HSCs and thus inhibition of KLF4 function would impair LSC self-renewal without altering blood production. Analyses of global gene expression in purified LSCs and genome-wide binding of KLF4 in a murine CML cell line revealed that KLF4 represses the gene encoding for the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). Immunoblots revealed elevated levels of DYRK2 protein in KLF4-deficient LSCs purified from the bone marrow of CML mice. Because phosphorylation of c-Myc and p53 by DYRK2 induces proteosomal degradation and cell death, respectively, we found that DYRK2 upregulation in KLF4-deficient LSCs was associated with a reduction of c-Myc protein and increased cleavage of PARP. As a proof-of-principle of the therapeutic potential of this finding, we explored the efficacy of vitamin K3 to eradicate LSCs by inhibiting the ubiquitin E3 ligase SIAH2 in charge of DYRK2 degradation. Vitamin K3 efficiently reduced cell viability in a panel of human-derived CML cell lines by inducing Dyrk2 expression and apoptosis. The identification of Dyrk2 as a critical mediator of LSC downfall is an innovative paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in CML patients. Citation Format: Chun Shik Park, Ye Shen, Koramit Suppipat, Julie Tomolonis, Monica Puppi, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Daniel Lacorazza. KLF4 promotes self-renewal by repressing DYRK2-mediated degradation of c-Myc in leukemic stem cells: development of targeted therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3334.

Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma

We aimed to investigate the molecular mechanisms of DYRK2 and the HCC sensitivity to Oxaliplatin in DYRK2-depleted HCC cells. HCC tissue specimens were obtained from 86 HCC patients during hepatectomy. We used immunohistochemistry and western blot to analyze DYRK2 expression in HCC tissues and cell lines, and used siRNA transfection to decrease DYRK2 expression in HCC cells. Flow cytometry and CCK-8 assay were detected in cell cycle progression, cell proliferation and the efficacy of Oxaliplatin, DYRK2 was down-regulated in HCC tissues, compared with adjacent nontumor ones. The significant correlation between DYRK2 expression and clinicopathologic factors was apparently shown in the immunohistochemical and statistical analyses. The expression of DYRK2 was significantly associated with histological grade of HCC patients. Univariate and multivariate survival analyses revealed that DYRK2 was a significant predictor for overall survival of HCC patients. The depletion of DYRK2 promoted HCC cell proliferation, and increased resistance to Oxaliplatin. These data showed that the downregulated expression of DYRK2 in HCC tumor tissues could promote the proliferation of HCC cells. In addition, reducing DYRK2 expression was associated with poor prognosis and Oxaliplatin resistance in HCC.

Effect of Danshen-containing serum on expression of SuFu and DYRK2 in HSCs

To observe the effects of Danshen-containing serum on SuFu and DYRK2 expression in the HSCs stimulated by leptin. SD rats (n = 60) were used to make danshen-containing serum by gastric perfusion for ten days with Danshen water decoction, normal saline and colchicine. The HSCs that were cultured in vitro would be stimulated for 24 hours by leptin (100 μg x L(-1)) except blank control group, after being intervened, the drug serum in each group would be cultured at 37 degrees C in 5% incubator. The cells would be collected after 24 hours, then the effects of danshen-containing serum on the proliferation of HSCs were detected by MTT, the expression of SuFu mRNA and DYRK2 mRNA were detected by RT-PCR, the expression of SuFu and DYRK2 proteins were tested by Western blot. Compared with blank control group, the expression of DYRK2 mRNA and DYRK2 proteins were enhanced obviously after stimulated the HSCs of rats by leptin (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, after cyclopamine group (Hh pathway inhibitor), Danshen-containing serum and colchicine were interfered, the expression of DYRK2 mRNA and DYRK2 proteins were decreased clearly (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were increased significantly (P < 0.01 or P < 0.05). Compared with model group, adding purmorphamine (Hh pathway agonist) to model group and making it activate could increase the expression of DYRK2 mRNA and DYRK2 proteins, but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, using the Danshen-containing serum to interfere the purmorphamine group could make the expression of DYRK2 mRNA and DYRK2 proteins decrease and the expression of SuFu mRNA and SuFu proteins increase significantly (P < 0.01). Danshen-containing serum would inhibition the activation and increment of HSCs by interfering the expression of SuFu and DYRK2 which were induced by leptin.

Silencing of DYRK2 increases cell proliferation but reverses CAM-DR in Non-Hodgkin's Lymphoma

DYRK2, a dual-specificity tyrosine-(Y)-phosphorylation regulated kinase gene, is involved in regulating many processes such as cell proliferation, cell differentiation and cytokinesis. DYRK2 also plays an important role in many cancers, such as breast cancer, non-small cell lung cancer and esophageal adenocarcinomas. In this study, we found that DYRK2 is associated with the proliferation of Non-Hodgkin's lymphoma (NHL) and cell adhesion mediated drug resistance (CAM-DR). Clinically, the mRNA and protein expression levels of DYRK2 are decreased in NHL tissues compared with reactive lymphoid hyperplasia tissues. Immunohistochemical analysis revealed that low expression of DYRK2 is associated with poor prognosis of NHL patients. Interestingly, knockdown of DYRK2 can promote cell proliferation via modulating cell cycle progression. Finally, we demonstrated that DYRK2 plays an important role in CAM-DR by regulating p27Kip1 expression. Importantly, DYRK2 knockdown reverses CAM-DR in NHL. Our research suggested that DYRK2 may be a novel therapeutic target for NHL.

Downregulation of DYRK2 can be a predictor of recurrence in early stage breast cancer.

This study investigated the potential of DYRK2, a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase gene, to predict disease-free survival for patients with early stage breast cancer. Two hundred and seventy-four patients with breast cancer underwent surgery from January 2000 to December 2009. All patients were in stage I or II. Immunohistochemical (IHC) analysis was used to determine the expression of DYRK2, which was examined for its association with clinicopathological factors or prognosis. A total of 85 of 274 cases (31%) were DYRK2 positive. No correlation was found between DYRK2 expression by IHC and clinicopathological factors such as tumor size, histological grade, hormone receptor status, and HER2 status; however, lymph node involvement was closely associated with DYRK2 expression. Ten-year disease-free survival in the DYRK2-positive group without node metastasis (95.9%) was significantly better than that in the DYRK2-negative group (87.3%, p = 0.015). These data show that DYRK2 expression is associated with lymph node involvement and is a possible predictive factor of breast cancer recurrence.

Clinical significance of expression of DYRK2 in pancreatic cancer

Clinical significance of expression of DYRK2 in pancreatic cancer

Abstract P5-04-02: DYRK2 regulates breast cancer invasion via Snail/E-cadherin pathway

Abstract Introduction: Epithelial-Mesenchymal Transition (EMT) is characterized by the loss of cellular adhesion molecule E-cadherin, and plays a fundamental role during early steps of breast cancer invasion. An important regulator of E-cadherin expression is Snail, a zinc finger transcriptional repressor. Snail is posphorylated by GSK3β and then degraded by βTrCP mediated ubiquitination. Here we found another kinase, DYRK2, regulates the stability of Snail. Knockdown of DYRK2 promoted the EMT and cancer invasion both in vitro and in vivo experiments. Methods: Cell lines: MCF-7 cells were transfected with pSuper vector (pSuper control) and pSuper shRNA DYRK2 (shRNA-DYRK2) with G418 to isolate stable cell lines. Immunoblotting: Expression of pertinent molecular markers was determined by western blotting. RT-PCR: RNA was extracted using TRIsure and 500 ng of total RNA was amplified using Primer Script One Step RT-PCR Kit Ver.2. Invasion assay: Various cancer cell lines were seeded on the top of the upper chamber with serum free medium while the bottom chambers were filled with medium containing 10% FBS. In vivo metastasis assay: Cells in PBS were injected into the left ventricle of 7-week-old female nude mice. Metastases to distant organs were confirmed by IVIS2000. Immunohistochemistry: We acquired paraffin-embedded tissue sections from primary breast tumor cores and from Jikei University of Medicine. Immunohistochemistry for DYRK2 was performed. Results: In MCF-7 cells, knockdown of DYRK2 increased Snail in the protein level but not in mRNA level. Upon DYRK2 knockdown, MG132 treatment had no effect on additional increase in Snail. DYRK2 knockdown decreased Snail ubiquitination. Stable DYRK2 depletion led to Snail accumulation and E-cadherin abrogation. Fibroblast marker, Vimentin, emerged in DYRK2 depleted cells. For further analysis, we carried out invasion assays. The invasion potential in DYRK2-depleted cells was substantially higher than that in control cells. In xenograft model, we used nude mice received intracardial injections of pSuper control or shRNA-DYRK2 cells. 6 weeks after injections, a significant increase in bone and lung metastasis was observed in shRNA-DYRK2 group. The patients with tumors expressed low DYRK2 showed significantly poor prognosis. Conclusion: In breast cancer cells, Snail is phosphorylated by DYRK2 and then acquires the ability to be ubiquitinated. In the absence of DYRK2, Snail is unable to be degraded by ubiquitin-proteasome machinery. Accumulation of Snail promotes EMT and cancer invasion, so low expression of DYRK2 leads to poor prognosis in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-02.

Abstract 4309: DYRK2 regulates cancer invasiveness via Snail/E-cadherin pathway

Abstract Epithelial-mesenchymal transition (EMT) is characterized by the loss of cellular adhesion molecule E-cadherin, and plays a fundamental role during early steps of invasion and metastasis. An important regulator of E-cadherin expression is Snail, a zinc-finger transcriptional repressor. Snail is phosphorylated by GSK3-α and then degraded by α-TrCP-mediated ubiquitination. Here we describe another kinase, DYRK2, acts as a priming kinase for snail whose phosphorylation is required for the subsequent GSK3&amp;gamma phosphorylation and proteasomal degradation. Inhibition of DYRK2 expression by siRNA results in up-regulation of Snail and down-regulation of E-cadherin. The expression of fibroblast mrakers, vimentin and fibronectin, emerged in these cells. Furthermore, the invasiveness of MCF7 cells was increased by stable DYRK2 silencing by shRNA. Tumors expressing DYRK2-shRNA robustly formed metastases in nude mice bones, while those expressing control-shRNA little if any. In human breast cancer tissues, DYRK2 exhibits an inverse correlation with the expression of Snail. Conversely, low level expression of DYRK2 is associated with down regulation of E-cadherin. The patiens with tumors expressed low DYRK2 showed significantly poor prognosis and short disease-free survival. These results demonstrate that DYRK2 regulates cancer invasiveness and metastasis via Snail/E-cadherin pathway in human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4309. doi:1538-7445.AM2012-4309

DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells

Dysregulation of the G(1)/S transition in the cell cycle contributes to tumor development. The oncogenic transcription factors c-Jun and c-Myc are indispensable regulators at this transition, and their aberrant expression is associated with many malignancies. Degradation of c-Jun/c-Myc is a critical process for the G(1)/S transition, which is initiated upon phosphorylation by glycogen synthase kinase 3 β (GSK3β). However, a specific kinase or kinases responsible for priming phosphorylation events that precede this GSK3β modification has not been definitively identified. Here, we found that the dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 functions as a priming kinase of c-Jun and c-Myc. Knockdown of DYRK2 in human cancer cells shortened the G(1) phase and accelerated cell proliferation due to escape of c-Jun and c-Myc from ubiquitination-mediated degradation. In concert with these results, silencing DYRK2 increased cell proliferation in human cancer cells, and this promotion was completely impeded by codeprivation of c-Jun or c-Myc in vivo. We also found marked attenuation of DYRK2 expression in multiple human tumor samples. Downregulation of DYRK2 correlated with high levels of unphosphorylated c-Jun and c-Myc and, importantly, with invasiveness of human breast cancers. These results reveal that DYRK2 regulates tumor progression through modulation of c-Jun and c-Myc.

Expression of dual-specificity tyrosine-(Y)-phosphorylation–regulated kinase 2 (DYRK2) can be a favorable prognostic marker in pulmonary adenocarcinoma

We investigated the possibility of DYRK2, a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase gene, to predict survival for patients with pulmonary adenocarcinoma. One hundred forty-four patients with pulmonary adenocarcinoma underwent surgery in our institute from 2000 to 2008. We used immunohistochemical analysis and real-time reverse-transcriptase polymerase chain reaction to determine the expression of DYRK2 and compared this with the clinicopathologic factors and survival. We found no correlation between DYRK2 expression by immunohistochemical and clinicopathologic factors; however, a negative nodal status and negative lymphatic invasion were significantly associated with DYRK2 expression by reverse-transcriptase polymerase chain reaction. Five-year disease-free survival in the DYRK2-positive group (75.4%) was significantly different from that in the negative group (55.4%; P = .03) by immunohistochemical analysis. The 5-year overall survival of 89.2% in the DYRK2-positive group was better than the 66.3% survival of the DYRK2-negative group (P = .01). Quantitative real-time reverse-transcriptase polymerase chain reaction analyses showed a significant difference between positive and negative expressions for disease-free survival (P = .003) and overall survival (P = .007). In multivariate Cox regression analysis, negative DYRK2 protein and messenger RNA expression showed a worse prognostic value of survival (hazard ratio [HR] = 4.7, 95% confidence intervals [CI] = 1.5-14.5, P=.007; HR = 2.5, 95% CI = 1.1-6.1, P = .04, respectively). When we analyzed adenocarcinoma cases except for bronchioloalveolar carcinoma, we found a close correlation between DYRK2 expression by immunohistochemical analysis and nodal status (P = .03). Furthermore, disease-free survivals between positive and negative groups of DYRK2 expression by immunohistochemistry (P = .03) and reverse-transcriptase polymerase chain reaction (P = .02) without bronchioloalveolar carcinoma were significantly different. Overall survivals in both groups showed significant differences by immunohistochemistry (P = .02) but not by reverse-transcriptase polymerase chain reaction (P = .08). These data showed that DYRK2 expression is associated with a favorable prognosis.