Dual-specificity Tyrosine-regulated Kinase 2 Research Articles

Dual-specificity tyrosine-regulated kinase 2 exerts anti-tumor effects by induction of G1 arrest in lung adenocarcinoma

ObjectivesLung cancer is a leading cause of cancer-related mortality and remains one of the most poorly prognosed disease worldwide. Therefore, it is necessary to identify novel molecular markers with potential therapeutic effects. Recent findings have suggested that dual-specificity tyrosine-regulated kinase 2 (DYRK2) plays a tumor suppressive role in colorectal, breast, and hepatic cancers; however, its effect and mechanism in lung cancer remain poorly understood. Therefore, this study aimed to investigate the tumor-suppressive role and molecular mechanism of DYRK2 in lung adenocarcinoma (LUAD) by in vitro experiments and xenograft models. Materials and methodsThe evaluation of DYRK2 expression was carried out using lung cancer cell lines and normal bronchial epithelial cells. Overexpression of DYRK2 was induced by an adenovirus vector, and cell proliferation was assessed through MTS assay and Colony Formation Assay. Cell cycle analysis was performed using flow cytometry. Additionally, proliferative capacity was evaluated in a xenograft model by subcutaneously implanting A549 cells into SCID mice (C·B17/Icr-scidjcl-scid/scid). ResultsImmunoblotting assays showed that DYRK2 was downregulated in most LUAD cell lines. DYRK2 overexpression using adenovirus vectors significantly suppressed cell proliferation compared with that in the control group. Additionally, DYRK2 overexpression suppressed tumor growth in a murine subcutaneous xenograft model. Mechanistically, DYRK2 overexpression inhibited the proliferation of LUAD cells via p21-mediated G1 arrest, which was contingent on p53. ConclusionTaken together, these findings suggest that DYRK2 may serve as potential prognostic biomarker and therapeutic target for LUAD.

DYRK2 promotes chemosensitivity via p53-mediated apoptosis after DNA damage in colorectal cancer.

Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates p53-Ser46 and induces apoptosis in response to DNA damage. However, the relationship between DYRK2 expression and chemosensitivity after DNA damage in colorectal cancer has not been well investigated. The aim of the present study was to examine whether DYRK2 could be a novel marker for predicting chemosensitivity after 5-fluorouracil- and oxaliplatin-induced DNA damage in colorectal cancer. Here we showed that DYRK2 knockout decreased the chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type colorectal cancer cells, whereas the chemosensitivity remained unchanged in p53-deficient/mutated colorectal cancer cells. In addition, no significant differences in chemosensitivity to 5-fluorouracil and oxaliplatin between scramble and siDYRK2 p53(-/-) colorectal cancer cells were observed. Conversely, the combination of adenovirus-mediated overexpression of DYRK2 with 5-fluorouracil or oxaliplatin enhanced apoptosis and chemosensitivity through p53-Ser46 phosphorylation in p53 wild-type colorectal cancer cells. Furthermore, DYRK2 knockout decreased chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type xenograft mouse models. Taken together, these findings demonstrated that DYRK2 expression was associated with chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type colorectal cancer, suggesting the importance of evaluating the p53 status and DYRK2 expression as a novel marker in therapeutic strategies for colorectal cancer.

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.

Abstract 3978: The growth inhibitory effect of MDV3100 on hormone receptor-positive, HER2-negative breast cancer cell lines with low DYRK2 expression

Abstract Introduction: Tumor progression is the main cause of death in patients with breast cancer. Accumulating evidence suggests that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. Recently, we reported for the first time that DYRK2 inhibits breast cancer stem cells and tumor cell proliferation through transcriptional downregulation of KLF4 and CDK14, respectively.Our previous study showed that Androgen receptor (AR) was a DYRK2-dependent transcriptional activator. In this context, we hypothesized that MDV3100, an inhibitor of AR, may be effective for breast cancer with low DYRK2 expression. Methods: We established stable DYRK2-depleted cells. MCF-7 cells, hormone receptor-positive, HER2-negative breast cancer cell line, were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) along with puromycin to isolate stable cell lines.These manipulated cells were compared with the control cells by various assays. We analyzed the activity of AR by Androgen receptor reporter assay. MTS assays were used to assess growth inhibition after treatment with MDV3100. Results: Androgen receptor reporter assay showed that knocking down DYRK2 induced AR transcription activity in MCF-7 cells.In MCF-7 DYRK2-depleted cells, MTS assays revealed that the treatment by MDV3100 was more effective than that in MCF-7 control cells. Conclusion: These findings demonstrate that reduced DYRK2 expression in hormone receptor-positive, HER2-negative breast cancer increases the activity of AR and significantly inhibits the growth by treatment with MDV3100. Citation Format: Yoshimi Imawari, Rei Mimoto, Kiyotsugu Yoshida. The growth inhibitory effect of MDV3100 on hormone receptor-positive, HER2-negative breast cancer cell lines with low DYRK2 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3978.

DYRK2 maintains genome stability via neddylation of cullins in response to DNA damage.

Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8), an ubiquitin-like protein, is an essential regulator of the DNA damage response. Numerous studies have shown that neddylation (conjugation of NEDD8 to target proteins) dysfunction causes several human diseases, such as cancer. Hence clarifying the regulatory mechanism of neddylation could provide insight into the mechanism of genome stability underlying the DNA damage response (DDR) and carcinogenesis. Here, we demonstrate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a novel regulator of neddylation and maintains genome stability. Deletion of DYRK2 leads to persistent DNA double-strand breaks (DSBs) and subsequent genome instability. Mechanistically, DYRK2 promotes neddylation through forming a complex with NAE1, which is a component of NEDD8-activating enzyme E1, and maintaining its protein level by suppressing polyubiquitylation. The present study is the first to demonstrate that DYRK2 controls neddylation and is necessary for maintaining genome stability. This article has an associated First Person interview with the first author of the paper.

Enforced dual-specificity tyrosine-regulated kinase 2 expression by adenovirus-mediated gene transfer inhibits tumor growth and metastasis of colorectal cancer.

Colorectal cancer is one of the most common gastrointestinal tumors with good outcomes; however, with distant metastasis, the outcomes are poor. Novel treatment methods are urgently needed. Our in vitro studies indicate that dual‐specificity tyrosine‐regulated kinase 2 (DYRK2) functions as a tumor suppressor in colorectal cancer by regulating cell survival, proliferation, and apoptosis induction. In addition, DYRK2 expression is decreased in tumor tissues compared to nontumor tissues in colorectal cancer, indicating a correlation with clinical prognosis. In this context, we devised a novel therapeutic strategy to overexpress DYRK2 in tumors by adenovirus‐mediated gene transfer. The present study shows that overexpression of DYRK2 in colon cancer cell lines by adenovirus inhibits cell proliferation and induces apoptosis in vitro. Furthermore, in mouse subcutaneous xenograft and liver metastasis models, enforced expression of DYRK2 by direct or intravenous injection of adenovirus to the tumor significantly inhibits tumor growth. Taken together, these findings show that adenovirus‐based overexpression of DYRK2 could be a novel gene therapy for liver metastasis of colorectal cancer.

The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress

To survive proteotoxic stress, cancer cells activate the proteotoxic-stress response pathway, which is controlled by the transcription factor heat shock factor 1 (HSF1). This pathway supports cancer initiation, cancer progression and chemoresistance and thus is an attractive therapeutic target. As developing inhibitors against transcriptional regulators, such as HSF1 is challenging, the identification and targeting of upstream regulators of HSF1 present a tractable alternative strategy. Here we demonstrate that in triple-negative breast cancer (TNBC) cells, the dual specificity tyrosine-regulated kinase 2 (DYRK2) phosphorylates HSF1, promoting its nuclear stability and transcriptional activity. DYRK2 depletion reduces HSF1 activity and sensitises TNBC cells to proteotoxic stress. Importantly, in tumours from TNBC patients, DYRK2 levels positively correlate with active HSF1 and associates with poor prognosis, suggesting that DYRK2 could be promoting TNBC. These findings identify DYRK2 as a key modulator of the HSF1 transcriptional programme and a potential therapeutic target.

Abstract P4-08-04: Downregulation of DYRK2 contributes to tumor cell proliferation by enhancing CDK14 expression in breast cancer

Abstract Introduction: Tumor progression is the main cause of death in patients with breast cancer. Accumulating evidence suggests that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. However, little is known about the mechanisms of transcriptional regulation by DYRK2 in cancer progression, particularly with respect to cancer proliferation and invasion. Here, using a comprehensive expression profiling approach, we show that cyclin-dependent kinase 14 (CDK14) is a target of DYRK2. The aim of this study is to clarify whether DYRK2 suppresses the proliferation of breast cancer cells through CDK14 expression. Methods: Cell lines: We established stable DYRK2-depleted cells. MCF-7 cells were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) along with puromycin to isolate stable cell lines. We generated stable DYRK2-overexpressing MDA-MB-231 cells using GFP-DYRK2 (GFP-DYRK2 cells). A GFP vector was transfected as a vehicle control (GFP control cells) with G418 to isolate stable cell lines. Moreover, we established both stable DYRK2- and CDK14-depleted cells. The shDYRK2 cells were transfected with the pSuper-neo vector (shDYRK2-pSuper control) or pSuper-neo CDK14 shRNAs (shDYRK2-shCDK14). The pSuper control cells were transfected with the pSuper-neo vector (pSuper control-pSuper control cells) as a vehicle control with puromycin and G418 to isolate stable cell lines. These manipulated cells were compared with the control cells by various assays. Immunohistological staining: Sixty samples from surgically treated breast cancer patients were obtained from the Surgery Department at the Jikei University Hospital between 2001 and 2002. The Jikei University School of Medicine Ethics Review Committee approved the study protocol, and informed consent was obtained. Results: We analyzed cancer cell proliferation by MTS and colony formation assays. We investigated the effects of CDK14 in DYRK2-depleted cells in a breast tumor xenograft model. We found that reduced DYRK2 expression increased CDK14 expression to promote cancer cell proliferation in vitro and tumorigenicity in vivo. We further identified androgen receptor (AR) as a candidate of DYRK2-dependent transcription factors to control CDK14. Real-time RT-PCR and western blotting revealed that inhibition of AR activity using an AR inhibitor MDV3100 decreased both mRNA and protein levels of CDK14 in shDYRK2 and GFP control cells. In contrast, knocking down DYRK2 induced AR transcription activity in MCF-7 cells. shDYRK2 cells were more sensitive to MDV3100 compared with pSuper control cells. Immunohistological staining of sixty samples from surgically treated breast cancer patients showed an inverse correlation between DYRK2 and CDK14 expression. Conclusion: These findings demonstrate that reduced DYRK2 expression in breast cancer promotes tumor cell proliferation by modulating CDK14 expression via AR. Citation Format: Yoshimi Imawari, Rei Mimoto, Noriko Yamaguchi, Makiko Kamio, Hiroko Nogi, Ken Uchida, Hiroshi Takeyama, Kiyotsugu Yoshida. Downregulation of DYRK2 contributes to tumor cell proliferation by enhancing CDK14 expression in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-04.

DNA Methylation of Dual-Specificity Tyrosine-Regulated Kinase 2 (DYRK2) Promoter Regulates Proliferation of Human Colorectal Cancer

DNA Methylation of Dual-Specificity Tyrosine-Regulated Kinase 2 (DYRK2) Promoter Regulates Proliferation of Human Colorectal Cancer

Forced expression of DYRK2 exerts anti-tumor effects via apoptotic induction in liver cancer

Liver cancer is highly aggressive and globally exhibits a poor prognosis. Therefore, the identification of novel molecules that can become targets for future therapies is urgently required. We have reported that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. However, the research into its clinical application as a molecular target has remained to be explored. Here we showed that DYRK2 knockdown enhanced tumor growth of liver cancer cells. Conversely and more importantly, adenovirus-mediated overexpression of DYRK2 resulted in inhibition of cell proliferation and tumor growth, and induction of apoptosis both in vitro and in vivo. Furthermore, we found that liver cancer patients with low DYRK2 expression had a significantly shorter overall survival. Given the findings that DYRK2 regulates proliferation and apoptosis of cancer cells, DYRK2 expression could be a promising predictive marker of the prognosis in liver cancer. Stabilized or forced expression of DYRK2 may become thus a potential target for novel gene therapy against liver cancer.

Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2

Curcumin, the active ingredient in Curcuma longa, has been in medicinal use since ancient times. However, the therapeutic targets and signaling cascades modulated by curcumin have been enigmatic despite extensive research. Here we identify dual-specificity tyrosine-regulated kinase 2 (DYRK2), a positive regulator of the 26S proteasome, as a direct target of curcumin. Curcumin occupies the ATP-binding pocket of DYRK2 in the cocrystal structure, and it potently and specifically inhibits DYRK2 over 139 other kinases tested in vitro. As a result, curcumin diminishes DYRK2-mediated 26S proteasome phosphorylation in cells, leading to reduced proteasome activity and impaired cell proliferation. Interestingly, curcumin synergizes with the therapeutic proteasome inhibitor carfilzomib to induce apoptosis in a variety of proteasome-addicted cancer cells, while this drug combination exhibits modest to no cytotoxicity to noncancerous cells. In a breast cancer xenograft model, curcumin treatment significantly reduces tumor burden in immunocompromised mice, showing a similar antitumor effect as CRISPR/Cas9-mediated DYRK2 depletion. These results reveal an unexpected role of curcumin in DYRK2-proteasome inhibition and provide a proof-of-concept that pharmacological manipulation of proteasome regulators may offer new opportunities for anticancer treatment.

Downregulation of dual-specificity tyrosine-regulated kinase 2 promotes tumor cell proliferation and invasion by enhancing cyclin-dependent kinase 14 expression in breast cancer.

Tumor progression is the main cause of death in patients with breast cancer. Accumulating evidence suggests that dual‐specificity tyrosine‐regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. However, little is known about the mechanisms of transcriptional regulation by DYRK2 in cancer progression, particularly with respect to cancer proliferation and invasion. Here, using a comprehensive expression profiling approach, we show that cyclin‐dependent kinase 14 (CDK14) is a target of DYRK2. We found that reduced DYRK2 expression increases CDK14 expression, which promotes cancer cell proliferation and invasion in vitro, in addition to tumorigenicity in vivo. CDK14 and DYRK2 expression inversely correlated in human breast cancer tissues. We further identified androgen receptor (AR) as a candidate of DYRK2‐dependent transcription factors regulating CDK14. Taken together, our findings suggest a mechanism by which DYRK2 controls CDK14 expression to regulate tumor cell proliferation and invasion in breast cancer. Targeting of this pathway may be a promising therapeutic strategy for treating breast cancer.

Regulating protein breakdown through proteasome phosphorylation.

The ubiquitin proteasome system degrades the great majority of proteins in mammalian cells. Countless studies have described how ubiquitination promotes the selective degradation of different cell proteins. However, there is a small but the growing literature that protein half-lives can also be regulated by post-translational modifications of the 26S proteasome. The present study reviews the ability of several kinases to alter proteasome function through subunit phosphorylation. For example, PKA (protein kinase A) and DYRK2 (dual-specificity tyrosine-regulated kinase 2) stimulate the proteasome's ability to degrade ubiquitinated proteins, peptides, and adenosine triphosphate, while one kinase, ASK1 (apoptosis signal-regulating kinase 1), inhibits proteasome function during apoptosis. Proteasome phosphorylation is likely to be important in regulating protein degradation because it occurs downstream from many hormones and neurotransmitters, in conditions that raise cyclic adenosine monophosphate or cyclic guanosine monophosphate levels, after calcium influx following synaptic depolarization, and during phases of the cell cycle. Beyond its physiological importance, pharmacological manipulation of proteasome phosphorylation has the potential to combat various diseases. Inhibitors of phosphodiesterases by activating PKA or PKG (protein kinase G) can stimulate proteasomal degradation of misfolded proteins that cause neurodegenerative or myocardial diseases and even reduce the associated pathology in mouse models. These observations are promising since in many proteotoxic diseases, aggregation-prone proteins impair proteasome function, and disrupt protein homeostasis. Conversely, preventing subunit phosphorylation by DYRK2 slows cell cycle progression and tumor growth. However, further research is essential to determine how phosphorylation of different subunits by these (or other) kinases alters the properties of this complex molecular machine and thus influence protein degradation rates.

Regulation of Glioma Cells Migration by DYRK2.

Dual-specificity tyrosine-regulated kinase 2 (DYRK2), a protein kinase that phosphorylates its substrates on serine/threonine, is expressed in numerous human tumors, but little is known about its role in the pathophysiology of glioma. In this study, we made an effort to explore the expression and function in human glioma. Western blot and immunohistochemistry analysis were performed to investigate the expression of DYRK2 protein in glioma tissues in 84 patients. Wound healing and transwell assay were carried out to determine the cell migration ability. We showed that the level of DYRK2 was significantly decreased in high-grade glioma tissues compared with low-grade tissues. In addition, the expression level of DYRK2 was positively correlated with glioma pathological grade and E-cadherin expression. Kaplane-Meier analysis revealed that low expression of DYRK2 was related to poor prognosis of glioma patients. Furthermore, wound healing and transwell assay revealed that DYRK2 could suppress cell migration and affect the expression levels of E-cadherin and vimentin through PI3K/AKT/GSK3β signaling pathway. Taken together, our results implied that DYRK2 could serve as a promising prognostic biomarker as well as a potential therapeutical target of glioma.

Dual-specificity tyrosine-regulated kinase 2 is a suppressor and potential prognostic marker for liver metastasis of colorectal cancer.

Colorectal cancer is a common cancer and a leading cause of cancer‐related death worldwide. The liver is a dominant metastatic site for patients with colorectal cancer. Molecular mechanisms that allow colorectal cancer cells to form liver metastases are largely unknown. Activation of epithelial–mesenchymal transition is the key step for metastasis of cancer cells. We recently reported that dual‐specificity tyrosine‐regulated kinase 2 (DYRK2) controls epithelial–mesenchymal transition in breast cancer and ovarian serous adenocarcinoma. The aim of this study is to clarify whether DYRK2 regulates liver metastases of colorectal cancer. We show that the ability of cell invasion and migration was abrogated in DYRK2‐overexpressing cells. In an in vivo xenograft model, liver metastatic lesions were markedly diminished by ectopic expression of DYRK2. Furthermore, we found that patients whose liver metastases expressed low DYRK2 levels had significantly worse overall and disease‐free survival. Given the findings that DYRK2 regulates cancer cell metastasis, we concluded that the expression status of DYRK2 could be a predictive marker for liver metastases of colorectal cancer.

Abstract P5-07-07: DYRK2 contributes to the generation of breast cancer stem cells through KLF4

Abstract <Introduction> Cancer stem cells (CSCs) have been defined by the potential to self-renew and to differentiate. CSCs pose a major hurdle in the treatment of cancer. However, the mechanisms by which cells acquire CSC properties such as drug resistance remain unclear. Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates its substrates on serine/threonine. Initially, we found that DYRK2 phosphorylates p53 at Ser 46 to regulate apoptotic cell death in response to DNA damage. Recently, we have shown that DYRK2 controls Snail degradation in breast cancer and ovarian serous adenocarcinoma. We also found that knockdown of DYRK2 in luminal-type breast cancer MCF-7 cells increased the cancer stem cell population. Kruppel-like factor 4 (KLF4) is one of the Yamanaka factors. It has been reported that pluripotent stem cells from mouse embryonic or adult fibroblasts are induced by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4. This finding led us to determine if KLF4 is indispensable for the maintenance of CSCs. The aim of this study is to clarify whether DYRK2 regulates CSCs through KLF4 in breast cancer. <Methods> Cell lines: MCF-7 (human mammary carcinoma: ATCC) cells were grown according to standard protocols. We established stable DYRK2-depleted cells. MCF-7 cells were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) with puromycin to isolate stable cell lines. In turn, we established both stable DYRK2- and KLF4-depleted cells. shDYRK2 cells were transfected with pSuper-neo vector (pSuper-neo control) or pSuper-neo KLF4 shRNAs (shKLF4) with puromycin and G418. Knockdown of DYRK2 or KLF4 was confirmed by real-time RT-PCR and immunoblotting. The depleted cells were compared with the control cells using real-time RT-PCR, immunoblotting, flow cytometric analysis, mammosphere assay, xenograft models and immunohistological staining. <Results> We analyzed the population of breast cancer stem cells by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of DYRK2 was associated with the increase of CD44+/CD24- cells. While pSuper control cells formed mammospheres, they did in a lesser extent compared to shDYRK2 cells. In real-time RT-PCR and immunoblotting analysis, stable DYRK2 depletion in MCF-7 cells induced KLF4 accumulation. We then investigated the effect of KLF4 on stemness by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of KLF4 in shDYRK2 cells reduced the proportion of CD44+/CD24- cells. Whereas shDYRK2/shKLF4 cells formed mammospheres, they did in a lesser extent compared to shDYRK2/pSuper-neo control cells. Moreover, the scale of the mammospheres formed in shDYRK2/shKLF4 cells was significantly smaller, as compared with that in shDYRK2/pSuper-neo control cells. In xenograft models, the loss of KLF4 protein expression significantly decreased tumor formation. Immunohistological staining of fifty-nine samples from surgically treated breast cancer patients showed an inverse correlation between DYRK2 and KLF4 expression. <Conclusion> These findings revealed that DYRK2 contributes to the generation of breast cancer stem cells through KLF4. Citation Format: Imawari Y, Mimoto RK, Yamaguchi N, Kamio M, Kato K, Nogi H, Toriumi Y, Uchida K, Takeyama H, Yoshida K. DYRK2 contributes to the generation of breast cancer stem cells through KLF4 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-07.

Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis

Despite the fundamental importance of proteasomal degradation in cells, little is known about whether and how the 26S proteasome itself is regulated in coordination with various physiological processes. Here we show that the proteasome is dynamically phosphorylated during cell cycle at Thr25 of the 19S subunit Rpt3. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrate that blocking Rpt3-Thr25 phosphorylation markedly impairs proteasome activity and impedes cell proliferation. Through a kinome-wide screen, we have identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates Rpt3-Thr25, leading to enhanced substrate translocation and degradation. Importantly, loss of the single phosphorylation of Rpt3-Thr25 or knockout of DYRK2 significantly inhibits tumor formation by proteasome-addicted human breast cancer cells in mice. These findings define an important mechanism for proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis.

DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma.

Epithelial-mesenchymal-transition (EMT) plays essential roles in ovarian cancer invasion, metastasis, and drug resistance. A hallmark of EMT is the loss of E-cadherin, which is regulated by Snail. Recently, it was shown that dual-specificity tyrosine-regulated kinase 2 (DYRK2) controls Snail degradation in breast cancer. The aim of this study is to clarify whether DYRK2 regulates EMT through Snail degradation in ovarian serous adenocarcinoma (SA). Expression of DYRK2 and Snail in two pairs of cisplatin-resistant and the original cisplatin-sensitive ovarian cancer cell line were analyzed by immunoblotting and real-time RT-PCR analysis. Morphological change, invasion ability, and chemosensitivity were evaluated by using DYRK2 stable knockdown cell line in 2008 (2008 shDYRK2). Immunohistochemical analyses for DYRK2 and Snail were performed with surgical specimens. The correlations between the expression of these proteins and the clinicopathological parameters, including prognosis, were determined. Moreover, we conducted a hypodermic administration test in nude mice and examined reproductive and cisplatin response activities. DYRK2 protein expression was posttranslationally reduced in cisplatin-resistant SA cell lines. 2008 shDYRK2 showed mesenchymal phenotype and resistant to cisplatin. Immunohistochemistry demonstrated that DYRK2 expression inversely correlated with Snail expression, and reduced expression of DYRK2 was associated with shorter overall survival in SA. DYRK2 may regulate EMT through Snail degradation in ovarian SA and might be a predictive marker for a favorable prognosis in the treatment of this cancer.