Rei Mimoto

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.

DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail

The epithelial-mesenchymal transition (EMT) plays a fundamental role in the early stages of breast cancer invasion. Snail, a zinc finger transcriptional repressor, is an important regulator of EMT. Snail is phosphorylated by GSK3β and is subsequently degraded by βTrCP-mediated ubiquitination. We identified an additional kinase, DYRK2, that regulates Snail stability. Knockdown of DYRK2 promoted EMT and cancer invasion in vitro and in vivo. Consistent with these results, DYRK2 was found to be down-regulated in human breast cancer tissue. Patients with low DYRK2-expressing tumors had a worse outcome than those with high DYRK2-expressing tumors. These findings revealed that DYRK2 regulates cancer invasion and metastasis by degrading Snail.

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.

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.

Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer

Whereas accumulating studies have supported the cancer stem cell theory, a specific therapy targeting a cancer stem cell subpopulation has not been established. Here, we show that dual-specificity tyrosine phosphorylation-kinase 2 (DYRK2) is a novel negative regulator for formation of breast cancer stem cells. Downregulation of DYRK2 promotes cancer stem-like traits in vitro, tumourigenesis in vivo and the proportion of the cancer stem cell population in human breast cancer tissues. We found that Krupple-like factor 4 (KLF4) serves as a key mediator of DYRK2’s control over the cancer stem phenotype. Reduced DYRK2 expression increases KLF4 expression, which induces cancer stem-like properties. We identified androgen receptor (AR) as a transcription factor binding to the KLF4 promoter region; this process is dependent on DYRK2 kinase activity. Our findings delineate a mechanism of cancer stem cell regulation by the DYRK2–AR–KLF4 axis in breast cancer. Targeting of this pathway may be a promising strategy against breast cancer stem cells.

Long-Term Follow-Up of Node-Negative Breast Cancer Patients Evaluated via Sentinel Node Biopsy After Neoadjuvant Chemotherapy

Micro‐Abstract The purpose of this study was to assess the usefulness of sentinel node biopsy (SNB) after neoadjuvant chemotherapy (NAC) in patients with clinically node‐negative breast cancer. SNB after NAC was as accurate as SNB without NAC. Axillary recurrence‐free survival rates were excellent regardless of whether NAC was performed before SNB. Background: Sentinel node biopsy (SNB) is used to accurately assess axillary lymph node status in patients with node‐negative breast cancer. However, its use after neoadjuvant chemotherapy (NAC) is controversial. We retrospectively assessed the usefulness of SNB after NAC by comparing axillary recurrence rates and other parameters in patients with clinically node‐negative breast cancer who underwent SNB after NAC or without NAC. Patients and Methods: At our hospital, 1179 patients with clinically node‐negative breast cancer underwent SNB from April 2007 to December 2013. The clinicopathological and survival data of patients who underwent SNB after NAC (the NAC group) and those who underwent SNB without NAC (the control group) were compared. Patients with a metastatic sentinel node underwent axillary lymph node dissection. Results: The number of patients in the NAC and control groups was 183 (15.5%) and 996 (84.5%), respectively. At diagnosis, tumors were significantly larger in the NAC group (P < .0001). Sentinel nodes were identified in almost all patients in both groups (99.5% in the NAC group vs. 99.8% in the control group). They were nonmetastatic in 147 (80.8%) patients in the NAC group and 849 (85.5%) patients in the control group. At the median follow‐up time of 51.1 months, 6 patients (0.6%) in the control group had axillary lymph node recurrence compared with no patients in the NAC group. Conclusion: SNB after NAC was as accurate as SNB without NAC in patients with clinically node‐negative breast cancer. Axillary recurrence‐free survival rates were excellent regardless of whether NAC was performed before SNB.

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.

Abstract 3060: DYRK2-mediated phosphorylation of c-Jun and c-Myc is requisite for proper control of the G1/S transition

Transcription factors c-Jun and c-Myc are indispensable regulators for the G1/S transition and their expressions were tightly regulated at transcriptional and post-translational levels. Dysregulation of these expressions leads to tumor development and progression. Degradation of c-Jun/c-Myc, a critical event for the G1/S transition, is triggered by sequential phosphorylations by unknown priming kinase(s) and GSK3-beta. Here we show that DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) functions as a priming kinase for c-Jun/c-Myc and modifies its expression at the by post-translational level. In the absence of priming phosphorylation, c-Jun and c-Myc escape from GSK-3-mediated sequential phosphorylation and Fbw-7-mediated ubiquitination. Therefore, depletion of DYRK2 results in aberrant expression of c-Jun and c-Myc. Furthermore, in DYRK2 knockdown cells, up-regulated c-Jun and c-Myc facilitate cell proliferation and shorten the length of the G1 phase and prompts the G1/S transition. Furthermore, DYRK2 inactivation contributes to cell proliferation and tumor progression in vitro and in vivo. Pathological analysis demonstrated that DYRK2 expression is decreased in multiple cancerous tissues. Taken together, DYRK2 controls cell cycle progression at G1/S phase via phosphorylation of c-Jun and c-Myc 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 3060. doi:1538-7445.AM2012-3060

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

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. 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. 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. 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.

Abstract P2-06-14: Clinical relevance and biological properties of oligometastatic breast cancer in lung; prognostic impact of CD44+/CD24−/low cells

Background: Metastatic breast cancer is a systemic disease. Our aim was to evaluate the clinical outcomes of pulmonary metastasectomy of recurrent breast tumors and to identify possible prognostic factors. Methods: We reviewed data from a registry of patients with lung metastases from breast tumors who received pulmonary metastasectomy in Jikei University Hospital between 2004 and 2011. We analyzed prognostic factors for overall survival (OS) and progression free survival (PFS) after metastasectomy. We also investigated lung metastases for the prevalence of CD44+/CD24−/low tumor cells and evaluated their prognostic significance. Results: Among 17 patients with lung metastasis of breast tumors, 5-year OS and PFS were 72% and 36%, respectively. Better OS was observed among patients with oligometastatic breast cancer (OMBC). Patients with OMBC, estrogen receptor (ER) positive cells, and disease free intervals (DFI) of >8 years had better PFS. The average prevalence of CD44+/CD24−/low tumor cells in lung metastases of breast cancer was 21%, ranging from 0 to 90%. The presence of CD44+/CD24−/low tumor cells influenced the progression after lung metastasectomy, with median PFS times of only 6 months in patients with high-prevalence of cancer-initiating cells. CD44+/CD24−/low cells with cancer-initiating properties were present in only 9% ± 12 of patients with OMBC but were found in 73% ± 21 of patients with non-OMBC. Conclusion: Pulmonary metastasectomy may be a treatment option for OMBC patients with lung metastases. Better prognosis of OMBC may be related to low levels of cancer-initiating cells. Citation Format: Rei Mimoto, Tadashi Kobayashi, Yoshimi Imawari, Makiko Kamio, Kumiko Kato, Hiroko Nogi, Yasuo Toriumi, Ken Uchida, Hiroshi Takeyama. Clinical relevance and biological properties of oligometastatic breast cancer in lung; prognostic impact of CD44+/CD24−/low cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-14.