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.

Background & Aims: 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. In vitro 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. Methods: To investigate the role of Dyrk2 during hepatocarcinogenesis, we developed liver-specific Dyrk2 conditional knockout mice and an in vivo 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. Results: 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. Conclusions: 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. Impact and Implications: 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.

PKCδ deficiency inhibits fetal development and is associated with heart elastic fiber hyperplasia and lung inflammation in adult PKCδ knockout mice.

Protein kinase C-delta (PKCδ) has a caspase-3 recognition sequence in its structure, suggesting its involvement in apoptosis. In addition, PKCδ was recently reported to function as an anti-cancer factor. The generation of a PKCδ knockout mouse model indicated that PKCδ plays a role in B cell homeostasis. However, the Pkcrd gene, which is regulated through complex transcription, produces multiple proteins via alternative splicing. Since gene mutations can result in the loss of function of molecular species required for each tissue, in the present study, conditional PKCδ knockout mice lacking PKCδI, II, IV, V, VI, and VII were generated to enable tissue-specific deletion of PKCδ using a suitable Cre mouse. We generated PKCδ-null mice that lacked whole-body expression of PKCδ. PKCδ+/- parental mice gave birth to only 3.4% PKCδ-/- offsprings that deviated significantly from the expected Mendelian ratio (χ2(2) = 101.7, P < 0.001). Examination of mice on embryonic day 11.5 (E11.5) showed the proportion of PKCδ-/- mice implanted in the uterus in accordance with Mendelian rules; however, approximately 70% of the fetuses did not survive at E11.5. PKCδ-/- mice that survived until adulthood showed enlarged spleens, with some having cardiac and pulmonary abnormalities. Our findings suggest that the lack of PKCδ may have harmful effects on fetal development, and heart and lung functions after birth. Furthermore, our study provides a reference for future studies on PKCδ deficient mice that would elucidate the effects of the multiple protein variants in mice and decipher the roles of PKCδ in various diseases.

Carbonic anhydrase 13 suppresses bone metastasis in breast cancer.

Metastatic progression is the leading cause of mortality in breast cancer. However, molecular mechanisms that govern this process remain unclear. In this study, we found that carbonic anhydrase 13 (CA13) plays a potential role in suppressing bone metastasis. iRFP713-labeled iCSCL-10A (iRFP-iCSCL-10A) breast cancer cells, which exhibit the hallmarks of cancer stem cells, exerted the ability of bone metastasis in hind legs after 5-week injections, whereas no metastasis was observed in control iRFP713-labeled MCF-10A (iRFP-MCF10A) cells. Transcriptome analysis indicated that the expression of several genes, including metabolism-related CA13, was reduced in bone metastatic iRFP-iCSCL-10A cells. In vitro and in vivo analyses demonstrated that overexpression of CA13 in iRFP-iCSCL-10A cells suppressed migration, invasion, and bone metastasis, together with the reduction of VEGF-A and M-CSF expression. Furthermore, we found that breast cancer patients with a low CA13 expression had significantly shorter overall survival and disease-free survival rates compared to those with higher CA13 expression. These findings suggest that CA13 may act as a novel prognostic biomarker and would be a therapeutic candidate for the prevention of bone metastasis in breast cancer.

Clinical implications of drug-screening assay for recurrent metastatic hormone receptor-positive, human epidermal receptor 2-negative breast cancer using conditionally reprogrammed cells.

Various new drugs have been developed for treating recurrent hormone receptor-positive (HR+)/human epidermal receptor 2-negative (HER2-) breast cancer. However, directly identifying effective drugs remains difficult. In this study, we elucidated the clinical relevance of cultured cells derived from patients with recurrent HR+/HER2- metastatic breast cancer. The recently established conditionally reprogrammed (CR) cell system enables us to examine heterogeneity, drug sensitivity and cell function using patient-derived tumour samples. The results of microarray analysis, DNA target sequencing and xenograft experiments indicated that the mutation status and pathological features were preserved in CR cells, whereas RNA expression was different from that in the primary tumour cells, especially with respect to cell adhesion-associated pathways. The results of drug sensitivity assays involving the use of primary breast cancer CR cells were consistent with gene expression profiling test data. We performed drug-screening assays using liver metastases, which were sensitive to 66 drugs. Importantly, the result reflected the actual clinical course of this patient. These results supported the use of CR cells obtained from the metastatic lesions of patients with HR+/HER2- breast cancer for predicting the clinical drug efficacy.

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.

Tumor suppressive role for kinases phosphorylating p53 in DNA damage-induced apoptosis.

Tumor suppressor p53 plays an important role in cancer prevention. Under normal conditions, p53 is maintained at a low level. However, in response to various cellular stresses, p53 is stabilized and activated, which, in turn, initiates DNA repair, cell-cycle arrest, senescence and apoptosis. Post-translational modifications of p53 including phosphorylation, ubiquitination, and acetylation at multiple sites are important to regulate its activation and subsequent transcriptional gene expression. Particularly, phosphorylation of p53 plays a critical role in modulating its activation to induce apoptosis in cancer cells. In this context, previous studies show that several serine/threonine kinases regulate p53 phosphorylation and downstream gene expression. The molecular basis by which p53 and its kinases induce apoptosis for cancer prevention has been extensively studied. However, the relationship between p53 phosphorylation and its kinases and how the activity of kinases is controlled are still largely unclear; hence, they need to be investigated. In this review, we discuss various roles for p53 phosphorylation and its responsible kinases to induce apoptosis and a new therapeutic approach in a broad range of cancers.

Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages.

Previous genetic studies in mice have shown that functional loss of activin receptor-like kinase 7 (ALK7), a type I transforming growth factor-ß receptor, increases lipolysis to resist fat accumulation in adipocytes. Although growth/differentiation factor 3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess conditions, it is unknown how GDF3 production is regulated. Here, we show that a physiologically low level of insulin converts CD11c- adipose tissue macrophages (ATMs) into GDF3-producing CD11c+ macrophages ex vivo and directs ALK7-dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and adipocytes represents a previously unrecognized mechanism by which insulin regulates both fat metabolism and mass.

Downregulation of CD24 suppresses bone metastasis of lung cancer.

Suppression of bone metastasis can improve patient quality of life. Current drugs for bone metastasis have been shown to prolong progression-free survival but not overall survival; therefore, other potential therapeutic targets for bone metastasis should be investigated. Cell-surface antigens, such as CD24, have been recently shown to be involved in the metastasis of various cancers. However, whether CD24 plays a role in bone metastasis of lung cancer remains unknown. To observe metastasis of lung cancer cells by imaging technology, we introduced a near-infrared fluorescent protein, iRFP720, into a bone-seeking subclone established from lung cancer cells, HARA-B4 cells. The anchorage-independent growth of these cells was then evaluated by colony formation assays. We also compared cancer cell tropism to bone tissue with HARA-B4 cells in the presence or absence of CD24 by cell adhesion assays. To clarify the role of CD24 in bone metastasis, we intracardially injected CD24-knockdown HARA-B4 cells into mice and monitored metastasis through detection of iRFP720 using an in vivo imaging system. CD24-knockdown HARA-B4 cells in vitro showed reduced anchorage-independent growth and cancer cell tropism to bone. Bone metastasis was diminished in mice inoculated with CD24-knockdown HARA-B4 cells, which was rescued by add-back of CD24 in cells. Our findings indicate that iRFP720 is effective for in vivo imaging analysis of bone metastasis and that downregulation of CD24 suppresses bone metastasis of lung cancer cells. These findings collectively indicate that CD24 may be considered a promising new therapeutic candidate for the prevention of bone metastasis of lung 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.

A Novel Near-infrared Fluorescent Protein, iRFP720, Facilitates Transcriptional Profiling of Prostate Cancer Bone Metastasis in Mice.

BACKGROUND: Bone represents a frequent site of prostate cancer metastasis. As the molecular mechanism remains unclear, an accessible animal model is required. MATERIALS AND METHODS: We established a novel murine metastasis model using near-infrared fluorescent protein iRFP720-labelled prostate cancer (PC3) cells. To clarify transcriptional alterations during metastasis, iRFP720-PC3 cells were intracardially injected into male mice. mRNA expression profiles of metastasis in bone using marrow cancer cells extracted by centrifugal separation and cell sorting were compared with those of parental cells by microarray. Differentially expressed genes were analyzed by pathway analysis. RESULTS: We identified 327 and 197 genes being up- and down-regulated, respectively. Pathway analysis revealed that the p53 signaling pathway, extracellular matrix receptor interaction, Mammalian target of rapamycin signaling pathway, cancer-related pathways, small cell lung cancer, and Escherichia coli infection response were altered. CONCLUSION: iRFP720 is useful for in vivo cell detection/isolation. The results of expression analysis may improve prostate cancer treatment strategies.

Interaction between RNF8 and DYRK2 is required for the recruitment of DNA repair molecules to DNA double-strand breaks.

The genome of eukaryotic cells is frequently exposed to damage by various genotoxins. Phosphorylation of histone H2AX at Serine 139 (γ-H2AX) is a hallmark of DNA damage. RNF8 monoubiquitinates γ-H2AX with the Lys63-linked ubiquitin chain to tether DNA repair molecules at DNA lesions. A high-throughput screening identified RNF8 as a binding partner of dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2). Notably, DNA damage-induced monoubiquitination of γ-H2AX is impaired in DYRK2-depleted cells. The foci formation of p53-binding protein 1 at DNA double-strand break sites is suppressed in DYRK2 knockdown cells, which fail to repair the DNA damage. A homologous recombination assay showed decreased repair efficiency in DYRK2-depleted cells. Our findings indicate direct interaction of DYRK2 with RNF8 in regulating response to DNA damage.

Transcriptional induction of protein kinase C delta by p53 tumor suppressor in the apoptotic response to DNA damage.

Genetic alterations and aberrant gene expression trigger malignant tumors. Tumor suppressor p53 is the most altered gene in human cancers. p53 induces apoptosis by promoting pro-apoptotic genes in response to DNA damage. Protein kinase C delta (PKCδ) also induces apoptosis via various mechanisms including modification of p53. The PKCδ-dependent apoptotic mechanism has been extensively studied; however, the transcriptional regulation of PKCδ remains obscure. The current study demonstrates the transcriptional regulation of PKCδ by p53 upon genotoxic stress. The p53-binding site in the promoter region of PKCδ was detected by the ChIP-sequencing assay. Notably, the expression of PKCδ was increased upon DNA damage, which is required for the stabilization of p53. More importantly, targeting single guide RNA-driven dead Cas9 to the p53-binding site of PKCδ disturbed p53-promoted PKCδ expression and suppressed apoptosis following DNA damage. Thus, our findings suggest that the transcriptional regulation of PKCδ is controlled by p53 in a positive feedback mechanism to induce apoptosis in response to DNA damage.

TFE3 controls lipid metabolism in adipose tissue of male mice by suppressing lipolysis and thermogenesis.

Transcription factor E3 (TFE3) is a transcription factor that binds to E-box motifs and promotes energy metabolism-related genes. We previously reported that TFE3 directly binds to the insulin receptor substrate-2 promoter in the liver, resulting in increased insulin response. However, the role of TFE3 in other tissues remains unclear. In this study, we generated adipose-specific TFE3 transgenic (aP2-TFE3 Tg) mice. These mice had a higher weight of white adipose tissue (WAT) and brown adipose tissue than wild-type (WT) mice under fasting conditions. Lipase activity in the WAT in these mice was lower than that in the WT mice. The mRNA level of adipose triglyceride lipase (ATGL), the rate-limiting enzyme for adipocyte lipolysis, was significantly decreased in aP2-TFE3 Tg mice. The expression of Foxo1, which directly activates ATGL expression, was also suppressed in transgenic mice. Promoter analysis confirmed that TFE3 suppressed promoter activities of the ATGL gene. In contrast, G0S2 and Perilipin1, which attenuate ATGL activity, were higher in transgenic mice than in WT mice. These results indicated that the decrease in lipase activity in adipose tissues was due to a decrease in ATGL expression and suppression of ATGL activity. We also showed that thermogenesis was suppressed in aP2-TFE3 Tg mice. The decrease in lipolysis in WAT of aP2-TFE3 Tg mice inhibited the supply of fatty acids to brown adipose tissue, resulting in the inhibition of the expression of thermogenesis-related genes such as UCP1. Our data provide new evidence that TFE3 regulates lipid metabolism by controlling the gene expression related to lipolysis and thermogenesis in adipose tissue.

Ubiquitylation and degradation of serum-inducible kinase by hVPS18, a RING-H2 type ubiquitin ligase.

Serum-inducible kinase (SNK) is a member of polo-like kinases that serve as regulators of multiple events during cell division. Rapid changes in the activity and abundance of SNK were reported after the serum stimulation and after the activation of synaptic transmission in the brain. Yet the detailed mechanisms that control the level of SNK protein have not been fully elucidated. In this report, we show that the RING-H2 domain of hVPS18 (human vacuolar protein sorting 18) has a genuine ubiquitin ligase (E3) activity. Using the yeast two-hybrid screening, we identify SNK as a candidate substrate of hVPS18. The half-life of SNK is increased in HeLa cells that down-regulated hVPS18 by lentivirus-mediated small hairpin RNA interference. Furthermore, the delayed entry into S phase is observed in HeLa cells overexpressing hVPS18. These results suggest that hVPS18 may play an important role in regulation of SNK activity through its ubiquitin ligase.

Mammalian Numb is a target protein of Mdm2, ubiquitin ligase.

Drosophila Numb protein functions as an antagonist against Notch signal. The expression of this protein is asymmetrical in divided cells and thought to be involved in the neural cell differentiation and/or cell fate. Human homologue of Numb (hNumb) was cloned as Mdm2-binding protein by yeast two-hybrid screening. Since Mdm2 is an oncoprotein and has ubiquitin ligase activity toward tumor suppressor p53, we assessed to find out whether Mdm2 ubiquitinylates the hNumb protein. The recombinant hNumb expressed in Sf-9 cells using baculovirus protein expression system bound to Mdm2 in vitro. When hNumb was subjected to in vitro ubiquitinylation assay system, which contains E1, E2, or UbcH5c, and Mdm2, hNumb was ubiquitinylated as efficiently as the p53 protein. However, when the Ring-finger domain mutant of Mdm2 was used in place of wild-type Mdm2, hNumb was not ubiquitinylated. Furthermore, when U2OS cells were co-transfected with hNumb and Mdm2, the hNumb protein was ubiquitinylated and degraded. These data strongly suggest that Mdm2 functions as the ubiquitin ligase toward hNumb and that it induces its degradation in intact cells.

Sumoylation of Mdm2 by protein inhibitor of activated STAT (PIAS) and RanBP2 enzymes.

Mdm2, a ubiquitin ligase that acts on p53, is regulated by sumoylation. In the current study, we identify the enzymes responsible for the sumoylation of Mdm2. When mammalian cells are co-transfected with cDNAs encoding Mdm2 and PIAS1 or PIASxbeta (protein inhibitor of activated STAT) as sumoylation enzymes, Mdm2 is highly sumoylated. Mdm2 is also sumoylated in an in vitro system containing PIASxbeta, PIAS1, and RanBP2. When several lysine residues of Mdm2 were sequentially mutated to arginine, the K182R mutant was not sumoylated in intact cells; however, in the in vitro system this mutant was sumoylated by PIAS1, PIASxbeta, and RanBP2 as efficiently as the wild-type Mdm2 protein. Lysine residues 182 and 185 map within the nuclear localization signal of Mdm2. A K185R mutant of Mdm2 is sumoylated in intact cells, whereas a K182R protein is not. Only a Mdm2 protein bearing the K182R mutation is localized exclusively in the cytoplasm. Because RanBP2 is a nuclear pore protein and PIAS proteins are localized within the nucleus, our data suggest that Mdm2 is sumoylated during nuclear translocation by RanBP2 and then further sumoylated once in the nucleus by PIASxbeta and PIAS1.