YAP1 is a potent driver of the onset and progression of oral squamous cell carcinoma.

Head-and-neck squamous cell carcinoma (HNSCC) is the sixth most common group of cancers in the world, and patients have a poor prognosis. Here, we present data indicating that YAP1 may be a strong driver of the onset and progression of oral SCC (OSCC), a major subtype of HNSCC. Mice with tongue-specific deletion of Mob1a/b and thus endogenous YAP1 hyperactivation underwent surprisingly rapid and highly reproducible tumorigenesis, developing tongue carcinoma in situ within 2 weeks and invasive SCC within 4 weeks. In humans, precancerous tongue dysplasia displays YAP1 activation correlating with reduced patient survival. Combinations of molecules mutated in OSCC may increase and sustain YAP1 activation to the point of oncogenicity. Strikingly, siRNA or pharmacological inhibition of YAP1 blocks murine OSCC onset in vitro and in vivo. Our work justifies targeting YAP1 as therapy for OSCC and perhaps HNSCC, and our mouse model represents a powerful tool for evaluating these agents.

Tyrosine-phosphorylated SOCS3 negatively regulates cellular transformation mediated by the myeloproliferative neoplasm-associated JAK2 V617F mutant.

In the majority of myeloproliferative neoplasms (MPNs) patients, a point mutation, V617F has been found in Janus kinase 2 (JAK2) gene, and this JAK2 mutant provoked aberrant signaling pathway. In the current study, we found that suppressor of cytokine signaling proteins 3 (SOCS3) possessed the tumor suppressive activity against the JAK2 V617F mutant-provoked cellular transformation. The knockdown of SOCS3 increased the expression level of the JAK2 V617F mutant, which enhanced the activation of signaling mediators, including signal transducer and activator of transcription 3 and 5 (STAT3, STAT5) and extracellular signal-regulated kinase (ERK), and also increased of the proliferation rate and tumorigenesis activity of Ba/F3 cells expressing the JAK2 V617F mutant and erythropoietin receptor (EpoR). In contrast, the enforced expression of SOCS3 significantly inhibited the JAK2 V617F mutant-induced activation of downstream signaling molecules, cell proliferation, and tumorigenesis by down-regulating the expression level of the JAK2 V617F mutant. SOCS3 interacted with the JAK2V617F mutant through its SH2 domain and was phosphorylated at Tyr-204 and Tyr-221 in its SOCS box by the JAK2V617F mutant. SOCS3 mutants carrying a mutation in the SH2 domain (R71E) and a substitution at Tyr-221 (Y221F) failed to exert inhibitory effects on JAK2V617F mutant-induced cellular transformation and tumorigenesis. Collectively, these results imply that SOCS3 plays a negative role in the JAK2 V617F mutant-induced oncogenic signaling pathway through its SH2 domain and the phosphorylation of Tyr-221 in its SOCS box.

Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Cell competition is involved in mammalian embryogenesis and tumor elimination and progression. It was previously shown that, whereas NIH3T3 mouse fibroblasts expressing high levels of the yes-associated protein 1(YAP1) target TEA domain family (TEAD) transcription factors become "winners" in cell competitions, Madin-Darby canine kidney cells expressing activated YAP1 become "losers" and are eliminated from culture monolayers. Thus, YAP1's role in cell competitions is clearly context dependent. Here, we show that keratinocytes overexpressing a constitutively activated YAP1 mutant lose in in vitro competitions with control cells conducted in standard tissue culture dishes and undergo apical extrusion. Similarly, cells in which endogenous YAP1 is activated by NF2 knockdown become losers. The YAP1-overexpressing cells exhibit a decrease in cell-matrix adhesion because of defective expression of adhesion molecules such as fibronectin-1. Cell adhesion-mediated proliferation is also impaired. However, because of intrinsic factors, YAP1-expressing cells proliferate faster than control cells when cocultured in dishes impeding cell adhesion. In vivo, Mob1a/b-deficient (YAP1-activated) epidermis, which shows decreased expression of type XVII collagen, cannot be engrafted successfully onto donor mice. YAP1-activated skin grafts shrink away from surrounding control skin, and the epidermis peels off the basement membrane. Our data show that YAP1 activation controls cell competition in part by decreasing cell adhesion.-Nishio, M., Miyachi, Y., Otani, J., Tane, S., Omori, H., Ueda, F., Togashi, H., Sasaki, T., Mak, T. W., Nakao, K., Fujita, Y., Nishina, H., Maehama, T., Suzuki, A. Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

Alpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK.

Anaplastic large cell lymphomas (ALCL) are mainly characterized by harboring the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The ALK inhibitor, crizotinib specifically induced apoptosis in Ba/F3 cells expressing NPM-ALK by inhibiting the activation of NPM-ALK and its downstream molecule, signal transducer and activator of transcription factor 3 (STAT3). We found that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. Although α-tocopherol suppressed the inhibitory effects of crizotinib on the signaling axis including NPM-ALK and STAT3, it had no influence on the intake of crizotinib into cells. Crizotinib also directly inhibited the kinase activity of NPM-ALK; however, this inhibitory effect was not altered by the co-treatment with α-tocopherol. Whereas the nuclear localization of NPM-ALK was disappeared by the treatment with crizotinib, the co-treatment with α-tocopherol swept the effect of crizotinib and caused the localization of NPM-ALK in nucleus. The administration of α-tocopherol attenuated the anti-tumor activity of crizotinib against NPM-ALK-provoked tumorigenesis in vivo. Furthermore, the α-tocopherol-induced inhibition of crizotinib-caused apoptosis was also observed in NPM-ALK-positive cells derived from ALCL patients, namely, SUDHL-1 and Ki-JK. Collectively, these results not only revealed the novel mechanism underlying crizotinib-induced apoptosis in NPM-ALK-positive cells, but also suggest that the anti-tumor effects of crizotinib are attenuated when it is taken in combination with vitamin E.

Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1.

Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein ß (C/EBPß) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPß activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.

Phosphorylated CIS suppresses the Epo or JAK2 V617F mutant-triggered cell proliferation through binding to EpoR.

The JAK2 V617F mutant-mediated aberrant signaling pathway is a hallmark of myeloproliferative neoplasms (MPNs). Although cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling (SOCS) are negative regulators of the JAK-STAT pathway, the functional role of CIS/SOCS family members in the JAK2 V617F mutant-induced oncogenic signaling pathway has not yet been elucidated. In this study, we found that the expression of CIS and SOCS1 was induced through the activation of signal transducer and activator of transcription 5 (STAT5) in not only the cells stimulated with Epo or IL-3 but also the cells transformed by the JAK2 V617F mutant. Cell proliferation and tumor formation in nude mice induced by the JAK2 V617F mutant were significantly enhanced when the expression of CIS was silenced using an RNA interference technique, whereas the knockdown of SOCS1 had no effect. The enforced expression of CIS caused apoptotic cell death in the transformed by JAK2 V617F mutant and drastically inhibited the JAK2 V617F mutant-induced tumor formation. CIS interacted with phosphorylated EpoR at Y401, which was critical for the activation of STAT5 and ERK. Whereas the activation of STAT5 and ERK in the transformed cells by JAK2 V617F mutant was increased by the knockdown of CIS, the enforced expression of CIS reduced the activation of these molecules. Furthermore, these anti-tumor effects of CIS required the function of SH2 domain and its tyrosine phosphorylation at Y253. We herein elucidated the mechanism by which CIS functions as a novel type of tumor suppressor in JAK2 V617F mutant-induced tumorigenesis.

Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis.

The erythropoietin receptor (EpoR) regulates development of blood cells, and its full activation normally requires the cytokine erythropoietin (Epo). In the case of myeloproliferative neoplasms (MPN), Epo-independent signaling through EpoR can be caused by a point mutation, V617F, in the EpoR-interacting tyrosine kinase Janus kinase 2 (JAK2). In cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellular domain of EpoR are phosphorylated, but the functional role of these phosphorylations in oncogenic signaling is incompletely understood. Here, to evaluate the functional consequences of the phosphorylation of these tyrosine residues, we constructed an EpoR-8YF mutant in which we substituted all eight tyrosine residues with phenylalanine. Co-expression of EpoR-8YF with the JAK2 V617F mutant failed to induce cytokine-independent cell proliferation and tumorigenesis, indicating that JAK2-mediated EpoR phosphorylation is the reason for JAK2 V617F mutant-induced oncogenic signaling. An exhaustive mutational analysis of the eight EpoR tyrosine residues indicated that three of these residues, Tyr-343, Tyr-460, and Tyr-464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity. We also showed that phosphorylation at these three residues was necessary for full activation of the transcription factor STAT5, which is a critical downstream factor of JAK2 V617F-induced oncogenic signaling. In contrast, Epo stimulation could moderately stimulate the proliferation of cells expressing wild type JAK2 and EpoR-8YF, suggesting that the requirement of the phosphorylation of these three tyrosine residues seems to be specific for the oncogenic proliferation provoked by V617F mutation. Collectively, these results have revealed that phosphorylation of Tyr-343, Tyr-460, and Tyr-464 in EpoR underlies JAK2 V617F mutant-induced tumorigenesis. We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.

Inhibitory effects of flavonoids extracted from Nepalese propolis on the LPS signaling pathway.

Flavonoids, particularly those derived from plants, harbor biological effects such as anti-inflammation and the inhibition of cancer progression. In the present study, we investigated the effects of 10 kinds of flavonoids isolated from Nepalese propolis on the LPS signaling pathway in order to clarify their anti-inflammatory activities. Five types of flavonoids: isoliquiritigenin, chrysin, 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, and cearoin, markedly inhibited inflammatory responses including LPS-induced NO production by suppressing the expression of iNOS mRNA and LPS-induced mRNA expression of TNFα and CCL2. Their inhibitory effects on LPS-induced inflammatory responses correlated with the intensities of these flavonoids to suppress the LPS-induced activation of nuclear factor κB (NF-κB), an essential transcription factor for the mRNA expression of iNOS, TNFα, and CCL2. Among these flavonoids, 3',4'-dihydroxy-4-methoxydalbergione and 4-methoxydalbergion markedly inhibited the LPS-induced activation of IKK, thereby abrogating the degradation of IκBα and nuclear localization of NF-κB. On the other hand, isoliquiritigenin, chrysin, and cearoin failed to inhibit these signaling steps, but suppressed the transcriptional activity of NF-κB, which caused their anti-inflammatory effects. The results of the present study revealed that these five kinds of flavonoids are the components of Nepalese propolis that exhibit anti-inflammatory activities with a different regulatory mechanism for the activation of NF-κB.

A bis-malonic acid fullerene derivative significantly suppressed IL-33-induced IL-6 expression by inhibiting NF-κB activation.

IL-33 functions as a ligand for ST2L, which is mainly expressed in immune cells, including mast cells. IL-33 is a potent inducer of pro-inflammatory cytokines, such as IL-6, and has been implicated in the pathogenesis of allergic inflammatory diseases. Therefore, IL-33 has recently been attracting attention as a new target for the treatment of inflammatory diseases. In the present study, we demonstrated that a water-soluble bis-malonic acid fullerene derivative (C60-dicyclopropane-1,1,1',1'-tetracarboxylic acid) markedly diminished the IL-33-induced expression of IL-6 in bone marrow-derived mast cells (BMMC). The bis-malonic acid fullerene derivative suppressed the canonical signaling steps required for NF-κB activation such as the degradation of IκBα and nuclear translocation of NF-κB by directly inhibiting the IL-33-induced IKK activation. Although p38 and JNK also contributed to IL-33-induced expression of IL-6, the bis-malonic acid fullerene derivative did not affect their activation. Furthermore, the bis-malonic acid fullerene derivative had no effect on the NF-κB activation pathway induced by TNFα and IL-1. These results suggest that the bis-malonic fullerene derivative has potential as a specific drug for the treatment of IL-33-induced inflammatory diseases by specifically inhibiting the NF-κB activation pathway.

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation.

Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPß, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.

Nepetaefuran and leonotinin isolated from Leonotis nepetaefolia R. Br. potently inhibit the LPS signaling pathway by suppressing the transactivation of NF-κB.

Leonotis nepetaefolia R. Br., also known as Klip Dagga or Lion's Ear, has traditionally been used as a folk medicine to treat inflammatory diseases such as rheumatism, bronchitis, and asthma; however, the components that exhibit its anti-inflammatory activity have not yet been identified. In the present study, we investigated the effects of three types of diterpenoids, nepetaefuran, leonotinin, and leonotin, which were isolated from L. nepetaefolia R. Br., on the LPS signaling pathway in order to elucidate the anti-inflammatory mechanism involved. Nepetaefuran more potently inhibited the LPS-induced production of NO and CCL2 than leonotinin by suppressing the expression of iNOS mRNA and CCL2 mRNA. On the other hand, leonotin failed to inhibit the production of NO and CCL2 induced by LPS. Although nepetaefuran and leonotinin had no effect on the LPS-induced degradation of IκBα or nuclear translocation of NF-κB p65, they markedly inhibited the transcriptional activity of NF-κB. Nepetaefuran and leonotinin also inhibited the transcriptional activity of the GAL4-NF-κB p65 fusion protein. On the other hand, nepetaefuran, leonotinin and leonotin did not affect the LPS-induced activation of MAP kinase family members such as ERK, p38, and JNK. In addition, inhibitory effect of nepetaefuran and leonotinin on NF-κB activation is well correlated with their ability to induce activation of Nrf2 and ER stress. Taken together, these results demonstrated that nepetaefuran and leonotinin could be the components responsible for the anti-inflammatory activity of L. nepetaefolia R. Br. by specifically inhibiting the LPS-induced activation of NF-κB.

Critical role of FANCC in JAK2 V617F mutant-induced resistance to DNA cross-linking drugs.

A point mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs) and an aberrant signaling pathway induced by constitutively active JAK2 V617F mutant is a hallmark of MPNs. Cells transformed by JAK2 V617F mutant exhibited resistance to anti-cancer drugs such as cisplatin (CDDP), mitomycin C (MMC) and bleomycin (BLM). We first found that the expression of FANCC, a member of the Fanconi anemia (FA) proteins, was significantly induced by JAK2 V617F mutant through activation of signal transducers and activators of transcription 5 (STAT5). In addition, monoubiqitination and foci formation of FANCD2, which are critical for activation of the FA pathway, were increased in cells transformed by JAK2 V617F mutant, compared to cells expressing wild-type JAK2. Interestingly, knockdown of FANCC in cells expressing JAK2 V617F mutant induced not only the reduction of monoubiqitination and foci formation of FANCD2 but also the enhancement of sensitivity to DNA damage induced by CDDP and MMC but not BLM. Taken together, FANCC is most likely to be critical for resistance to DNA cross-linking drug-induced DNA damage in cells transformed by JAK2 V617F mutant.

Dynamin isoforms decode action potential firing for synaptic vesicle recycling.

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.