Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice shows the implication of Rassf6 in the regulation of NF-κB signaling.

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised, and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis showed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.

The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity.

YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP's functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP's co-activation of TEAD-mediated CTGF transcription.

A pancreatic cancer mouse model with human immunity.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a tumor immune microenvironment (TIME) that promotes resistance to immunotherapy. A preclinical model system that facilitates studies of the TIME and its impact on the responsiveness of human PDAC to immunotherapies remains an unmet need. We report a novel mouse model, which develops metastatic human PDAC that becomes infiltrated by human immune cells recapitulating the TIME of human PDAC. The model may serve as a versatile platform to study the nature of human PDAC TIME and its response to various treatments.

Tumor-resident regulatory T cells in pancreatic cancer express the αvß5 integrin as a targetable activation marker.

Pancreatic ductal adenocarcinoma (PDAC) has abundant immunosuppressive regulatory T cells (Tregs), which contribute to a microenvironment resistant to immunotherapy. Here, we report that Tregs in the PDAC tissue, but not those in the spleen, express the αvß5 integrin in addition to neuropilin-1 (NRP-1), which makes them susceptible to the iRGD tumor-penetrating peptide, which targets cells positive for αv integrin- and NRP-1. As a result, long-term treatment of PDAC mice with iRGD leads to tumor-specific depletion of Tregs and improved efficacy of immune checkpoint blockade. αvß5 integrin + Tregs are induced from both naïve CD4 + T cells and natural Tregs upon T cell receptor stimulation, and represent a highly immunosuppressive subpopulation of CCR8 + Tregs. This study identifies the αvß5 integrin as a marker for activated tumor-resident Tregs, which can be targeted to achieve tumor-specific Treg depletion and thereby augment anti-tumor immunity for PDAC therapy.

Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver.

After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.

Tumor-penetrating therapy for ß5 integrin-rich pancreas cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia and drug resistance due, in part, to poor drug delivery to extravascular tumor tissue. Here, we report that carcinoma-associated fibroblasts (CAFs) induce ß5 integrin expression in tumor cells in a TGF-ß dependent manner, making them an efficient drug delivery target for the tumor-penetrating peptide iRGD. The capacity of iRGD to deliver conjugated and co-injected payloads is markedly suppressed when ß5 integrins are knocked out in the tumor cells. Of note, ß5 integrin knock-out in tumor cells leads to reduced disease burden and prolonged survival of the mice, demonstrating its contribution to PDAC progression. iRGD significantly potentiates co-injected chemotherapy in KPC mice with high ß5 integrin expression and may be a powerful strategy to target an aggressive PDAC subpopulation.

Effect of Diphtheria Toxin-Based Gene Therapy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a major global malignancy, responsible for >90% of primary liver cancers. Currently available therapeutic options have poor performances due to the highly heterogeneous nature of the tumor cells; recurrence is highly probable, and some patients develop resistances to the therapies. Accordingly, the development of a novel therapy is essential. We assessed gene therapy for HCC using a diphtheria toxin fragment A (DTA) gene-expressing plasmid, utilizing a non-viral hydrodynamics-based procedure. The antitumor effect of DTA expression in HCC cell lines (and alpha-fetoprotein (AFP) promoter selectivity) is assessed in vitro by examining HCC cell growth. Moreover, the effect and safety of the AFP promoter-selective DTA expression was examined in vivo using an HCC mice model established by the hydrodynamic gene delivery of the yes-associated protein (YAP)-expressing plasmid. The protein synthesis in DTA transfected cells is inhibited by the disappearance of tdTomato and GFP expression co-transfected upon the delivery of the DTA plasmid; the HCC cell growth is inhibited by the expression of DTA in HCC cells in an AFP promoter-selective manner. A significant inhibition of HCC occurrence and the suppression of the tumor marker of AFP and des-gamma-carboxy prothrombin can be seen in mice groups treated with hydrodynamic gene delivery of DTA, both 0 and 2 months after the YAP gene delivery. These results suggest that DTA gene therapy is effective for HCC.

Novel YAP1 Activator, Identified by Transcription-Based Functional Screen, Limits Multiple Myeloma Growth.

Yes-associated protein 1 (YAP1) interacts with numerous transcription factors, including TEA-domain family proteins (TEAD) and p73. YAP1 is negatively regulated by the tumor suppressor Hippo pathway. In human cancers, the deregulation of the Hippo pathway and YAP1 gene amplification lead to the activation of YAP1, which induces epithelial-mesenchymal transition (EMT) and drug resistance. YAP1 inhibitors are expected to be useful in cancer therapy. On the other hand, in certain cancers, YAP1 upregulates p73-dependent gene transcription and behaves as a tumor suppressor. Moreover, as YAP1 regulates self-renewal and differentiation of tissue stem cells and plays an important role in tissue homeostasis, YAP1 activators may contribute to the regenerative medicine. With this in our mind, we screened for YAP1 activators by using human retinal pigment epithelial ARPE-19 cells expressing the TEAD-responsive fluorescence reporter under the coexpression of YAP1. From an extensive chemical compound library (n = 18,606) 47 candidate YAP1 activators were identified. These compounds were characterized to determine whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 activator was effective against the human multiple myeloma IM-9 cells and chronic myeloid leukemia K562 cells.Implications: YAP1 activation limits growth, induces apoptosis, and may be useful at suppressing hematological cancers. Mol Cancer Res; 16(2); 197-211. ©2017 AACR.

Effect of a neural relay on liver regeneration in mice: activation of serotonin release from the gastrointestinal tract.

The development of therapeutic options to promote hepatic regeneration following severe liver injury is essential. While humoral factors have been reported as mechanisms of liver regeneration, the contributions of interorgan communication to liver regeneration have not been reported. In this study, we examined the effect of a neural relay on liver regeneration via activation of serotonin release from the gastrointestinal (GI) tract. Our results demonstrated that the afferent visceral nerve from the liver activates the efferent vagus nerve from the brain, leading to activation of serotonin release from the GI tract and contributing to liver regeneration. While it is difficult to apply these results directly to human health, we believe that this study may represent a step toward developing essential therapeutics to promote liver regeneration.

Erratum: YAP determines the cell fate of injured mouse hepatocytes in vivo.

This corrects the article DOI: 10.1038/ncomms16017.

YAP determines the cell fate of injured mouse hepatocytes in vivo.

The presence of senescent, transformed or damaged cells can impair tissue function or lead to tumorigenesis; therefore, organisms have evolved quality control mechanisms to eliminate them. Here, we show that YAP activation induced by inactivation of the Hippo pathway specifically in damaged hepatocytes promotes their selective elimination by using in vivo mosaic analysis in mouse liver. These damaged hepatocytes migrate into the hepatic sinusoids, undergo apoptosis and are engulfed by Kupffer cells. In contrast, YAP activation in undamaged hepatocytes leads to proliferation. Cellular stresses such as ethanol that damage both liver sinusoidal endothelial cells and hepatocytes switch cell fate from proliferation to migration/apoptosis in the presence of activated YAP. This involves the activation of CDC42 and Rac that regulate cell migration. Thus, we suggest that YAP acts as a stress sensor that induces elimination of injured cells to maintain tissue and organ homeostasis.

Age-dependent motor dysfunction due to neuron-specific disruption of stress-activated protein kinase MKK7.

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family and controls various physiological processes including apoptosis. A specific upstream activator of JNKs is the mitogen-activated protein kinase kinase 7 (MKK7). It has been reported that MKK7-JNK signaling plays an important regulatory role in neural development, however, post-developmental functions in the nervous system have not been elucidated. In this study, we generated neuron-specific Mkk7 knockout mice (MKK7 cKO), which impaired constitutive activation of JNK in the nervous system. MKK7 cKO mice displayed impaired circadian behavioral rhythms and decreased locomotor activity. MKK7 cKO mice at 8 months showed motor dysfunctions such as weakness of hind-limb and gait abnormality in an age-dependent manner. Axonal degeneration in the spinal cord and muscle atrophy were also observed, along with accumulation of the axonal transport proteins JNK-interacting protein 1 and amyloid beta precursor protein in the brains and spinal cords of MKK7 cKO mice. Thus, the MKK7-JNK signaling pathway plays important roles in regulating circadian rhythms and neuronal maintenance in the adult nervous system.

Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells.

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.

MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status.

Cell competition is a cell-cell interaction by which a cell compares its fitness to that of neighboring cells. The cell with the relatively lower fitness level is the "loser" and actively eliminated, while the cell with the relatively higher fitness level is the "winner" and survives. Recent studies have shown that cells with high Yes-associated protein (YAP) activity win cell competitions but the mechanism is unknown. Here, we report the unexpected finding that cells overexpressing constitutively active YAP undergo apical extrusion and are losers, rather than winners, in competitions with normal mammalian epithelial cells. Inhibitors of metabolism-related proteins such as phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR), or p70S6 kinase (p70S6K) suppressed this apical extrusion, as did knockdown of vimentin or filamin in neighboring cells. Interestingly, YAP-overexpressing cells switched from losers to winners when co-cultured with cells expressing K-Ras (G12V) or v-Src. Thus, the role of YAP in deciding cell competitions depends on metabolic factors and the status of neighboring cells.

The mevalonate pathway regulates primitive streak formation via protein farnesylation.

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.

A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs on Early Embryogenesis.

Mammalian fetal development is easily disrupted by exogenous agents, making it essential to test new drug candidates for embryotoxicity and teratogenicity. To standardize the testing of drugs that might be used to treat pregnant women, the U.S. Food and Drug Administration (FDA) formulated special grade categories, labeled A, B, C, D and X, that define the level of risk associated with the use of a specific drug during pregnancy. Drugs in categories (Cat.) D and X are those with embryotoxic and/or teratogenic effects on humans and animals. However, which stages of pregnancy are affected by these agents and their molecular mechanisms are unknown. We describe here an embryonic stem cell test (EST) that classifies FDA pregnancy Cat.D and Cat.X drugs into 4 classes based on their differing effects on primitive streak formation. We show that ~84% of Cat.D and Cat.X drugs target this period of embryogenesis. Our results demonstrate that our modified EST can identify how a drug affects early embryogenesis, when it acts, and its molecular mechanism. Our test may thus be a useful addition to the drug safety testing armamentarium.

CLOCK:BMAL-independent circadian oscillation of zebrafish Cryptochrome1a gene.

In the vertebrate circadian feedback loop, CLOCK:BMAL heterodimers induce the expression of Cry genes. The CRY proteins in turn inhibit CLOCK:BMAL-mediated transcription closing the negative feedback loop. Four CRYs, which all inhibit CLOCK:BMAL-mediated transcription, exist in zebrafish. Although these zebrafish Crys (zCry1a, 1b, 2a, and 2b) show a circadian pattern of expression, previous studies have indicated that the circadian oscillation of zCry1a could be CLOCK:BMAL-independent. Here we show that abrogation of CLOCK:BMAL-dependent transcription in zebrafish cells by the dominant negative zCLOCK3-DeltaC does not affect the circadian oscillation of zCry1a. Moreover, we provide several lines of evidence indicating that the extracellular signal-regulated kinase (ERK) signaling cascade modulates the circadian expression of zCry1a gene in constant darkness. Taken together, our data strongly support the notion that circadian oscillation of zCry1a is CLOCK:BMAL-independent and further indicate that mechanisms involving non-canonical clock genes could contribute to the circadian expression of zCry1a gene in a cell autonomous manner.

Common light signaling pathways controlling DNA repair and circadian clock entrainment in zebrafish.

UV radiation causes a number of harmful events including growth delay, cell death and ultimately cancer. The reversal of such effects by concomitant exposure to visible light is a conserved mechanism which has been uncovered in many multi-cellular organisms. Here we show that light-dependent UV-tolerance is a cell autonomous phenomenon in zebrafish. In addition, we provide several lines of evidence indicating that light induction of 64PHR, a DNA repair enzyme, and the subsequent light-dependent DNA repair mediated by this enzyme are prerequisites for light-mediated UV tolerance. 64PHR is evolutionary related to and has a high degree of structural similarity to animal CRY, an essential circadian regulator. The zebrafish circadian clock is controlled by a cell-autonomous and light-dependent oscillator, where zCRY1a functions as an important mediator of light entrainment of the circadian clock. In this study, we show that light directly activates MAPK signaling cascades in zebrafish cells and we provide evidence that light-induced activation of these pathways controls the expression of two evolutionary-related genes, z64Phr and zCry1a, revealing that light-dependent DNA repair and the entrainment of circadian clock share common regulatory pathways.