SPTAN1/NUMB axis senses cell density to restrain cell growth and oncogenesis through Hippo signaling.

The loss of contact inhibition is a key step during carcinogenesis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density-dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane-stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity-regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20-like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling.

Glycogen accumulation and phase separation drives liver tumor initiation.

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.

miR-29a-3p directly targets Smad nuclear interacting protein 1 and inhibits the migration and proliferation of cervical cancer HeLa cells.

Smad nuclear interacting protein 1 (SNIP1) is a nuclear protein and involved in essential biological processes. MicroRNAs are effective regulators of tumorigenesis and cancer progression via targeting multiple genes. In present study, we aimed to investigate the function of SNIP1 and identify novel miRNA-SNIP1 axis in the development of cervical cancer. The results showed for the first time that silencing of the SNIP1 gene inhibited the migration and proliferation in HeLa cells significantly. Bioinformatics analysis and dual luciferase reporter assay demonstrated that miR-29a-3p could target 3' UTR of SNIP1 directly. The mRNA and protein expression levels of SNIP1 were negative regulated by miR-29a-3p according to the RT-qPCR and Western blot analysis, respectively. Furthermore, functional studies showed that over-expression of miR-29a-3p restrained HeLa cells migration and proliferation, and the mRNA expression of SNIP1 downstream genes (HSP27, c-Myc, and cyclin D1) were down-regulated by miR-29a-3p. Together, we concluded that miR-29a-3p suppressed the migration and proliferation in HeLa cells by directly targeting SNIP1. The newly identified miR-29a-3p/SNIP1 axis could provide new insight into the development of cervical cancer.

FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis.

The external factors that modulate Hippo signaling remain elusive. Here, we report that FGF15 activates Hippo signaling to suppress bile acid metabolism, liver overgrowth, and tumorigenesis. FGF15 is induced by FXR in ileal enterocytes in response to increased amounts of intestinal bile. We found that circulating enterohepatic FGF15 stimulates hepatic receptor FGFR4 to recruit and phosphorylate NF2, which relieves the inhibitory effect of Raf on the Hippo kinases Mst1/2, thereby switching FGFR4's role from pro-oncogenic to anti-tumor signaling. The activated Mst1/2 subsequently phosphorylates and stabilizes SHP to downregulate the key bile acid-synthesis enzyme Cyp7a1 expression, thereby limiting bile acid synthesis. In contrast, Mst1/2 deficiency impairs bile acid metabolism and remarkably increases Cyp7a1 expression and bile acid production. Importantly, pharmacological depletion of intestinal bile abrogates Mst1/2-mutant-driven liver overgrowth and oncogenesis. Therefore, FGF15-Hippo signaling along the gut-liver axis acts as a sensor of bile acid availability to restrain liver size and tumorigenesis.

Expression of a wheat ß-1,3-glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel.

ß-1,3-glucanases, the plant PR-2 family of pathogenesis-related (PR) proteins, can be constitutively expressed and induced in wheat crop to enhance its anti-fungal pathogen defense. This study aimed to investigate the inhibitory effect of wheat ß-1,3-glucanase on fungi most commonly associated with wheat kernel. A ß-1,3-glucanase from wheat was successfully expressed in Pichia pastoris X-33 and its biochemical and antifungal properties were characterized herein. The molecular weight of recombinant ß-1,3-glucanase is approximately 33 kDa. ß-1,3-glucanase displays optimal activity at pH 6.5, remaining relatively high at pH 5.5-8.0. The optimal reaction temperature of ß-1,3-glucanase is 50 °C, retaining approximately 84.0% residual activity after heat-treated at 50 °C for 1 h. The steady-state kinetic parameters of ß-1,3-glucanase against laminarin was determined and the Km and Vmax were 1.32 ±â€¯0.20 mg/ml and 96.4 ±â€¯4.4 U mg-1 protein, respectively. The inhibitory effect of purified ß-1,3-glucanase against the seven fungi commonly associated with wheat kernel was assessed in vitro. ß-1,3-glucanase exerted differential inhibitory effects on hyphal growth of Fusarium graminearum, Alternaria sp., A. glaucus, A. flavus, A. niger, and Penicillium sp. Spore formation and mycelial morphology of Alternaria sp., A. flavus, and A. niger were significantly affected by ß-1,3-glucanase (1U). The present results would help elucidate the mechanism underlying the inhibition of wheat ß-1,3-glucanases on pathogens.

Hippo Signaling Suppresses Cell Ploidy and Tumorigenesis through Skp2.

Polyploidy can lead to aneuploidy and tumorigenesis. Here, we report that the Hippo pathway effector Yap promotes the diploid-polyploid conversion and polyploid cell growth through the Akt-Skp2 axis. Yap strongly induces the acetyltransferase p300-mediated acetylation of the E3 ligase Skp2 via Akt signaling. Acetylated Skp2 is exclusively localized to the cytosol, which causes hyper-accumulation of the cyclin-dependent kinase inhibitor p27, leading to mitotic arrest and subsequently cell polyploidy. In addition, the pro-apoptotic factors FoxO1/3 are overly degraded by acetylated Skp2, resulting in polyploid cell division, genomic instability, and oncogenesis. Importantly, the depletion or inactivation of Akt or Skp2 abrogated Hippo signal deficiency-induced liver tumorigenesis, indicating their epistatic interaction. Thus, we conclude that Hippo-Yap signaling suppresses cell polyploidy and oncogenesis through Skp2.

Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration.

Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay-based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.

[New osmo-regulational promoters in the industrial yeast].

OBJECTIVE: The work was aimed at selecting osmo-regulated prompters possessing excellent performance for further research of the industrial yeast Candida glycerinogenes. METHODS: Promoters PCgPGI, PCgTPI, PCgZWF, PCgSTL1, PCgSTL2 and PCgSTL3 were amplified by PCR and their bioinformatics analysis of stress response elements (STREs) were conducted. We constructed integrative plasmids containing 5.8S rDNA, a fluorescence protein gene gfp and a promoter PCgPGI, PCgTPI, PCgZWF, PCgSTL1, PCgSTL2 or PCgSTL3. The promoters' activities and osmo-regulations were compared according to the results of fluorescence and qRT-PCR. RESULTS: PCgSTL3 had more STREs, higher transcription level, lager gfp expression and it was more sensitive to stress. CONCLUSION: PCgSTL3 is an excellent induced promoter responding to hyperosmotic stress. Controlled expression of target genes can be realized using PCgSTL3 in the industrial yeast.