Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin†A1.

Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1-resistant cells were analyzed by high-throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1-resistant cells, SQLE, which encodes squalene epoxidase, was identified as a candidate gene. SQLE-depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild-type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.

Cadmium-coordinated supramolecule suppresses tumor growth of T-cell leukemia in mice.

Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 µM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 µM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 µM, which is comparable to that of 6.5 µM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia.

Anti-melanogenesis effect of Glechoma hederacea L. extract on B16 murine melanoma cells.

Glechoma hederacea L. (Labiatae) has been used in folk medicine to treat various ailments for centuries. We investigated the effects of G. hederacea extract on melanogenesis in B16 melanoma cells. It significantly reduced both the cellular melanin content and tyrosinase activity in a concentration-dependent manner. An MTT assay did not reveal any obvious cytotoxicity. Furthermore, we found that G. hederacea extract decreased tyrosinase and microphthalmia-associated transcription factor protein expression, but did not inhibit tyrosinase-related protein-1 and tyrosinase-related protein-2 expression. RT-PCR analysis indicated that the antimelanogenic effect of G. hederacea extract might be due to inhibition of tyrosinase gene transcription. Moreover, this effect is regulated via suppression of microphthalmia-associated transcription factor protein expression. Our data indicate that G. hederacea extract inhibits melanin synthesis in B16 melanoma cells but is not cytotoxic. Hence it might prove a useful therapeutic agent for treating hyperpigmentation and an effective component of whitening cosmetics.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity.

Pharmacological interventions to enhance fibrinolysis are effective for treating thrombotic disorders. Utilizing the in vitro U937 cell line-based fibrin degradation assay, we had previously found a cyclic pentapeptide malformin A1 (MA1) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA1-induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA1 showed that MA1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA1-induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA1-enhanced fibrinolysis in U937 cells. Synthetic active MA1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA1-induced phosphorylation of RSK1 and ERK1/2, indicating that MA1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA1 upregulated the expression of urokinase-type plasminogen activator (uPA) and increased uPA secretion. These inductions were abrogated in RSK1 knockout cells. These results indicate that RSK1 is a key regulator of MA1-induced extracellular fibrinolytic activity.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function.

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

Effective clearance of intracellular Leishmania major in vivo requires Pten in macrophages.

Leishmaniases are a major international public health problem, and macrophages are crucial for host resistance to this parasite. To determine if phosphatase and tensin homologue deleted on chromosome ten (Pten), a negative regulator of the PI3K pathway, plays a role in macrophage-mediated resistance to Leishmania, we generated C57BL/6 mice lacking Pten specifically in macrophages (LysMCrePten(flox/flox) mice). Examination of lesions resulting from Leishmania major infection showed that LysMCrePten(flox/flox) mice were more susceptible to the parasite than wild-type (WT) mice in the early phase of the infection, but were eventually able to eliminate the pathogen. In vitro Pten-deficient macrophages showed a reduced ability to kill parasites in response to IFN-gamma treatment, possibly because the mutant cells exhibited decreased TNF secretion that correlated with reductions in inducible nitric oxide synthase expression and nitric oxide production. In response to various TLR ligands, Pten-deficient macrophages produced less TNF and IL-12 but more IL-10 than WT cells. However, analysis of cells in the lymph nodes draining L. major inoculation sites indicated that both LysMCrePten(flox/flox) and WT mice developed normal Th1 responses following L. major infection, in line with the ability of LysMCrePten(flox/flox) mice to eventually eliminate the parasite. Our results indicate that the efficient clearance of intracellular parasites requires Pten in macrophages.

The Pten/PI3K pathway governs the homeostasis of Valpha14iNKT cells.

The tumor suppressor PTEN is mutated in many human cancers. We previously used the Cre-loxP system to generate mice (LckCrePten mice) with a Pten mutation in T-lineage cells. Here we describe the phenotype of Pten-deficient Valpha14iNKT cells. A failure in the development of Valpha14iNKT cells occurs in the LckCrePten thymus between stage 2 (CD44(high)NK1.1(-)) and stage 3 (CD44(high)NK1.1(+)), resulting in decreased numbers of peripheral Valpha14iNKT cells. In vitro, Pten-deficient Valpha14iNKT cells show reduced proliferation and cytokine secretion in response to alphaGalCer stimulation but enhanced inhibitory Ly49 receptor expression. Following interaction with dendritic cells (DCs) loaded with alphaGalCer, Pten-deficient Valpha14iNKT cells demonstrate activation of PI3K. Indeed, the effects of the Pten mutation require intact function of the PI3K subunits p110gamma and p110delta. In vivo, LckCrePten mice display reduced serum IFNgamma after alphaGalCer administration. Importantly, Valpha14iNKT cell-mediated protection against the metastasis of melanoma cells to the lung was impaired in the absence of Pten. Thus, the Pten/PI3K pathway is indispensable for the homeostasis and antitumor surveillance function of Valpha14iNKT cells.

Hyperplasia and carcinomas in Pten-deficient mice and reduced PTEN protein in human bladder cancer patients.

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate an urothelium-specific null mutation of Pten in mice [FabpCrePten(flox/flox) (FPten(flox/flox)) mice]. Histologic examination revealed that all FPten(flox/flox) mice exhibited urothelial hyperplasia in which component cells showed enlarged nuclei and increased cell size. With time, 10% of FPten(flox/flox) mice spontaneously developed pedicellate papillary transitional cell carcinomas (TCC). This type of tumor also arose in FPten(flox/flox) mice treated with the chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. FPten(flox/flox) urothelial cells were hyperproliferative and showed increased activation of the survival signaling molecules Akt and extracellular signal-regulated kinase. In humans, 53% of primary bladder cancer patients exhibited decreased or absent expression of PTEN protein in either the cytoplasm or nucleus of tumor cells. In early bladder cancers, PTEN expression was repressed in 42% of superficial papillary TCC but in only 8% of cases of carcinoma in situ (CIS). In advanced bladder cancers, PTEN protein was significantly reduced (particularly in the nucleus) in 94% of cases, and this decrease in PTEN correlated with disease stage and grade. Thus, PTEN deficiency may contribute to bladder cancer both by initiating superficial papillary TCC and by promoting the progression of CIS to advanced invasive and metastatic forms.

The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis.

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.