PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

Contact reductions from live poultry market closures limit the epidemic of human infections with H7N9 influenza.

An early steep increase in the number of humans infected with avian influenza A(H7N9) virus was observed in China, raising great public concern domestically and internationally. Little is known about the dynamics of the transmission contacts between poultry and human populations, although such understanding is essential for developing effective strategies to control this zoonosis. In this study, we evaluated the effects of contact reductions from live poultry markets (LPMs) closures on the transmission of H7N9 virus during epidemics in Guangdong Province, China. A mathematical model of the poultry-to-person transmission dynamics of H7N9 virus was constructed. The parameters in the model were estimated from publicly available data on confirmed cases of human infection and information on LPMs closure during 2013-2017. By fitting the model, we measured the time-dependent contact quantity of the susceptible population to LPMs. The results showed that periodic intervention strategies can greatly reduce the magnitude of outbreaks, and the earlier interventions for policy are implemented, the smaller is the outbreak. The control efforts for LPMs to decrease the contact quantity are critical in preventing epidemics in the long term. This model should provide important insights for the development of a national intervention strategy for the long-term control of avian influenza virus epidemics.

An Integrative Analysis Reveals a Central Role of P53 Activation via MDM2 in Zika Virus Infection Induced Cell Death.

Zika virus (ZIKV) infection is an emerging global threat that is suspected to be associated with fetal microcephaly. However, the molecular mechanisms underlying ZIKV disease pathogenesis in humans remain elusive. Here, we investigated the human protein interaction network associated with ZIKV infection using a systemic virology approach, and reconstructed the transcriptional regulatory network to analyze the mechanisms underlying ZIKV-elicited microcephaly pathogenesis. The bioinformatics findings in this study show that P53 is the hub of the genetic regulatory network for ZIKV-related and microcephaly-associated proteins. Importantly, these results imply that the ZIKV capsid protein interacts with mouse double-minute-2 homolog (MDM2), which is involved in the P53-mediated apoptosis pathway, activating the death of infected neural cells. We also found that synthetic mimics of the ZIKV capsid protein induced cell death in vitro and in vivo. This study provides important insight into the relationship between ZIKV infection and brain diseases.

Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR.

Single tumor-initiating cells evade immune clearance by recruiting type II macrophages.

Tumor infiltrated type II (M2) macrophages promote tumorigenesis by suppressing immune clearance, promoting proliferation, and stimulating angiogenesis. Interestingly, macrophages were also found to enrich in small foci of altered hepatocytes containing liver tumor-initiating cells (TICs). However, whether and how TICs specifically recruit macrophages and the function of these macrophages in tumor initiation remain unknown due to technical difficulties. In this study, by generating genetically defined liver TICs, we demonstrate that TICs actively recruit M2 macrophages from as early as the single-cell stage. Elimination of TIC-associated macrophages (TICAMs) abolishes tumorigenesis in a manner dependent on the immune system. Mechanistically, activation of the Hippo pathway effector Yes-associated protein (YAP) underlies macrophage recruitment by TICs. These results demonstrate for the first time that macrophages play a decisive role in the survival of single TICs in vivo and provide a proof of principle for TIC elimination by targeting YAP or M2 macrophages.

A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis.

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

RNA interference mediated JAM-A gene silencing promotes human epidermal stem cell proliferation.

The objective of the study was to explore the influence of junctional adhesion molecule A (JAM-A) gene decoration on proliferation and differentiation of human epidermal stem cells (hEpSCs). JAM-A gene and JAM-A interference gene lentivirus eukaryotic expression vectors were established. The recombinant lentivirus was introduced into hEpSCs to observe and detect viral transfection by fluorescence microscopy and Western blot, respectively. After confirmation of successful introduction of the target gene, cell growth curves were mapped out by cytometry to detect cell proliferation in different groups. The expression of hEpSCs labeled molecules was detected by immunofluorescence, and cell safety was detected by teratoma test in all groups. (1) Fluorescence microscopy showed that in the JAM-A over-expression (JAM-A(ov) EpSCs) group, the green fluorescence was mainly distributed in the cell membrane; in the JAM-A interference (JAM-A(kd) EpSCs) group and blank vector (GFP EpSCs) group, all cell bodies were luminous. Western blot showed that JAM-A protein was up-regulated in JAM-A(ov) EpSCs and down-regulated in JAM-A(kd) EpSCs. (2) Growth curves showed that hEpSCs entered the quick-growing phase 4 days after inoculation and reached the platform phase at day 7. JAM-A(ov) EpSCs proliferated more slowly than GFP EpSCs, while JAM-A(kd) EpSCs proliferated significantly faster than GFP EpSCs. (3) Immunofluorescence showed that the expression of transient amplification epidermal marker keratin 14, hEpSCs marker keratin I9 and ß-integrin was down-regulated in JAM-A(kd) EpSCs group as compared to that in the GFP EpSCs group, and the expression of epidermal terminal differentiation marker K10 was negative in the JAM-A(kd) EpSCs group. There was no significant difference in the expression of specific molecules between JAM-A(ov) EpSCs and hEpSCs. (4) The result of teratoma test was negative in all groups. The proliferative ability of hEpSCs was increased markedly after down-regulation of JAM-A. Cells presented initial differentiation, but retained their stem cell characteristics without evidence of tumorigenesis.

Mesenchymal stem cells with modification of junctional adhesion molecule a induce hair formation.

The junctional adhesion molecule A (JAM-A) has been shown to serve a crucial role in the proliferation, differentiation, and tube-like formation of epithelial cells during angiogenesis. The role of JAM-A in hair follicle (HF) regeneration has not yet been reported. In this study, we used human JAM-A-modified human mesenchymal stem cells (MSCs) to repair HF abnormalities in BALB/c nu/nu mice. The JAM-A gene and JAM-A short hairpin RNA were transfected into cultured human MSCs to generate the JAM-A overexpression MSCs (JAM-A(ov) MSCs) and JAM-A knockdown MSCs (JAM-A(kd) MSCs), respectively. These cells were injected intradermally into the skin of nude mice during the first telogen phase of the HF that occurs 21 days postnatally. We found that JAM-A(ov) MSCs migrated into the HF sheath and remodeled HF structure effectively. The HF abnormalities such as HF curve and HF zigzag were remodeled, and hair formation was improved 7 days following injection in both the JAM-A(ov) MSC and MSC groups, compared with the JAM-A(kd) MSC group or negative control group. Furthermore, the JAM-A(ov) MSC group showed enhanced hair formation in contrast to the MSC group, and the number of curved and zigzagged HFs was reduced by 80% (p < .05). These results indicated that JAM-A(ov) MSCs improved hair formation in nude mice through HF structure remodeling.

Phosphorylation of angiomotin by Lats1/2 kinases inhibits F-actin binding, cell migration, and angiogenesis.

The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.

Par-1 regulates tissue growth by influencing hippo phosphorylation status and hippo-salvador association.

The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. Here, we reported the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevated Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminished Hpo target gene expression and reduced organ size. We demonstrated that par-1 functioned downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, we also found that Par-1 physically interacted with Hpo and Sav and regulated the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibited the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, we provided evidence that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, our findings identified Par-1 as a novel component of the Hpo signaling network.

Integration of mechanical and chemical signals by YAP and TAZ transcription coactivators.

YAP and TAZ are transcription coactivators and effectors of the Hippo pathway, which play a key role in organ size control. Through interaction with transcription factors such as TEADs, they activate gene transcription and thus promote cell proliferation, inhibit apoptosis, and regulate cell differentiation. Dysregulation of YAP/TAZ was found to correlate with human cancers. The oncogenic roles of these proteins were also demonstrated in animal models. The growth promoting activity of YAP/TAZ is limited by the Hippo tumor suppressor pathway through phosphorylation-induced cytoplasmic retention and destabilization. Recently, it was found that YAP and TAZ mediate responses to several extracellular signals including mechanical stress, GPCR signaling, and the Wnt signaling pathway. All these growth-regulating signals play important roles in normal development and cancer. In this review, we would like to discuss the function of YAP and TAZ as effectors of these physiological signals.

Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal.

The embryonic stem cell (ESC) transcriptional and epigenetic networks are controlled by a multilayer regulatory circuitry, including core transcription factors (TFs), posttranscriptional modifier microRNAs (miRNAs), and some other regulators. However, the role of large intergenic noncoding RNAs (lincRNAs) in this regulatory circuitry and their underlying mechanism remains undefined. Here, we demonstrate that a lincRNA, linc-RoR, may function as a key competing endogenous RNA to link the network of miRNAs and core TFs, e.g., Oct4, Sox2, and Nanog. We show that linc-RoR shares miRNA-response elements with these core TFs and that linc-RoR prevents these core TFs from miRNA-mediated suppression in self-renewing human ESC. We suggest that linc-RoR forms a feedback loop with core TFs and miRNAs to regulate ESC maintenance and differentiation. These results may provide insights into the functional interactions of the components of genetic networks during development and may lead to new therapies for many diseases.

MicroRNA-181 regulates CARM1 and histone arginine methylation to promote differentiation of human embryonic stem cells.

As a novel epigenetic mechanism, histone H3 methylation at R17 and R26, which is mainly catalyzed by coactivator-associated protein arginine methyltransferase 1 (CARM1), has been reported to modulate the transcription of key pluripotency factors and to regulate pluripotency in mouse embryos and mouse embryonic stem cells (mESCs) in previous studies. However, the role of CARM1 in human embryonic stem cells (hESCs) and the regulatory mechanism that controls CARM1 expression during ESCs differentiation are presently unknown. Here, we demonstrate that CARM1 plays an active role in the resistance to differentiation in hESCs by regulating pluripotency genes in response to BMP4. In a functional screen, we identified the miR-181 family as a regulator of CARM1 that is induced during ESC differentiation and show that endogenous miR-181c represses the expression of CARM1. Depletion of CARM1 or enforced expression of miR-181c inhibits the expression of pluripotency genes and induces differentiation independent of BMP4, whereas overexpression of CARM1 or miR-181c inhibitor elevates Nanog and impedes differentiation. Furthermore, expression of CARM1 rescue constructs inhibits the effect of miR-181c overexpression in promoting differentiation. Taken together, our findings demonstrate the importance of a miR-181c-CARM1 pathway in regulating the differentiation of hESCs.

N-acetylcysteine-pretreated human embryonic mesenchymal stem cell administration protects against bleomycin-induced lung injury.

INTRODUCTION: The transplantation of mesenchymal stem cells (MSCs) has been reported to be a promising approach in the treatment of acute lung injury. However, the poor efficacy of transplanted MSCs is one of the serious handicaps in the progress of MSC-based therapy. Therefore, the purpose of this study was to investigate whether the pretreatment of human embryonic MSCs (hMSCs) with an antioxidant, namely N-acetylcysteine (NAC), can improve the efficacy of hMSC transplantation in lung injury. METHODS: In vitro, the antioxidant capacity of NAC-pretreated hMSCs was assessed using intracellular reactive oxygen species (ROS) and glutathione assays and cell adhesion and spreading assays. In vivo, the therapeutic potential of NAC-pretreated hMSCs was assessed in a bleomycin-induced model of lung injury in nude mice. RESULTS: The pretreatment of hMSCs with NAC improved antioxidant capacity to defend against redox imbalances through the elimination of cellular ROS, increasing cellular glutathione levels, and the enhancement of cell adhesion and spreading when exposed to oxidative stresses in vitro. In addition, the administration of NAC-pretreated hMSCs to nude mice with bleomycin-induced lung injury decreased the pathological grade of lung inflammation and fibrosis, hydroxyproline content and numbers of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid and apoptotic cells, while enhancing the retention and proliferation of hMSCs in injured lung tissue and improving the survival rate of mice compared with results from untreated hMSCs. CONCLUSIONS: The pretreatment of hMSCs with NAC could be a promising therapeutic approach to improving cell transplantation and, therefore, the treatment of lung injury.

hMSCs possess the potential to differentiate into DP cells in vivo and in vitro.

DP (dermal papilla) is a mesenchyme-derived structure situated at the base of the HF (hair follicle) that plays an important role in embryonic hair morphogenesis and maintenance of the hair growth cycle. hMSCs (human mesenchymal stem cells) have gained widespread attention in the field of tissue engineering, but not much is known about the differentiation of hMSCs into DP cells. hMSCs involved in HF formation were examined in our previous study. Here, we have explored the differentiation potential of hMSCs into DP cells by co-culturing hMSCs with DP cells, which proved to be the case. During the differentiation process, the expression of versican, CD133, SCF (stem cell factor), ET-1 (endothelin-1) and bFGF (basic fibroblast growth factor) increased. Compared with hMSCs alone, the aggregate number clearly increased when co-cultured with DP cells. The expression in vivo of HLA-I (human leucocyte antigen class I) was confined to DP of the newly formed HF. The data suggest that hMSCs possess the potential to differentiate into DP cells in vivo and in vitro.