Lixin Hong

The miR-17-92 Cluster of MicroRNAs Confers Tumorigenicity by Inhibiting Oncogene-Induced Senescence

In mammalian cells, activation of oncogenes usually triggers innate tumor-suppressing defense mechanisms, including apoptosis and senescence, which are compromised by additional mutations before cancers are developed. The miR-17-92 gene cluster, a polycistron encoding six microRNAs (miRNA), is frequently overexpressed in human cancers and has been shown to promote several aspects of oncogenic transformation, including evasion of apoptosis. In the current study, we show a new role of miR-17-92 in inhibiting oncogenic ras-induced senescence. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family accounts for this antisenescence activity. miR-17 and miR-20a are both necessary and sufficient for conferring resistance to ras-induced senescence by directly targeting p21(WAF1), a key effector of senescence. By contrast, these components are not essential for the ability of miR-17-92 to evade Myc-induced apoptosis. Moreover, disruption of senescence by miR-17-92 or its miR-17/20a components leads to enhanced oncogenic transformation by activated ras in primary human cells. Taken together with previous reports that miR-17-92 inhibits apoptosis by suppressing Pten via the miR-19 components, our results indicate that this miRNA cluster promotes tumorigenesis by antagonizing both tumor-suppressing mechanisms, apoptosis, and senescence, through the activities of different miRNA components encoded in this cluster.

Participation of the p38 pathway in Drosophila host defense against pathogenic bacteria and fungi

The signaling network of innate immunity in Drosophila is constructed by multiple evolutionarily conserved pathways, including the Toll- or Imd-regulated NF-κB and JNK pathways. The p38 MAPK pathway is evolutionarily conserved in stress responses, but its role in Drosophila host defense is not fully understood. Here we show that the p38 pathway also participates in Drosophila host defense. In comparison with wild-type flies, the sensitivity to microbial infection was slightly higher in the p38a mutant, significantly higher in the p38b mutant, but unchanged in the p38c mutant. The p38b;p38a double-mutant flies were hypersensitive to septic injury. The immunodeficiency of p38b;p38a mutant flies was also demonstrated by hindgut melanization and larvae stage lethality that were induced by microbes naturally presented in fly food. A canonical MAP3K-MKK cascade was found to mediate p38 activation in response to infection in flies. However, neither Toll nor Imd was required for microbe-induced p38 activation. We found that p38-activated heat-shock factor and suppressed JNK collectively contributed to host defense against infection. Together, our data demonstrate that the p38 pathway-mediated stress response contribute to Drosophila host defense against microbial infection.

Attenuation of TORC1 signaling delays replicative and oncogenic RAS-induced senescence

Numerous stimuli, including oncogenic signaling, DNA damage or eroded telomeres trigger proliferative arrest, termed cellular senescence. Accumulating evidence suggests that cellular senescence is a potent barrier to tumorigenesis in vivo, however oncogene induced senescence can also promote cellular transformation.1,2 Several oncogenes, whose overexpression results in cellular senescence, converge on the TOR (target of rapamycin) pathway. We therefore examined whether attenuation of TOR results in delay or reversal of cellular senescence. By using primary human fibroblasts undergoing either replicative or oncogenic RAS-induced senescence, we demonstrated that senescence can be delayed, and some aspects of senescence can be reversed by inhibition of TOR, using either the TOR inhibitor rapamycin or by depletion of TORC1 (TOR Complex 1). Depletion of TORC2 fails to affect the course of replicative or RAS-induced senescence. Overexpression of REDD1 (Regulated in DNA Damage Response and Development), a negative regulator of TORC1, delays the onset of replicative senescence. These results indicate that TORC1 is an integral component of the signaling pathway that mediates cellular senescence.

p38α and p38γ Mediate Oncogenic ras-induced Senescence through Differential Mechanisms

Oncogene-induced senescence is a tumor-suppressive defense mechanism triggered upon activation of certain oncogenes in normal cells. Recently, the senescence response to oncogene activation has been shown to act as a bona fide barrier to cancer development in vivo. Multiple previous studies have implicated the importance of the p38 MAPK pathway in oncogene-induced senescence. However, the contribution of each of the four p38 isoforms (encoded by different genes) to senescence induction is unclear. In the current study, we demonstrated that p38α and p38γ, but not p38β, play an essential role in oncogenic ras-induced senescence. Both p38α and p38γ are expressed in primary human fibroblasts and are activated upon transduction of oncogenic ras. Small hairpin RNA-mediated silencing of p38α or p38γ expression abrogated ras-induced senescence, whereas constitutive activation of p38α and p38γ caused premature senescence. Furthermore, upon activation by oncogenic ras, p38γ stimulated the transcriptional activity of p53 by phosphorylating p53 at Ser33, suggesting that the ability of p38γ to mediate senescence is at least partly achieved through p53. However, p38α contributed to ras-inducted senescence via a p53-indepdendent mechanism in cells by mediating ras-induced expression of p16INK4A, another key senescence effector. These findings have identified p38α and p38γ as essential components of the signaling pathway that regulates the tumor-suppressing senescence response, providing insights into the molecular mechanisms underlying the differential involvement of the p38 isoforms in senescence induction.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.Summary Mitochondria need to be juxtaposted to phagosomes to synergistically produce ample reactive oxygen species (ROS) in phagocytes for pathogens killing. However, how phagosomes transmit signal to recruit mitochondria remains unclear. Here, we report that the kinases Mst1 and Mst2 function to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activate Rac GTPase to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for mitochondrial recruitment to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupt the TRAF6-ECSIT complex by sequestering TRAF6, and severely dampen ROS production and greatly increase susceptibility to bacterial infection. These findings demonstrate the TLR-Mst1-Mst2-Rac signalling axis to be critical for effective phagosome-mitochondrion function and bactericidal activity.

Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt.

Akt activation is a hallmark of human cancers. Here, we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that downregulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase) to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is downregulated in some cancer contexts and upregulated in others.

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

Manipulation of the Hippo signaling pathway with a reversible and selective small-molecule inhibitor of Hippo kinase MST1/2 provides a therapeutic option for tissue injury and repair. Drug-induced regeneration Popping a pill to repair an organ may eventually become reality. Turning away from conventional scaffolds, materials, and cell-based regenerative medicine strategies, Fan and colleagues sought a small molecule that could specifically target a critical signaling molecule in the Hippo pathway. Loss of kinases in this pathway, MST1/2, increases cell proliferation during development; thus, the authors hypothesized that inhibiting their activity in mature organs could help repair any damage. They discovered a drug, XMU-MP-1, that blocked MST1/2 activity and found that it promoted liver repair and regeneration in four different mouse models of acute and chronic injuries, including acetaminophen-induced injury, which is a common cause of liver failure worldwide. Such a pharmacological strategy could make tissue regeneration easier for many, compared to complex biomaterial and cell therapies. 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.

Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis

The role of the unfolded protein response (UPR) in tissue homeostasis remains largely unknown. Here we find that loss of Mst1/2, the mammalian Hippo orthologues, or their regulator WW45, leads to a remarkably enlarged endoplasmic reticulum (ER) size-associated UPR. Intriguingly, attenuation of the UPR by tauroursodeoxycholic acid (TUDCA) diminishes Mst1/2 mutant-driven liver overgrowth and tumorigenesis by promoting nuclear exit and degradation of Hippo downstream effector Yap. Yap is required for UPR activity and ER expansion to alleviate ER stress. During the adaptive stage of the UPR, PERK kinase-eIF2α axis activates Yap, while prolonged ER stress-induced Hippo signalling triggers assembly of the GADD34/PP1 complex in a negative feedback loop to inhibit Yap and promote apoptosis. Significantly, the deregulation of UPR signals associated with Yap activation is found in a substantial fraction of human hepatocellular carcinoma (HCC). Thus, we conclude Yap integrates Hippo and UPR signalling to control liver size and tumorigenesis.

Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling.

The receptor-interacting protein kinase 3 (RIPK3) plays crucial roles in programmed necrosis and innate inflammatory responses. However, a little is known about the involvement of RIPK3 in NKT cell-mediated immune responses. Here, we demonstrate that RIPK3 plays an essential role in NKT cell function via activation of the mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5). RIPK3-mediated activation of PGAM5 promotes the expression of cytokines by facilitating nuclear translocation of NFAT and dephosphorylation of dynamin-related protein 1 (Drp1), a GTPase is essential for mitochondrial homoeostasis. Ripk3−/− mice show reduced NKT cell responses to metastatic tumour cells, and both deletion of RIPK3 and pharmacological inhibition of Drp1 protects mice from NKT cell-mediated induction of acute liver damage. Collectively, the results identify a crucial role for RIPK3-PGAM5-Drp1/NFAT signalling in NKT cell activation, and further suggest that RIPK3-PGAM5 signalling may mediate crosstalk between mitochondrial function and immune signalling.

A Novel Function of p38-Regulated/Activated Kinase in Endothelial Cell Migration and Tumor Angiogenesis

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.