Specific Chemical Inhibitors Research Articles

P0051 Is NKCC1, a marker of dysplasia associated with inflammation and CAC, playing a role in colorectal carcinogenesis?

Abstract Background Colitis-associated cancer (CAC) is a distinct entity from sporadic colorectal cancer (CRC)1. Previous studies have demonstrated that Solute Carrier Family 12 Member 2 (SLC12A2/NKCC1), a Na-K-Cl cotransporter, is a tissue marker able to discriminate dysplasia from inflammatory and normal tissues2. NKCC1 is overexpressed in both pre-tumoral and tumoral lesions of CAC and CRC. It is located at the cell membrane and in the cytoplasm and is mainly expressed in fluid-secreting cells. In IBD, NKCC1 mutations cause defects in mucus secretion and chronic inflammation3. Our objective is to study its specific role in colorectal carcinogenesis. Methods Oxidative stress was induced in 3 CRC cell lines using H2O2 concomitantly with bumetanide (BMT), a chemical inhibitor of NKCC1, or by silencing its expression via shRNA. Cell viability, the mTORC1 pathway, and markers of stemness, proliferation, and differentiation were assessed by Western blot and qPCR. Human intestinal organoids deriving form patient tumours and normal margins, grown under the proliferative or differentiated states were used to characterize NKCC1 at the transcriptional and protein levels. Finally, NKCC1 expression in CRC was investigated using public single-cell datasets (GEO: GSE178341) to determine its specific expression within epithelial and tumoral cell-clusters4. Results Exposure of Caco-2 cells to 1 mM H2O2 for 24h resulted in 68% cell viability. However, when co-treated with 200 µM bumetanide cell viability dropped to 18%. Similarly, HT-29 cell viability dropped from 62% to 36%, while HCT116 cells showed a reduction from 12% to 5% with BMT and was shown to be BMT concentration-dependent. BMT-treated organoids/tumoroids revealed an activation of the mTORC1 pathway. Silencing NKCC1 expression via shRNA significantly increased cancer stem cell markers (LGR5, CD44, ALDH1A1). Upon differentiation, human intestinal organoids showed a marked decrease in NKCC1 expression mirroring the reduction of stem cell markers. This was confirmed by the single-cell data exploration, revealing that NKCC1 is highly expressed in both intestinal normal and cancer stem cells. Conclusion We confirmed that NKCC1 is expressed in both intestinal and cancer stem cells of colorectal tumours. Inhibition of NKCC1 transporter activity reduced cell viability in CRC cell lines only under oxidative stress exposure. NKCC1 silencing enhanced cancer stem cell marker expression, while its specific chemical inhibition activated the mTORC1 pathway. These results suggest that NKCC1 could be an important player in carcinogenesis at least by its ability to regulate oxidative stress in tumoral cells. Investigation of its role in ulcerative colitis and CAC remains to be clarified.

α4 Nicotinic Acetylcholine Receptors in Lipopolysaccharide-Related Lung Inflammation

Sepsis remains an important healthcare challenge. The lungs are often affected in sepsis, resulting in acute lung injury characterized by inflammation. Mechanisms involving lipopolysaccharide (LPS) stimulation of toll-like receptor (TLR) signaling with induction of proinflammatory pathways have been implicated in this process. To date, however, studies targeting these pathways have failed to improve outcomes. We have found that LPS may also promote lung injury through the activation of α4 nicotinic acetylcholine receptors (α4 nAChRs) in immune cells. We observed increased expression of α4 nAChRs in human THP-1 monocytic cells exposed to LPS (100 ng/mL, 24 h). We also observed that LPS stimulated the expression of other relevant genes, including tumor necrosis factor-α, interleukin-1β, plasminogen activator inhibitor-1, the solute carrier family 7 member 11, extracellular superoxide dismutase, and transforming growth factor-β1. Of interest, dihydro-β-erythroidine hydrobromide (DHβE), a specific chemical inhibitor of α4 nAChRs, inhibited the LPS-induced expression of these genes. We generated mice with a global knockout mutation of the α4 nAChR subunit in the C57BL/6 background using CRISPR/Cas9 technology. The lungs of these LPS-treated animals demonstrated a reduction in the expression of the above-mentioned genes when compared with the lungs of wild-type animals. In support of the role of oxidative stress, we observed that LPS induced expression of the cystine transporter Slc7a11 in both THP-1 cells and in wild-type mouse lungs. The effects of LPS on THP-1 cells were blocked by the thiol antioxidant N-acetylcysteine and mimicked by redox stress. Importantly, the induction of IL-1β by redox stress was inhibited by the α4 nAChR inhibitor DHβE. Finally, we showed that LPS stimulated calcium influx in THP-1 cells, which was blocked by the α4 nAChR inhibitor. Our observations suggest that LPS promotes lung injury by stimulating redox stress, which activates α4 nAChR signaling and drives proinflammatory cytokine expression.

The class III phosphatidylinositol 3-kinase VPS34 supports EV71 replication by promoting viral replication organelle formation.

Enterovirus 71 (EV71) belongs to the family of Picornaviridae; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development.

Abstract 569: Targeting UHRF1’s proteasomal destruction as a novel approach for small cell lung cancer treatment

Abstract Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with a five-year survival rate of less than 8% due to rapid tumor growth, early metastasis, and a “cold” immune environment. Genetic alteration at the RB transcriptional corepressor 1 (RB1) is nearly universally present in SCLC patients. However, the therapeutic development was impeded by a lack of druggable oncogenic mutations. Studies in our laboratory identified ubiquitin-like, containing PHD and RING Finger domains 1 (UHRF1) as a critical protein overexpressed in many cancers with RB-pathway inactivation, including SCLC. Despite UHRF1 overexpression correlates with poor survival in SCLC patients, the roles of UHRF1 in SCLC progression remain to be elucidated. We hypothesized that UHRF1 overexpression is essential for SCLC tumor progression and can be targeted through endogenous proteasomal degradation. To define the role of UHRF1 in SCLC, we generated UHRF1 knockout and knockdown human SCLC cell lines. Using these mutants, we assessed changes in proliferation, clonogenicity, migration, and invasion capabilities. We report that UHRF1 overexpression present in human SCLC cell lines contributes to a more aggressive tumor presentation, affecting both tumor growth and metastasis. Besides human cell line models, we also generated genetically engineered mouse models of SCLC to study the role of Uhrf1 in tumor development, especially in an immune environment. Our results indicate that Uhrf1 overexpression promotes SCLC proliferation, angiogenesis, and pro-tumoral macrophage polarization, overall conditioning a tumor microenvironment that supports a more aggressive phenotype. Loss of Uhrf1 in this system results in a robust increase in overall survival. These results strongly support a critical role of UHRF1 in SCLC tumor progression and provide guidance for future SCLC therapeutics targeting UHRF1 or its key downstream effectors. One approach we apply is to utilize UHRF1’s endogenous degradative machinery by inhibiting its deubiquitinase USP7. In normal cells, UHRF1 abundance oscillates through cell cycle due to the regulation of ubiquitin machinery. USP7 cleaves ubiquitin marks on UHRF1, antagonizing UHRF1 degradation. We report that same as UHRF1, knocking out USP7 leads to decreased clonogenicity, migration, and invasion in human SCLC cell lines. Highly specific USP7 chemical inhibitors, XL-177A and FT671, were also tested and the same effects as USP7 knockout were observed. Collectively, our findings describe the oncogenic role of UHRF1 in SCLC proliferation, metastasis, and immune recognition and provide a therapeutic strategy of inhibiting USP7 for UHRF1 destruction. Citation Format: Yijun Gu, Claudia A. Benavente. Targeting UHRF1’s proteasomal destruction as a novel approach for small cell lung cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 569.

Targeting the PHF8/YY1 axis suppresses cancer cell growth through modulation of ROS.

High levels of mitochondrial reactive oxygen species (mROS) are linked to cancer development, which is tightly controlled by the electron transport chain (ETC). However, the epigenetic mechanisms governing ETC gene transcription to drive mROS production and cancer cell growth remain to be fully characterized. Here, we report that protein demethylase PHF8 is overexpressed in many types of cancers, including colon and lung cancer, and is negatively correlated with ETC gene expression. While it is well known to demethylate histones to activate transcription, PHF8 demethylates transcription factor YY1, functioning as a co-repressor for a large set of nuclear-coded ETC genes to drive mROS production and cancer development. In addition to genetically ablating PHF8, pharmacologically targeting PHF8 with a specific chemical inhibitor, iPHF8, is potent in regulating YY1 methylation, ETC gene transcription, mROS production, and cell growth in colon and lung cancer cells. iPHF8 exhibits potency and safety in suppressing tumor growth in cell-line- and patient-derived xenografts in vivo. Our data uncover a key epigenetic mechanism underlying ETC gene transcriptional regulation, demonstrating that targeting the PHF8/YY1 axis has great potential to treat cancers.

Pharmacological inhibition of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) induces ferroptosis in vascular smooth muscle cells

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE−/−) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.

3D environment controls H3K4 methylation and the mechanical response of the nucleus in acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, and the infiltration of leukemic cells is critical for disease progression and relapse. Nuclear deformability plays a critical role in cancer cell invasion through confined spaces; however, the direct impact of epigenetic changes on the nuclear deformability of leukemic cells remains unclear. Here, we characterized how 3D collagen matrix conditions induced H3K4 methylation in ALL cell lines and clinical samples. We used specific shRNA and chemical inhibitors to target WDR5 (a core subunit involved in H3K4 methylation) and determined that targeting WDR5 reduced the H3K4 methylation induced by the 3D environment and the invasiveness of ALL cells in vitro and in vivo. Intriguingly, targeting WDR5 did not reduce the adhesion or the chemotactic response of leukemia cells, suggesting a different mechanism by which H3K4 methylation might govern ALL cell invasiveness. Finally, we conducted biochemical, and biophysical experiments to determine that 3D environments promoted the alteration of the chromatin, the morphology, and the mechanical behavior of the nucleus in ALL cells. Collectively, our data suggest that 3D environments control an upregulation of H3K4 methylation in ALL cells, and targeting WDR5 might serve as a promising therapeutic target against ALL invasiveness in vivo.

Tolerized Microglia Protect Neurons Against Endotoxin-Induced TNF-α Production via an LBP-Dependent Intracellular p38 MAPK Signaling Pathway.

The development of microglial endotoxin tolerance (ET) is a critical event in protecting neurons against excessive immune responses when microglia are administered two consecutive lipopolysaccharide (LPS) challenges. However, the intrinsic mechanisms of microglia shape ET programs and protect neurons remain unclear. This study aimed to determine whether extracellular autocrine cascades or intracellular signaling pathways are involved in ET microglia-mediated tumor necrosis factor-alpha (TNF-α) reduction and neuroprotection. Neuron-glia cultures composed of astroglia, neurons, and microglia were performed in different conditions: with or without serum or LPS-binding proteins (LBP), along with an induction approach of ET. Enzyme-linked immunosorbent assay results revealed that LPS induced TNF-α tolerance of microglia in an LBP-dependent manner. Furthermore, we determined whether the early pro-inflammatory cytokines induced by LPS might contribute to the development of microglial ET. Our data showed that the neutralization of TNF-α using an anti-TNF-α antibody had no change in the TNF-α tolerance of microglia during the ET challenge. Furthermore, pre-incubation of TNF-α, interleukin-1 beta, and prostaglandin E2 failed to induce any TNF-α tolerance in microglia after LPS treatment. Moreover, using three specific chemical inhibitors that respectively blocked the activities of the mitogen-activated protein kinases (MAPKs) namely p38, c-Jun N-terminal kinase and extracellular signal-related kinases revealed that inhibition of p38 MAPK by SB203580 disrupted the tolerated microglia-mediated TNF-α reduction and neuroprotection. In summary, our findings demonstrated that the LPS pre-treatment immediately programmed the microglial ET to prevent endotoxin-induced TNF-α production and neuronal damage through the intracellular p38 MAPK signaling pathway.

Impact of Short-Term (+)-JQ1 Exposure on Mouse Aorta: Unanticipated Inhibition of Smooth Muscle Contractility.

(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.

Identification of Mutated in colorectal cancer (MCC) protein as a novel partner of PDZRN3 during Blood brain barrier development

Blood brain barrier (BBB) disruption is a critical component of the physiopathology of neurological disorder. Wnt signalling is crucial for BBB development and its stability. We have reported that an endothelial excessive activation of the ubiquitin ligase PDZRN3, a partner in Wnt signalling, destabilizes the BBB. However, the molecular pathway is still poorly understood. Using a differential screening strategy by BioID, MCC was discovered as a potential PDZRN3 interacting partner. This study focused on understanding how MCC/PDZRN3 interaction regulates Wnt signalling in vitro and in vivo. We then investigated the role of MCC in endothelial function. PDZRN3 and MCC protein partners were identified by a proteomic screening strategy (bioID). MCC functional role in Wnt signalling was studied in brain endothelial cells (HBMEC) using siRNA strategy, and specific chemical inhibitors. MCC in vivo expression was analysed in extracted blood vessels from wild type and transgenic mouse brains. MCC is a direct target of the kinase CKIɛ, a key regulator of the canonical Wnt pathway. We demonstrated that MCC is constantly phosphorylated by this kinase. We then reported that PDZRN3 prevents CKIɛ-induced phosphorylation of MCC. During post-natal mouse BBB development, MCC undergoes a switch from un- to hyper-phosphorylated state, correlated with a decrease in PDZRN3 expression level. Interestingly, in mouse mutants, specific ectopic Pdzrn3 over-expression in brain endothelial cells impairs MCC phosphorylation switch. We then investigated MCC involvement in endothelial function. MCC knock down impairs HBMEC directed migration under flow condition and planar MTOC polarization. Finally, using a bioID screening, we have identified several centrosomal proteins, such as CEP131, as potential MCC partners. Centrosome being a key organelle in cell polarization, we propose that MCC may regulate its organisation during HBMEC planar migration. By forming a stable complex with MCC, PDZRN3 prevents CKIɛ-induced phosphorylation of MCC. MCC would then regulate centrosome polarisation required for endothelial planar migration, making MCC an actor of BBB dynamic maturation.

Combined contributions of cytochrome P450s (CYPs) and non-enzymatic metabolism in the in vitro biotransformation of anaprazole, a novel proton pump inhibitor.

Anaprazole, a new proton pump inhibitor (PPI), is designed for the treatment of acid-related diseases, such as gastric ulcers and gastroesophageal reflux. This study explored the in vitro metabolic transformation of anaprazole. The metabolic stabilities of anaprazole in human plasma and human liver microsomes (HLM) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, the contribution (%) of non-enzymatic and cytochrome P450s (CYPs) enzyme-mediated anaprazole metabolism was assessed. To obtain the metabolic pathways of anaprazole, the metabolites generated in HLM, thermal deactivated HLM, and cDNA-expressed recombinant CYPs incubation systems were identified by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Results showed thatanaprazole was very stable in human plasma and unstable in HLM. The contribution (%) of non-enzymatic vs. CYPs enzyme-mediated metabolism was 49% vs. 51%. CYP3A4 was the major enzyme (48.3%), followed by CYP2C9 (17.7%) and CYP2C8 (12.3%), in responsible for the metabolism of anaprazole. Specific chemical inhibitors targeting CYP enzymes notably blocked the metabolic transformation of anaprazole. Six metabolites of anaprazole were identified in the non-enzymatic system, whereas 17 metabolites were generated in HLM. The biotransformation reactions mainly included sulfoxide reduction to thioether, sulfoxide oxidation to sulfone, deoxidation, dehydrogenation, O-dealkylation or O-demethylation of thioether, O-demethylation and dehydrogenation of thioether, O-dealkylation and dehydrogenation of thioether, thioether O-dealkylation and dehydrogenation of thioether, and O-dealkylation of sulfone. Both enzymatic and non-enzymatic metabolisms contribute to the clearance of anaprazole in human. Anaprazole is less likely to develop drug-drug interactions in clinical use compared to other PPIs.

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells.

Sapoviruses belonging to the genus Sapovirus within the family Caliciviridae are commonly responsible for severe acute gastroenteritis in both humans and animals. Caliciviruses are known to induce intrinsic apoptosis in vitro and in vivo, however, calicivirus-induced necroptosis remains to be fully elucidated. Here, we demonstrate that infection of porcine kidney LLC-PK cells with porcine sapovirus (PSaV) Cowden strain as a representative of caliciviruses induces receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis and acts as proviral compared to the antiviral function of PSaV-induced apoptosis. Infection of LLC-PK cells with PSaV Cowden strain showed that the interaction of phosphorylated RIPK1 (pRIPK1) with RIPK3 (pRIPK3), mixed lineage kinase domain-like protein (pMLKL) increased in a time-dependent manner, indicating induction of PSaV-induced RIPK1-dependent necroptosis. Interfering of PSaV-infected cells with each necroptotic molecule (RIPK1, RIPK3, or MLKL) by treatment with each specific chemical inhibitor or knockdown with each specific siRNA significantly reduced replication of PSaV but increased apoptosis and cell viability, implying proviral action of PSaV-induced necroptosis. In contrast, treatment of PSaV-infected cells with pan-caspase inhibitor Z-VAD-FMK increased PSaV replication and necroptosis, indicating an antiviral action of PSaV-induced apoptosis. These results suggest that PSaV-induced RIPK1-dependent necroptosis and apoptosis‒which have proviral and antiviral effects, respectively‒counterbalanced each other in virus-infected cells. Our study contributes to understanding the nature of PSaV-induced necroptosis and apoptosis and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.

Decreased Innate Migration of Pro-Inflammatory M1 Macrophages through the Mesothelial Membrane Is Affected by Ceramide Kinase and Ceramide 1-P.

The retrograde flow of endometrial tissues deposited into the peritoneal cavity occurs in women during menstruation. Classically (M1) or alternatively (M2) activated macrophages partake in the removal of regurgitated menstrual tissue. The failure of macrophage egress from the peritoneal cavity through the mesothelium leads to chronic inflammation in endometriosis. To study the migration differences of macrophage phenotypes across mesothelial cells, an in vitro model of macrophage egress across a peritoneal mesothelial cell monolayer membrane was developed. M1 macrophages were more sessile, emigrating 2.9-fold less than M2 macrophages. The M1 macrophages displayed a pro-inflammatory cytokine signature, including IL-1α, IL-1β, TNF-α, TNF-β, and IL-12p70. Mass spectrometry sphingolipidomics revealed decreased levels of ceramide-1-phosphate (C1P), an inducer of migration in M1 macrophages, which correlated with its poor migration behavior. C1P is generated by ceramide kinase (CERK) from ceramide, and blocking C1P synthesis via the action of NVP231, a specific CERK chemical inhibitor, prohibited the emigration of M1 and M2 macrophages up to 6.7-fold. Incubation with exogenously added C1P rescued this effect. These results suggest that M1 macrophages are less mobile and have higher retention in the peritoneum due to lower C1P levels, which contributes to an altered peritoneal environment in endometriosis by generating a predominant pro-inflammatory cytokine environment.

Crosstalk between heat shock factor 1 and signal transducer and activator of transcription 3 mediated by interleukin-8 autocrine signaling maintains the cancer stem cell phenotype in liver cancer.

Liver cancer stem cells (LCSCs) cause therapeutic refractoriness and relapse in hepatocellular carcinoma. Heat shock factor 1 (HSF1) plays versatile roles in multiple cancers. However, the role of HSF1 in LCSCs is not well understood. This study investigated the function and signal mechanisms of HSF1 in maintaining LCSC phenotypes. We established two LCSC lines, HepG2-R and HuH-7-R. Constitutive activation of HSF1 was observed in these LCSCs. Specific short hairpin RNAs (shRNAs) and chemical inhibitors were used to identify the relationship between HSF1 expression and LCSCs phenotypes. We revealed a concomitant activation modality involving HSF1 and STAT3 in LCSCs and liver cancer tissues. We also found that liver cancer patients whose HSF1 and STAT3 mRNA expression levels were high presented with unfavorable clinicopathological characteristics. Moreover, the secretion of interleukin-8 (IL-8) was elevated in the LCSC medium and was directly regulated by HSF1 at the transcriptional level. In turn, IL-8 activated HSF1 and STAT3 signaling, and a neutralizing IL-8 antibody inhibited HSF1 and STAT3 activity, reduced cancer stem cell marker expression, and decreased LCSC microsphere formation. Simultaneous intervention with HSF1 and STAT3 led to synergistically suppressed stemness acquisition and growth suppression in the LCSCs in vivo and in vitro. Our study indicates that IL-8 mediates the crosstalk between the HSF1 and Stat3 signaling pathways in LCSCs and that the combined targeting of HSF1 and STAT3 is a promising treatment strategy for patients with advanced liver cancer.

ROCK1/MLC2 inhibition induces decay of viral mRNA in BPXV infected cells

Rho-associated coiled-coil containing protein kinase 1 (ROCK1) intracellular cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. We observed the activation of ROCK1/myosin light chain (MLC2) signaling pathway in buffalopox virus (BPXV) infected Vero cells. ROCK1 depletion by siRNA and specific small molecule chemical inhibitors (Thiazovivin and Y27632) resulted in a reduced BPXV replication, as evidenced by reductions in viral mRNA/protein synthesis, genome copy numbers and progeny virus particles. Further, we demonstrated that ROCK1 inhibition promotes deadenylation of viral mRNA (mRNA decay), mediated via inhibiting interaction with PABP [(poly(A)-binding protein] and enhancing the expression of CCR4-NOT (a multi-protein complex that plays an important role in deadenylation of mRNA). In addition, ROCK1/MLC2 mediated cell contraction, and perinuclear accumulation of p-MLC2 was shown to positively correlate with viral mRNA/protein synthesis. Finally, it was demonstrated that the long-term sequential passage (P = 50) of BPXV in the presence of Thiazovivin does not select for any drug-resistant virus variants. In conclusion, ROCK1/MLC2 cell signaling pathway facilitates BPXV replication by preventing viral mRNA decay and that the inhibitors targeting this pathway may have novel therapeutic effects against buffalopox.

Autophagy promotes GSDME-mediated pyroptosis via intrinsic and extrinsic apoptotic pathways in cobalt chloride-induced hypoxia reoxygenation-acute kidney injury

Cobalt is a transition element that abundantly exists in the environment. Besides direct hypoxia stress, cobalt ions indirectly induce hypoxia-reoxygenation injury (HRI), the main cause of acute kidney injury (AKI), a life-threatening clinical syndrome characterized by the necrosis of the proximal tubular epithelial cells (PTECs) and inflammation. Pyroptosis, a type of inflammatory programmed cell death, might play an essential role in HRI-AKI. However, whether pyroptosis is involved in cobalt chloride (CoCl2)-induced HRI-AKI remains unknown. Autophagy is a cellular biological process maintaining cell homeostasis that is involved in cell damage in AKI, yet the underlying regulatory mechanism of autophagy on pyroptosis has not been fully understood. In this study, the in vitro and in vivo models of CoCl2-induced HRI-AKI were established with HK-2 cell line and C57BL/6J mouse. Pyroptosis-related markers were detected with western blotting and immunofluorescence assays, and results showed that gasdermin E (GSDME)-mediated pyroptosis was involved in the cell damage in HRI-AKI. Specific chemical inhibitors of caspase 3, caspase 8, and caspase 9 significantly inhibited GSDME-mediated pyroptosis, verifying that GSDME-mediated pyroptosis was induced via the activation of caspase 3/8/9. The western blotting and immunofluorescence assays were adopted to detect the accumulation of the autophagosomes, and results suggested that HRI increased the autophagic level. The effects of autophagy on apoptosis and pyroptosis were evaluated using lentivirus transfection assays to knock down autophagy-specific genes atg5 and fip200, and results demonstrated that autophagy induced GSDME-mediated pyroptosis via apoptotic pathways in HRI-AKI. Our results revealed the involvement of GSDME-mediated pyroptosis in CoCl2-induced HRI-AKI and promoted the understanding of the regulatory mechanism of GSDME cleavage. Our study might provide a potential therapeutic target for HRI-AKI, and will be helpful for the risk evaluation of cobalt exposure.

The canonical Wnt/β-catenin signaling pathway facilitates pseudorabies virus proliferation and enhances virus-induced autophagy

Pseudorabies virus (PRV) is a swine herpesvirus with a broad host range that causes significant economic losses worldwide. The Wnt/β-catenin signaling pathway is reportedly involved in multiple viruses' proliferation. In this study, we demonstrated that PRV infection significantly activated the Wnt/β-catenin signaling and promoted the nuclear translocation of β-catenin. Applying specific chemical inhibitors (FH535 and iCRT14) caused a remarkable decrease in PRV titers in various cell lines. Knockdown of β-catenin by siRNA also reduced the proliferation of PRV. On the contrary, treatment with lithium chloride (LiCl), an inhibitor of GSK3β, stimulated the Wnt/β-catenin signaling pathway and enhanced the PRV proliferation. Similarly, overexpression of β-catenin promoted PRV proliferation and reversed the antiviral effect of FH535. Moreover, LiCl promoted PRV-induced autophagy, whereas FH535 and iCRT14 showed converse effects. These findings suggest that PRV infection stimulates the canonical Wnt/β-catenin signaling pathway, facilitating PRV proliferation and regulating virus-induced autophagy. These data also provide potential targets for developing antiviral agents against PRV.

Regulation of Metabolism by Mitochondrial MUL1 E3 Ubiquitin Ligase.

MUL1 is a multifunctional E3 ubiquitin ligase that is involved in various pathophysiological processes including apoptosis, mitophagy, mitochondrial dynamics, and innate immune response. We uncovered a new function for MUL1 in the regulation of mitochondrial metabolism. We characterized the metabolic phenotype of MUL1(−/−) cells using metabolomic, lipidomic, gene expression profiling, metabolic flux, and mitochondrial respiration analyses. In addition, the mechanism by which MUL1 regulates metabolism was investigated, and the transcription factor HIF-1α, as well as the serine/threonine kinase Akt2, were identified as the mediators of the MUL1 function. MUL1 ligase, through K48-specific polyubiquitination, regulates both Akt2 and HIF-1α protein level, and the absence of MUL1 leads to the accumulation and activation of both substrates. We used specific chemical inhibitors and activators of HIF-1α and Akt2 proteins, as well as Akt2(−/−) cells, to investigate the individual contribution of HIF-1α and Akt2 proteins to the MUL1-specific phenotype. This study describes a new function of MUL1 in the regulation of mitochondrial metabolism and reveals how its downregulation/inactivation can affect mitochondrial respiration and cause a shift to a new metabolic and lipidomic state.

Absorption, Metabolism, and Excretion of ACT-1004-1239, a First-In-Class CXCR7 Antagonist: In Vitro, Preclinical, and Clinical Data.

ACT-1004-1239 is a potent, selective, first-in-class CXCR7 antagonist, which shows a favorable preclinical and clinical profile. Here we report the metabolites and the metabolic pathways of ACT-1004-1239 identified using results from in vitro and in vivo studies. Two complementary in vitro studies (incubation with human liver microsomes in the absence/presence of cytochrome P450- [CYP] specific chemical inhibitors and incubation with recombinant CYPs) were conducted to identify CYPs involved in ACT-1004-1239 metabolism. For the in vivo investigations, a microtracer approach was integrated in the first-in-human study to assess mass balance and absorption, distribution, metabolism, and excretion (ADME) characteristics of ACT-1004-1239. Six healthy male subjects received orally 100 mg non-radioactive ACT-1004-1239 together with 1 μCi 14C-ACT-1004-1239. Plasma, urine, and feces samples were collected up to 240 h post-dose and 14C-drug-related material was measured with accelerator mass spectrometry. This technique was also used to construct radiochromatograms of pooled human samples. Metabolite structure elucidation of human-relevant metabolites was performed using high performance liquid chromatography coupled with high resolution mass spectrometry and facilitated by the use of rat samples. CYP3A4 was identified as the major CYP catalyzing the formation of M1 in vitro. In humans, the cumulative recovery from urine and feces was 84.1% of the dose with the majority being eliminated via the feces (69.6%) and the rest via the urine (14.5%). In human plasma, two major circulating metabolites were identified, i.e., M1 and M23. Elimination via M1 was the only elimination pathway that contributed to ≥25% of ACT-1004-1239 elimination. M1 was identified as a secondary amine metabolite following oxidative N-dealkylation of the parent. M23 was identified as a difluorophenyl isoxazole carboxylic acid metabolite following central amide bond hydrolysis of the parent. Other metabolites observed in humans were A1, A2, and A3. Metabolite A1 was identified as an analog of M1 after oxidative defluorination, whereas both, A2 and A3, were identified as a reduced analog of M1 and parent, respectively, after addition of two hydrogen atoms at the isoxazole ring. In conclusion, CYP3A4 contributes to a relevant extent to ACT-1004-1239 disposition and two major circulating metabolites were observed in humans. Clinical Trial Registration: (https://clinicaltrials.gov/ct2/show/NCT03869320) ClinicalTrials.gov Identifier NCT03869320.

Differential regulation of Hippo signaling pathway components between 8-cell and blastocyst stages of bovine preimplantation embryogenesis.

The Hippo signaling pathway is an important regulator of lineage segregation (trophectoderm and inner cell mass) during blastocyst formation in the mouse embryos. However, the role and regulation of Hippo signaling pathway components during bovine embryonic development is not completely understood. This study was thus designed to interpret the roles of Hippo cell signaling pathway components using two different yet specific chemical inhibitors (Cerivastatin and XMU-MP-1). A significant decrease in the blastocyst rates were observed on treatment with Cerivastatin and XMU-MP-1 inhibitors for the treatment groups, in comparison to the control groups. At the 8-cell stage, a significant decrease was observed in the gene expression and nuclear protein localization of YAP1 (Yes Associated Protein 1) and pYAP1 components of Hippo signaling pathway. However, no such effect of Cerivastatin treatment was observed on the localization of TAZ at this cell stage. On the contrary, during bovine blastocyst formation a significant decrease in the gene expression and nuclear localization of both YAP1 and TAZ suggest differences in the regulation of these components at 8-cell and blastocyst stages of embryonic development. Furthermore, XMU-MP-1 mediated chemical inhibition of Mst1 at the blastocyst stage also suggests differences in the regulation of Yap1 and Taz components of Hippo signaling pathway. Overall, this study indicates novel differences in the regulation of Hippo signaling transcript levels and protein localization between the 8-cell and blastocyst stages of bovine preimplantation embryonic development.