Unveiling the intracellular dynamics of α4ß2 nAChR-mediated ERK activation through the interplay of arrestin, Gßγ, and PKCßII.

AIMS: In contrast to G protein-coupled receptors or receptor tyrosine kinases, the mechanism underlying ERK activation through nicotine acetylcholine receptors (nAChRs), members of the ligand-gated ion channel family, remains poorly elucidated. This study aimed to delineate the signaling pathway responsible for ERK activation by the α4ß2 nAChR subtype, which is implicated in nicotine addiction and various mental disorders. MATERIALS AND METHODS: Loss-of-function strategies and mutants of arrestin2/PKCßII with distinct functional characteristics were employed to identify the cellular components and processes involved in ERK activation. KEY FINDINGS: ERK activation via α4ß2 nAChR was observed within the nucleus and necessitated the nuclear translocation of arrestin2 and PKCßII, which exhibited mutual augmentation. Activation of PKCßII by α4ß2 nAChR stimulation facilitated the nuclear translocation of arrestin2 by enhancing its interaction with importin ß1. Apart from scaffolding ERK activation in the nucleus, arrestin2, in cooperation with GRK2, facilitated the activation of the Src/Syk/PKCßII signaling cascade, leading to the nuclear entry of PKCßII in a Gßγ-dependent manner. Upon nuclear localization, PKCßII underwent ubiquitination by Mdm2 and interacted with MEK1, resulting in ERK activation. In summary, α4ß2 nAChR-mediated ERK activation in the nucleus involves the nuclear translocation of arrestin2 and PKCßII, which is reciprocally facilitated via positive feedback augmentation. SIGNIFICANCE: As α4ß2 nAChRs play a pivotal role in various cellular processes including drug addiction and mental disorders, our findings will offer insights into understanding the pathogenesis of α4ß2 nAChR-related disorders and may facilitate the development of targeted therapeutic interventions.

Protective effect of irbesartan against hepatic ischemia-reperfusion injury in rats: role of ERK, STAT3, and PPAR-γ inflammatory pathways in rats.

This study aimed to elucidate the possible hepatocellular protective role of irbesartan during hepatic ischemia-reperfusion injury (HIRI) and the probable underlying mechanisms. Wistar rats were allocated into four groups: sham; HIRI (control); irbesartan (50 mg/kg) + HIRI; irbesartan (100 mg/kg) + HIRI; irbesartan + GW9662 (1 mg/kg, i.p.) + HIRI. Rats pretreated orally with irbesartan or vehicle for 14 days underwent 45-min hepatic ischemia followed by 60-min reperfusion. Irbesartan preconditioning diminished alanine transaminase (ALT) and aspartate transaminase (AST) serum levels, and reduced extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3). Irbesartan decreased proapoptotic BAX (bcl-2-like protein 4), increased anti-apoptotic B-cell lymphoma 2 (BCL2) hepatic content, and thereby reduced BAX/BCL2 ratio. Moreover, irbesartan preconditioning reduced autophagy-related proteins Beclin1 and LC3 II, and elevated p62 (protein responsible for autophagosome degradation). It elevated proliferator-activated receptor γ (PPAR-γ), and reduced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) hepatic gene expression. Also, hepatic protein expressions of nuclear factor kappa-B p65 (NF-κB p65) and caspase-3 were lessoned by irbesartan pretreatment in HIRI rats. However, GW9662 abrogated irbesartan's effect on HIRI. The protective effect of irbesartan on HIRI may be mediated by alleviation of ERK, STAT3, and PPAR-γ inflammatory pathways, exerting anti-apoptotic and anti-autophagic effects in HIRI in rats.

Electroacupuncture negatively regulates the Nesfatin-1/ERK/CREB pathway to alleviate HPA axis hyperactivity and anxiety-like behaviors caused by surgical trauma.

BACKGROUND: Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis constitutes a pivotal response by surgical trauma, manifesting as a critical aspect of the acute stress reaction. This hyperactivity resulted in adverse surgical outcomes and is often associated with increased postoperative anxiety. Increased evidence suggests that Nesfatin-1 plays a crucial role in stress responses and stress-related psychiatric disorders. Electroacupuncture (EA) is widely used to alleviate stress responses and anxiety, although its mechanism of action remains unclear. This study aimed to assess the mechanisms by which hypothalamic Nesfatin-1 contribute to the alleviation of HPA axis hyperactivity and anxiety by EA. METHODS: Partial hepatectomy (HT) was performed to simulate surgical trauma, and EA was applied at Zusanli (ST36) and Sanyinjiao (SP6). The levels of hypothalamic Nesfatin-1, c-Fos, and corticotropin-releasing hormone (CRH) were detected, and serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were regarded as indicators of HPA axis activity. Anxiety levels were assessed through open field tests (OFT), elevated plus maze (EPM), and light-dark box tests (LDBT). To investigate the role of Nesfatin-1, its expression was modulated using stereotactic viral injections or plasmid transfections. Transcriptome sequencing was employed to explore the downstream signaling pathways of Nesfatin-1. Additionally, brain cannula implantation was performed to facilitate targeted drug administration. RESULTS: Our findings demonstrated that EA reduced the hypothalamic overexpression of CRH and Nesfatin-1, as well as serum levels of ACTH and CORT. Additionally, it alleviated anxiety-like behaviors resulting from surgical trauma. We observed that overexpression of Nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) triggered hyperactivity of the HPA axis and anxiety. Conversely, knocking down Nesfatin-1 in the PVN reversed these effects caused by surgical trauma. Transcriptome sequencing identified the extracellular regulated protein kinases (ERK)/cAMP-response element binding protein (CREB) pathway as a key mediator in the impacts of surgical trauma and EA on the hypothalamus. Both in vivo and in vitro studies showed that overexpression of Nesfatin-1 activated the ERK/CREB pathway. Furthermore, administering ERK or CREB inhibitors into the PVN mitigated HPA axis hyperactivity and anxiety-like behaviors induced by surgical trauma. Finally, EA was observed to decrease the phosphorylation levels of ERK and CREB in the PVN. CONCLUSION: EA alleviates HPA axis hyperactivity and anxiety-like behaviors caused by surgical trauma through inhibition of Nesfatin-1/ERK/CREB pathway in the hypothalamus.

BAP1-mediated MAFF deubiquitylation regulates tumor growth and is associated with adverse outcomes in colorectal cancer.

BACKGROUND: Despite improvements in colorectal cancer (CRC) treatment, the prognosis for advanced CRC patients remains poor. Disruption of protein stability is one of the important factors in cancer development and progression. In this study, we aim to identify and analyze novel dysregulated proteins in CRC, assessing their significance and the mechanisms. METHODS: Using quantitative proteomics, expression pattern analysis, and gain-of-function/loss-of-function experiments, we identify novel functional protein dysregulated by ubiquitin-proteasome axis in CRC. Prognostic significance was evaluated in a training cohort of 546 patients and externally validated in 794 patients. Mechanistic insights are gained through molecular biology experiments, deubiquitinating enzymes (DUBs) expression library screening, and RNA sequencing. RESULTS: MAFF protein emerged as the top novel candidate substrate regulated by ubiquitin-proteasome in CRC. MAFF protein was preferentially downregulated in CRC compared to adjacent normal tissues. More importantly, multicenter cohort study identified reduced MAFF protein expression as an independent predictor of overall and disease-free survival in CRC patients. The in vitro and vivo assays showed that MAFF overexpression inhibited CRC growth, while its knockdown had the opposite effect. Intriguingly, we found the abnormal expression of MAFF protein was predominantly regulated via ubiquitination of MAFF, with K48-ubiquitin being dominant. BAP1 as a nuclear deubiquitinating enzyme (DUB), bound to and deubiquitinated MAFF, thereby stabilizing it. Such stabilization upregulated DUSP5 expression, resulting in the inhibition of ERK phosphorylation. CONCLUSIONS: This study describes a novel BAP1-MAFF signaling axis which is crucial for CRC growth, potentially serving as a therapeutic target and a promising prognostic biomarker for CRC.

ERK5 promotes autocrine expression to sustain mitogenic balance for cell fate specification in human pluripotent stem cells.

The homeostasis of human pluripotent stem cells (hPSCs) requires the signaling balance of extracellular factors. Exogenous regulators from cell culture medium have been widely reported, but little attention has been paid to the autocrine factor from hPSCs themselves. In this report, we demonstrate that extracellular signal-related kinase 5 (ERK5) regulates endogenous autocrine factors essential for pluripotency and differentiation. ERK5 inhibition leads to erroneous cell fate specification in all lineages even under lineage-specific induction. hPSCs can self-renew under ERK5 inhibition in the presence of fibroblast growth factor 2 (FGF2) and transforming growth factor ß (TGF-ß), although NANOG expression is partially suppressed. Further analysis demonstrates that ERK5 promotes the expression of autocrine factors such as NODAL, FGF8, and WNT3. The addition of NODAL protein rescues NANOG expression and differentiation phenotypes under ERK5 inhibition. We demonstrate that constitutively active ERK5 pathway allows self-renewal even without essential growth factors FGF2 and TGF-ß. This study highlights the essential contribution of autocrine pathways to proper maintenance and differentiation.

Combined swimming with melatonin protects against behavioural deficit in cerebral ischemia-reperfusion injury induced rats associated with modulation of Mst1- MAPK -ERK signalling pathway.

BACKGROUND: The inconvenience of social and behavioural deficits after cerebral ischaemia reperfusion (I/R) injury is still not well documented. AIM: We aimed to study the protective effect of preconditioning swimming exercise combined with melatonin against cerebral I/R induced injury. METHODOLOGY: Sixty rats were allocated into 6 groups; groups I and II served as control. Groups 3,4,5,6 subjected to bilateral carotid ligation for 30 minutes (min.) followed by reperfusion. Group 3 left untreated while groups 4 and 6; underwent swimming exercise 30 min/day, five days a week for three weeks before the surgery. Groups 5 and 6 treated with melatonin 30 minutes before the operation, then, all rats in groups 4, 5,6 were subjected to I/R. After that, groups 5 and 6 treated with 2nd dose of melatonin 30 minutes after reperfusion. RESULTS: Combined strategy exhibited the most neuroprotective effect through prevention of cerebral I/R induced inflammation, oxidative stress and apoptosis with subsequent improvement in socio behaviour deficits and enhanced Glial cell proliferative capacity. CONCLUSION: The protective contribution of combined strategy is associated with modulation in Macrophage-stimulating 1/mitogen-activated protein kinase/extracellular signal-regulated kinase (MST1/MAPK/ERK) pathway which may explain, at least in part, its protective potential.

Preconditioning swimming exercise combined with melatonin protected against cerebral I/R induced socio-behavioural deficit.Cerebral I/R induced pathophysiological alterations in Prefrontal cortical neurons (PFC) are prevented by combined Preconditioning swimming exercise and melatoninCombined Preconditioning swimming exercise/melatonin in cerebral I/R modulates MST1/MAPK/ERK signalling pathwayCombined Preconditioning swimming exercise/melatonin inhibit inflammation, oxidative stress and apoptosis, thus enhance Glial cell proliferative capacity in I/R induced injury in PFC neurons.

PPARß/δ attenuates hepatic fibrosis by reducing SMAD3 phosphorylation and p300 levels via AMPK in hepatic stellate cells.

The role of peroxisome proliferator-activated receptor (PPAR)ß/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARß/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-ß (TGF-ß). The PPARß/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard-/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARß/δ activation abrogated TGF-ß1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-ß1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARß/δ agonist reduces TGF-ß1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels.

REPURPOSING OF NICLOSAMIDE, AN ANTHELMINTIC, IN AUTISM SPECTRUM DISORDER BY TARGETING ERK/MAPK SIGNALING PATHWAY IN RATS.

AIM: The current study explores niclosamide's neuroprotective potential in an animal model of autism spectrum disorder (ASD) and goes further to understand how the ERK/MAPK signaling pathway is thought to contribute to this activity. METHODS: In order to create an autism-like phenotype in rats, 4 µl of 1 M PPA was infused intracerebroventricularly. The oral treatment with niclosamide (50 and 100 mg/kg) and risperidone (1 mg/kg) (used as standard) was given from 3rd to 30th day. Between the 14th and 28th day, behavioral assessments were made for sociability, stereotypy, anxiety, depression, novelty preference, repetitive behavior, and perseverative behavior. The animals were euthanized on the 29th day, and oxidative stress markers were assessed in the brain homogenate. The levels of neuroinflammatory cytokines such as TNF-α, IL-6, NF-κB, IFN-γ and glutamate were estimated using ELISA kits. To assess the involvement of the ERK/MAPK signaling pathway, levels of Nrf2 and ERK2 were also measured. KEY FINDINGS: Niclosamide therapy significantly restored behavioral, biochemical, neurological, and molecular impairments. Hence, niclosamide could be a potential neurotherapeutic candidate for further studies for use in ASD.

PRSS50-mediated inhibition of MKP3/ERK signaling is crucial for meiotic progression and sperm quality.

Serine protease 50 (PRSS50/TSP50) is highly expressed in spermatocytes. Our study investigated its role in testicular development and spermatogenesis. Initially, PRSS50 knockdown was observed to impair DNA synthesis in spermatocytes. To further explore this, we generated PRSS50 knockout ( Prss50 -/- ) mice ( Mus musculus), which exhibited abnormal spermatid nuclear compression and reduced male fertility. Furthermore, dysplastic seminiferous tubules and decreased sex hormones were observed in 4-week-old Prss50 -/- mice, accompanied by meiotic progression defects and increased apoptosis of spermatogenic cells. Mechanistic analysis indicated that PRSS50 deletion resulted in increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and elevated levels of MAP kinase phosphatase 3 (MKP3), a specific ERK antagonist, potentially accounting for testicular dysplasia in adolescent Prss50 -/- mice. Taken together, these findings suggest that PRSS50 plays an important role in testicular development and spermatogenesis, with the MKP3/ERK signaling pathway playing a significant role in this process.

17ß-Estradiol reduces inhibitory synaptic currents in entorhinal cortex neurons through G protein-coupled estrogen receptor-1 activation of extracellular signal-regulated kinase.

Estrogens are believed to modulate cognitive functions in part through the modulation of synaptic transmission in the cortex and hippocampus. Administration of 17ß-estradiol (E2) can rapidly enhance excitatory synaptic transmission in the hippocampus and facilitate excitatory synaptic transmission in rat lateral entorhinal cortex via activation of the G protein-coupled estrogen receptor-1 (GPER1). To assess the mechanisms through which GPER1 activation facilitates synaptic transmission, we assessed the effects of acute 10 nM E2 administration on pharmacologically isolated evoked excitatory and inhibitory synaptic currents in layer II/III entorhinal neurons. Female Long-Evans rats were ovariectomized between postnatal day (PD) 63 and 74 and implanted with a subdermal E2 capsule to maintain continuous low levels of E2. Electrophysiological recordings were obtained between 7 and 20 days after ovariectomy. Application of E2 for 20 min did not significantly affect AMPA or NMDA receptor-mediated excitatory synaptic currents. However, GABA receptor-mediated inhibitory synaptic currents (IPSCs) were markedly reduced by E2 and returned towards baseline levels during the 20-min washout period. The inhibition of GABA-mediated IPSCs was blocked in the presence of the GPER1 receptor antagonist G15. GPER1 can modulate protein kinase A (PKA), but blocking PKA with intracellular KT5720 did not prevent the E2-induced reduction in IPSCs. GPER1 can also stimulate extracellular signal-regulated kinase (ERK), a negative modulator of GABAA receptors, and blocking activation of ERK with PD90859 prevented the E2-induced reduction of IPSCs. E2 can therefore result in a rapid GPER1 and ERK signaling-mediated reduction in GABA-mediated IPSCs. This provides a novel mechanism through which E2 can rapidly modulate synaptic excitability in entorhinal layer II/III neurons and may also contribute to E2 and ERK-dependent alterations in synaptic transmission in other brain areas.

Aquaporin-3 is down-regulated by LMP1 in nasopharyngeal carcinoma cells to regulate cell migration and affect EBV latent infection.

Epstein-Barr virus (EBV) infection has a strong correlation with the development of nasopharyngeal carcinoma (NPC). Aquaporin 3 (AQP3), a member of the aquaporin family, plays an important role in tumor development, especially in epithelial-mesenchymal transition. In this study, the expression of AQP3 in EBV-positive NPC cells was significantly lower than that in EBV-negative NPC cells. Western blot and qRT-PCR analysis showed that LMP1 down-regulated the expression of AQP3 by activating the ERK pathway. Cell biology experiments have confirmed that AQP3 affects the development of tumor by promoting cell migration and proliferation in NPC cells. In addition, AQP3 can promote the lysis of EBV in EBV-positive NPC cells. The inhibition of AQP3 expression by EBV through LMP1 may be one of the mechanisms by which EBV maintains latent infection-induced tumor progression.

CRH upregulates supervillin through ERK and AKT pathways to promote bladder cancer cell migration.

Corticotropin-releasing hormone (CRH) has been well documented playing a role in the regulation of cellular processes, immune responses, and inflammatory processes that can influence the occurrence and development of tumors. Supervillin (SVIL) is a membrane-associated and actin-binding protein, which is actively involved in the proliferation, spread, and migration of cancer cells. This work investigated CRH's influence on bladder cancer cells' migration and relevant mechanisms. By using human bladder cancer cells T24 and RT4 in wound healing experiments and transwell assay, we found that the migration ability of the T24 cells was significantly increased after CRH treatment. In vivo experiments showed that CRH significantly promoted the metastases of T24 cells in cell line-derived xenograft (CDX) mouse model. Interestingly, downregulation of SVIL by SVIL-specifc small hairpin RNAs significantly reduced the promoting effect of CRH on bladder cancer cell migration. Furthermore, CRH significantly increased SVIL messenger RNA and protein expression in T24 cells, accompanied with AKT and ERK phosphorylation in T24 cells. Pretreatment with AKT inhibitor (MK2206) blocked the CRH-induced SVIL expression and ERK phosphorylation. Also, inhibition of ERK signaling pathway by U0126 significantly reduced the CRH-induced SVIL expression and AKT phosphorylation. It suggested that cross-talking between AKT and ERK pathways was involved in the effect of CRH on SVIL. Taken together, we demonstrated that CRH induced migration of bladder cancer cells, in which AKT and ERK pathways -SVIL played a key role.

Sulfenylation of ERK1/2: A novel mechanism for SO2-mediated inhibition of cardiac fibroblast proliferation.

Background: Endogenous sulfur dioxide (SO2) plays a crucial role in protecting heart from myocardial fibrosis by inhibiting the excessive growth of cardiac fibroblasts. This study aimed to investigate potential mechanisms by which SO2 suppressed myocardial fibrosis. Methods and results: Mouse model of angiotensin II (Ang II)-induced cardiac fibrosis and cell model of Ang II-stimulated cardiac fibroblast proliferation were employed. Our findings discovered that SO2 mitigated the aberrant phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by Ang II, leading to a reduction of fibroblast proliferation. Mechanistically, for the first time, we found that SO2 sulfenylated ERK1/2, and inhibited ERK1/2 phosphorylation and cardiac fibroblast proliferation, while a sulfhydryl reducing agent dithiothreitol (DTT) reversed the above effects of SO2. Furthermore, mutant ERK1C183S (cysteine 183 to serine) abolished the sulfenylation of ERK by SO2, thereby preventing the inhibitory effects of SO2 on ERK1 phosphorylation and cardiac fibroblast proliferation. Conclusion: Our study suggested that SO2 inhibited cardiac fibroblast proliferation by sulfenylating ERK1/2 and subsequently suppressing ERK1/2 phosphorylation. These new findings might enhance the understanding of the mechanisms underlying myocardial fibrosis and emphasize the potential of SO2 as a novel therapeutic target for myocardial fibrosis.

Corrigendum: Fucoidan ameliorates renal injury-related calcium-phosphorus metabolic disorder and bone abnormality in the CKD-MBD model rats by targeting FGF23-Klotho signaling axis.

[This corrects the article DOI: 10.3389/fphar.2020.586725.].

IL-1ß-activated PI3K/AKT and MEK/ERK pathways coordinately promote induction of partial epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a cellular process in embryonic development, wound healing, organ fibrosis, and cancer metastasis. Previously, we and others have reported that proinflammatory cytokine interleukin-1ß (IL-1ß) induces EMT. However, the exact mechanisms, especially the signal transduction pathways, underlying IL-1ß-mediated EMT are not yet completely understood. Here, we found that IL-1ß stimulation leads to the partial EMT-like phenotype in human lung epithelial A549 cells, including the gain of mesenchymal marker (vimentin) and high migratory potential, without the complete loss of epithelial marker (E-cadherin). IL-1ß-mediated partial EMT induction was repressed by PI3K inhibitor LY294002, indicating that the PI3K/AKT pathway plays a significant role in the induction. In addition, ERK1/2 inhibitor FR180204 markedly inhibited the IL-1ß-mediated partial EMT induction, demonstrating that the MEK/ERK pathway was also involved in the induction. Furthermore, we found that the activation of the PI3K/AKT and MEK/ERK pathways occurred downstream of the epidermal growth factor receptor (EGFR) pathway and the IL-1 receptor (IL-1R) pathway, respectively. Our findings suggest that the PI3K/AKT and MEK/ERK pathways coordinately promote the IL-1ß-mediated partial EMT induction. The inhibition of not one but both pathways is expected yield clinical benefits by preventing partial EMT-related disorders such as organ fibrosis and cancer metastasis.

Update on the Genetics of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous heritable skeletal dysplasia characterized by bone fragility and deformity, growth deficiency, and other secondary connective tissue defects. OI is now understood as a collagen-related disorder caused by defects of genes whose protein products interact with collagen for folding, post-translational modification, processing and trafficking, affecting bone mineralization and osteoblast differentiation. This review provides the latest updates on genetics of OI, including new developments in both dominant and rare OI forms, as well as the signaling pathways involved in OI pathophysiology. There is a special emphasis on discoveries of recessive mutations in TENT5A, MESD, KDELR2 and CCDC134 whose causality of OI types XIX, XX, XXI and XXI, respectively, is now established and expends the complexity of mechanisms underlying OI to overlap LRP5/6 and MAPK/ERK pathways. We also review in detail new discoveries connecting the known OI types to each other, which may underlie an eventual understanding of a final common pathway in OI cellular and bone biology.

Glypican-3 deficiency in liver cancer upregulates MAPK/ERK pathway but decreases cell proliferation.

Glypican-3 (GPC3) is overexpressed in hepatocellular carcinomas and hepatoblastomas and represents an important therapeutic target but the biologic importance of GPC3 in liver cancer is unclear. To date, there are limited data characterizing the biological implications of GPC3 knockout (KO) in liver cancers that intrinsically express this target. Here, we report on the development and characterization of GPC3-KO liver cancer cell lines and compare to them to parental lines. GPC3-KO variants were established in HepG2 and Hep3B liver cancer cell lines using a lentivirus-mediated CRISPR/Cas9 system. We assessed the effects of GPC3 deficiency on oncogenic properties in vitro and in murine xenograft models. Downstream cellular signaling pathway changes induced by GPC3 deficiency were examined by RNAseq and western blot. To confirm the usefulness of the models for GPC3-targeted drug development, we evaluated the target engagement of a GPC3-selective antibody, GC33, conjugated to the positron-emitting zirconium-89 (89Zr) in subcutaneous murine xenografts of wild type (WT) and KO liver cancer cell lines. Deletion of GPC3 significantly reduced liver cancer cell proliferation, migration, and invasion compared to the parental cell lines. Additionally, the tumor growth of GPC3-KO liver cancer xenografts was significantly slower compared with control xenografts. RNA sequencing analysis also showed GPC3-KO resulted in a reduction in the expression of genes associated with cell cycle regulation, invasion, and migration. Specifically, we observed the downregulation of components in the AKT/NFκB/WNT signaling pathways and of molecules related to cell cycle regulation with GPC3-KO. In contrast, pMAPK/ERK1/2 was upregulated, suggesting an adaptive compensatory response. KO lines demonstrated increased sensitivity to ERK (GDC09994), while AKT (MK2206) inhibition was more effective in WT lines. Using antibody-based positron emission tomography (immunoPET) imaging, we confirmed that 89Zr-GC33 accumulated exclusively in GPC3-expression xenografts but not in GPC3-KO xenografts with high tumor uptake and tumor-to-liver signal ratio. We show that GPC3-KO liver cancer cell lines exhibit decreased tumorigenicity and altered signaling pathways, including upregulated pMAPK/ERK1/2, compared to parental lines. Furthermore, we successfully distinguished between GPC3+ and GPC3- tumors using the GPC3-targeted immunoPET imaging agent, demonstrating the potential utility of these cell lines in facilitating GPC3-selective drug development.

FASN promotes anoikis resistance in colorectal liver metastases through the ERK1/2 pathway.

PURPOSE: Colorectal cancer (CRC) is recognized as the third most common form of malignancy, with the liver frequently serving as the main site for metastasis. Anoikis resistance (AR) is critical in colorectal cancer liver metastases (CRLM). Fatty acid synthase (FASN), essential in lipid synthesis, mediates AR in many cancers. The present research examines the function of FASN in ERK1/2-mediated AR in CRLM and evaluates its therapeutic potential. METHODS: We performed scratch and migration experiment to evaluate the migration capacity of the LoVo cells. Flow cytometry was employed to identify cell apoptosis. The levels of FASN, p-ERK1/2, and proteins related to apoptosis was analyzed by Western blot. The mRNA level of FASN was determined by q-PCR after FASN silencing. In addition, we used an intrasplenic liver metastasis model of nude to assess the effect of FASN on CRLM. RESULTS: In vitro experiments showed that after FASN silencing, the cell apoptosis rate was increased, migration capability was notably decreased, the expression of p-ERK1/2, the proteins related to anti-apoptotic were significantly decreased, and the proteins related to apoptosis were significantly increased. In vivo experiments showed that AR significantly increased the number of liver metastatic foci, whereas FASN silencing significantly inhibited CRLM. CONCLUSION: These results suggest that FASN silencing suppressed AR through the ERK 1/2 pathway, which in turn suppressed CRLM.

Antrodia camphorata Supplementation during Early Life Alters Gut Microbiota and Inhibits Young-Onset Intestinal Tumorigenesis in APC1638N Mice Later in Life.

Young-onset colorectal cancer is an increasing concern worldwide due to the growing prevalence of Westernized lifestyles in childhood and adolescence. Environmental factors during early life, particularly early-life nutrition, significantly contribute to the increasing incidence. Recently, there have been reports of beneficial effects, including anti-inflammation and anti-cancer, of a unique fungus (Antrodia camphorate, AC) native to Taiwan. The objective of this study is to investigate the impact of AC supplementation in early life on the development of young-onset intestinal tumorigenesis. APC1638N mice were fed with a high-fat diet (HF) at 4-12 weeks of age, which is equivalent to human childhood/adolescence, before switching to a normal maintenance diet for an additional 12 weeks up to 24 weeks of age, which is equivalent to young to middle adulthood in humans. Our results showed that the body weight in the HF groups significantly increased after 8 weeks of feeding (p < 0.05). Following a switch to a normal maintenance diet, the change in body weight persisted. AC supplementation significantly suppressed tumor incidence and multiplicity in females (p < 0.05) and reduced IGF-1 and Wnt/ß-catenin signaling (p < 0.05). Moreover, it altered the gut microbiota, suppressed inflammatory responses, and created a microenvironment towards suppressing tumorigenesis later in life.

Methylsulfinyl Hexyl Isothiocyanate (6-MSITC) from Wasabi Is a Promising Candidate for the Treatment of Cancer, Alzheimer's Disease, and Obesity.

Methylsulfinyl hexyl isothiocyanate (6-MSITC) isolated from Eutrema japonicum is a promising candidate for the treatment of breast cancer, colorectal and stomach cancer, metabolic syndrome, heart diseases, diabetes, and obesity due to its anti-inflammatory and antioxidant properties. Also, its neuroprotective properties, improving cognitive function and protecting dopaminergic neurons, make it an excellent candidate for treating neurodegenerative diseases like dementia, Alzheimer's, and Parkinson's disease. 6-MSITC acts on many signaling pathways, such as PPAR, AMPK, PI3K/AKT/mTOR, Nrf2/Keap1-ARE, ERK1/2-ELK1/CHOP/DR5, and MAPK. However, despite the very promising results of in vitro and in vivo animal studies and a few human studies, the molecule has not yet been thoroughly tested in the human population. Nonetheless, wasabi should be classified as a "superfood" for the primary and secondary prevention of human diseases. This article reviews the current state-of-the-art research on 6-MSITC and its potential clinical uses, discussing in detail the signaling pathways activated by the molecule and their interactions.