S6 Kinase Research Articles

Diosmin alleviates NLRP3 inflammasome-dependent cellular pyroptosis after stroke through RSK2/CREB pathway.

In the context of our previous analyses on the main active ingredients of Jieyudan, a classic formula targeting aphasia in stroke, we further delve into the function and mechanisms of its active ingredient, Diosmin (DM), which may exert neuroprotective effects, in ischemic stroke. Herein, bioinformatics analysis revealed targets of DM and their intersection with differentially expressed genes in ischemic stroke. Middle cerebral artery occlusion (MCAO) rats and oxygen-glucose deprivation (OGD) cells were used to construct in vivo and in vitro models of ischemic stroke. The effects of DM on MCAO rats were assessed by Zea-Longa score, Morris water maze, TTC staining, Nissl staining, immunohistochemistry, and Western blot. At the cellular level, cell counting kit-8 assay and Western blot were carried out to verify the mechanism of DM in ischemic stroke. In vivo, DM decreased neurological deficit score, cerebral infarct volume and neuronal damage, and improved cognitive function in MCAO rats. In vitro, DM increased the viability of OGD-treated cells. In addition, DM down-regulated the expressions of NLR family pyrin domain containing 3 (NLRP3) and pyroptosis-associated proteins, while up-regulating ribosomal protein S6 kinase A3 (RSK2) level and activating cyclic-AMP response element-binding protein (CREB) signaling. Conversely, RSK2 inhibitor LJH685 reduced the viability and promoted pyroptosis-associated protein levels, which also partially reversed the effects of DM in vitro. Collectively, DM plays a therapeutic role in ischemic stroke by inhibiting NLRP3 inflammasome-mediated cellular pyroptosis via the RSK2/CREB pathway.

CSIG-30. EXPLORING THE ROLE OF CAPICUA (CIC) IN RESISTANCE MECHANISMS OF MEK INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, resistant to conventional therapy. Despite its heterogeneous nature, a key feature of GBM is aberrant kinase signaling. Specifically, hyperactivation of receptor tyrosine kinases (RTK) and their downstream tumorigenic effectors Ras/MEK/Extracellular Signal Regulated Kinase (ERK) pathway are attributed to the aggressive nature of GBM. Capicua (CIC) is a High Mobility Group (HMG) box transcriptional repressor that directly binds to DNA and counteracts the transcription of genes typically expressed only in response to RTK activation such as oncogenic transcription factors ETV1/4/5. Hyperactive MEK/ERK signaling in GBM causes CIC degradation. The mechanism of CIC’s repressor function is not well understood, however, transcriptional repressors (like CIC) typically form co-repressor complexes to repress target genes. We discovered a novel interaction between CIC and the transcriptional regulator yin yang 1 (YY1), a context-dependent transcription factor that can repress or activate gene expression. We uncovered that 90-kDa ribosome S6 protein kinase (p90RSK), a downstream effector of ERK and a known CIC regulator, mediates the CIC/YY1 interaction. Interestingly, we found re-activation of p90RSK following MEK/ERK inhibition implicating p90RSK in the resistance mechanism of RTK/MEK/ERK inhibition in GBM. We hypothesize that p90RSK inhibition will stabilize the CIC/YY1 co-repressor complex and re-sensitize towards MEK/ERK inhibition. Patient-derived glioma stem-like cells (GSCs) were treated with increasing doses of BI-D1870 or LJI308 (p90RSK inhibitors) with and without selumetinib (MEK inhibitor) and various functional assays were performed to examine the effect of p90RSK inhibition on the CIC/YY1 complex and sensitization to MEK/ERK inhibition. Treatment with BI-D1870 and LJI308 markedly reduced viability and mRNA expression of ETV1/4/5 indicating that p90RSK inhibition may mediate CIC /YY1 DNA binding ability. Importantly, inhibiting p90RSK sensitized GSCs to MEK/ERK inhibition. These results highlight the importance of downstream p90RSK inhibition in stabilizing CIC/YY1 repressor complex function and sensitizing cells to RTK/MEK/ERK inhibition.

P21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2

Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK.

Regulatory-Associated Protein of mTOR-Mediated Signaling: A Nexus Between Tumorigenesis and Disease

RAPTOR (regulatory-associated protein of mTOR) is a pivotal component of the mammalian target of rapamycin complex 1 (mTORC1), playing a central role in regulating cell growth, metabolism and stress responses. As a scaffold protein, RAPTOR recruits key substrates such as eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 kinase (S6K), facilitating their phosphorylation by mTORC1, which in turn drives protein synthesis, lipid metabolism and cellular proliferation. Its regulatory function becomes especially crucial under conditions of nutrient deprivation or stress, where it enhances the stability of the mTORC1 complex, allowing cells to adapt to fluctuating environmental cues. The hyperactivation of mTORC1, largely mediated by RAPTOR, is frequently observed in various cancers, contributing to uncontrolled cell proliferation and tumorigenesis. Moreover, RAPTOR’s modulation of immune responses and metabolic pathways extends its influence beyond oncogenesis, impacting inflammatory diseases and metabolic disorders. This review meticulously elucidates RAPTOR’s structure, post-translational modifications as well as its indispensable role within the mTORC1 complex, emphasizing its regulatory functions in cellular growth, metabolic adaptation, immune response and disease pathology including oncogenesis. Furthermore, it explores emergent therapeutic avenues targeting RAPTOR-mediated mTORC1 signaling, underscoring their potential to revolutionize cancer treatment and the management of related pathophysiological conditions.

The novel secretome ST266 activates Akt and protects against oxidative stress-mediated injury in human RPE and Müller cells

Oxidative stress-mediated retinal pigment epithelial (RPE) cell damage is associated with age-related macular degeneration (AMD). ST266 is the biological secretome produced by a novel population of amnion-derived multipotent progenitor cells. Herein, we investigated the effect of ST266 on RPE cell injury induced by hydroquinone (HQ), a cigarette smoke related oxidant, hydrogen peroxide (H2O2) and all-trans retinal (atRal), a pro-oxidant component of the retinoid cycle. We additionally investigated its effect on Müller cell injury induced by H2O2.Cultured human RPE cells were pre-treated for 1 hour in the presence or absence of MK-2206, a protein kinase B (Akt) inhibitor, then treated with varying concentrations of HQ, H2O2, or atRal for 1.5 hours. Cultured human Müller cells (MIO-M1) were pre-treated for 1 hour in the presence or absence of MK-2206, then treated with varying concentrations of H2O2 for 1.5 hours. Media were then replaced with STM100 (control media into which the ST266 secretome proteins were collected) or ST266 at various times. Cell viability was determined with WST-1 reagent. Mitochondrial membrane potential (Δψm) was quantified by a fluorescence plate reader. The protein phosphorylation levels of Akt, glycogen synthase kinase 3 beta (GSK-3β), and p70 ribosomal S6 kinase (p70S6K) were measured by Western blot.ST266 significantly improved RPE and MIO-M1 cell viability that was reduced by oxidant exposure and improved oxidant-disrupted Δψm. In both cell types, ST266 induced phosphorylation of Akt, GSK-3β, and p70S6K. MK-2206 significantly eliminated ST266-mediated protein phosphorylation of Akt, GSK-3β, and p70S6K and abolished the ST266-protective effect on cell viability. In conclusion, ST266 activates Akt, protects against oxidative stress-mediated cell injury in an Akt-dependent manner, and improves Δψm, suggesting a potential role for ST266 therapy in treating retinal diseases such as AMD.

Research on Function of Ribosomal Protein S6 Kinases, 1α and β, Based on Molecular Cloning and siRNA-Based Interference in Juvenile Blunt Snout Bream (Megalobrama amblycephala)

The aim of this study was to investigate the effects of S6K1α and β on the expression of glycolysis- and gluconeogenesis-related genes in juvenile blunt snout bream. Two isoforms, α and β, of ribosomal protein S6 kinase 1 in blunt snout bream were cloned and characterized, and their expression patterns were examined in vivo. The sequence analysis showed that s6k1α and s6k1β contain open reading frames of 2217 and 1497 bp, encoding 738 and 498 amino acids, respectively. Both S6K1α and S6K1β consist of an S_TKc domain and an extended S_TK_X domain. s6k1α and s6k1β were abundantly expressed in the heart and gonads. siRNAs were designed, and the experiment showed that α-siRNA inhibited s6k1α and s6k1β expression, but β-siRNA exclusively inhibited s6k1α expression (p < 0.05). α-siRNA upregulated the expression levels of gk and pk, while β-siRNA upregulated pepck and g6p expression (p < 0.05). The expression of g6pdh was found to be downregulated, but the gs mRNA level was overexpressed after treatment with α-siRNA and β-siRNA (p < 0.05). In the present experiment, S6K1α was more intimately involved in the regulation of gluconeogenesis when only S6K1α was inhibited, whereas the inhibition of both S6K1α and S6K1β collectively co-regulated glycolysis.

Far-infrared irradiation inhibits proliferation of human upper airway epithelial cells via protein phosphatase 2A-promoted dephosphorylation of p70 S6 kinase.

Far-infrared (FIR) ray, an invisible electromagnetic radiation with a wavelength of 3‒1000μm, elicits various biological effects. Excessive proliferation of human upper airway epithelial cells (HUAEpCs) contributes to the development and exacerbation of nasal narrowing diseases, including nasal polyposis and chronic rhinosinusitis with nasal polyps (CRSwNP). Here, we investigated the molecular mechanisms through which FIR irradiation inhibits the proliferation of HUAEpCs. FIR irradiation significantly inhibited the proliferation of NCI-H292 cells without alteration in cell viability. The anti-proliferative effect of FIR radiation was accompanied by decreased phosphorylation of p70S6K at Thr389 (p-p70S6K-Thr389), without changes in the phosphorylation of mammalian target of rapamycin and adenosine monophosphate-activated protein kinase (AMPK). Overexpression of p70S6K-T389E mutant gene, not dominant negative-AMPKα1 gene, significantly reversed FIR irradiation-inhibited p-p70S6K-Thr389 and cell proliferation. Cotreatment with okadaic acid or knockdown of protein phosphatase 2A catalytic subunit (PP2Ac) gene expression significantly reversed FIR irradiation-decreased p-p70S6K-Thr389 and cell proliferation. FIR irradiation remarkably promoted the physical association of p70S6K and PP2Ac without change in total PP2Ac expression. Hyperthermal stimulus (39°C) did not alter p-p70S6K-Thr389 and cell proliferation. In line with NCI-H292 cell results, FIR irradiation, not hyperthermal stimulus, significantly decreased p-p70S6K-Thr389 and cell proliferation in primary human nasal turbinate and polyp epithelial cells. These results demonstrated that FIR irradiation decreased the proliferation of HUAEpCs through PP2A-mediated inhibition of p70S6K phosphorylation, independent of its hyperthermal effect. Our data suggest that FIR therapy can be used to treat upper airway narrowing epithelial hyperplastic diseases, including nasal polyposis and CRSwNP.

ALYREF recruits ELAVL1 to promote colorectal tumorigenesis via facilitating RNA m5C recognition and nuclear export

ALYREF can recognize 5-methylcytosine (m5C) decoration throughout RNAs to regulate RNA metabolism. However, its implications in cancer and precise regulatory mechanisms remain largely elusive. Here, we demonstrated that ALYREF supported colorectal cancer (CRC) growth and migration. Integrated analysis of ALYREF-RIP-Bis-seq and transcriptome profiles identified ribosomal protein S6 kinase B2 (RPS6KB2) and regulatory-associated protein of mTOR (RPTOR) as ALYREF’s possible downstream effectors. Mechanistically, ALYREF formed a complex with ELAV like RNA binding protein 1 (ELAVL1) to cooperatively promote m5C recognition and nuclear export of the two mRNAs. Moreover, ALYREF protein was highly expressed in tumor tissues of CRC patients, which predicted their poor prognosis. E2F transcription factor 6 (E2F6)-mediated transactivation gave a molecular insight into ALYREF overexpression. Collectively, ALYREF recruits ELAVL1 to collaboratively facilitate m5C recognition and nuclear export of RPS6KB2 and RPTOR transcripts for colorectal tumorigenesis, providing RNA m5C methylation as promising therapeutic targets and prognostic biomarkers for CRC.

Reciprocal regulation of mTORC1 signaling and ribosomal biosynthesis determines cell cycle progression in activated T cells.

Ribosomal biosynthesis in nucleoli is an energy-demanding process driven by all RNA polymerases and hundreds of auxiliary proteins. We investigated how this process is regulated in activated T lymphocytes by T cell receptor (TCR) signals and the multiprotein complexes mTORC1 and mTORC2, both of which contain the kinase mTOR. Deficiency in mTORC1 slowed the proliferation of T cells, with further delays in each consecutive division, an effect not seen with deficiency in mTORC2. mTORC1 signaling was stimulated by components of conventional TCR signaling, and, reciprocally, TCR sensitivity was decreased by mTORC1 inhibition. The substantial increase in the amount of RNA per cell induced by TCR activation was reduced by 50% by deficiency in mTORC1, but not in mTORC2 or in S6 kinases 1 and 2, which are activated downstream of mTORC1. RNA-seq data showed that mTORC1 deficiency reduced the abundance of all RNA biotypes, although rRNA processing was largely intact in activated T cells. Imaging cytometry with FISH probes for nascent pre-rRNA revealed that deletion of mTORC1, but not that of mTORC2, reduced the number and expansion of nucleolar sites of active transcription. Protein translation was consequently decreased by 50% in the absence of mTORC1. Inhibiting RNA polymerase I blocked not only proliferation but also mTORC1 signaling. Our data show that TCR signaling, mTORC1 activity, and ribosomal biosynthesis in the nucleolus regulate each other during biomass production in clonally expanding T cells.

Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B

BackgroundEpigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.MethodsKDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.ResultsRSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.ConclusionsOverall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.

Key Anabolic Markers in Human M. Soleus after 21-Day Head-Down Tilt Bedrest

Prolonged bed rest can have a significant negative effect on skeletal muscle, leading to muscle wasting and reduced strength. This process can occur in as little as 10 days in healthy individuals, with the loss of muscle mass and strength being particularly pronounced during the first week of immobilization. Head-down tilt bed rest (HDT) is a method used to simulate the physiological changes that occur in weightlessness during spaceflight. This technique involves lying in bed with the head tilted downward. This paper is dedicated to the analysis of key anabolic markers of the soleus muscle during 21 days of HDT BR. The HDT BR experiment was conducted at the Institute of Biomedical Problems, Russian Academy of Sciences. Six healthy male volunteers, aged 25–35 years, were subjected to 21 days of strict bed rest with a tilt angle of –6°. A needle biopsy of the m.soleus was performed using the Bergström method before the start of HDT BR and on day 21 of HDT BR. The biopsy material was immediately frozen in liquid nitrogen for further Western blot and PCR analysis. Examination of mTORC1 substrates showed a significant decrease in p70 and 4EBP1 phosphorylation after HDT BR. We also observed a significant decrease in the phosphorylation of another ribosomal kinase, p90RSK, a significant increase in eEF2 phosphorylation and an increase in eEF2k mRNA expression. In addition, the phosphorylation of AMPK and its substrate ACC decreased after HDT BR. The data obtained in this work support the hypothesis that a decrease in protein synthesis, together with an increase in proteolysis, contributes to the development of human m. soleus atrophy after 21 days of HDT BR.

Pleiotrophin Activates cMet- and mTORC1-Dependent Protein Synthesis through PTPRZ1-The Role of ανβ3 Integrin.

Pleiotrophin (PTN) is a secreted factor that regulates endothelial cell migration through protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) and αvβ3 integrin. Genetic deletion of Ptprz1 results in enhanced endothelial cell proliferation and migration, due to the decreased expression of β3 integrin and the subsequent, enhanced cMet phosphorylation. In the present study, we investigated the effect of PTN and PTPRZ1 on activating the mTORC1 kinase and protein synthesis and identified part of the implicated signaling pathway in endothelial cells. PTN or genetic deletion of Ptprz1 activates protein synthesis in a mTORC1-dependent manner, as shown by the enhanced phosphorylation of the mTORC1-downstream targets ribosomal protein S6 kinase 1 (SK61) and 4E-binding protein 1 (4EBP1) and the upregulation of HIF-1α. The cMet tyrosine kinase inhibitor crizotinib abolishes the stimulatory effects of PTN or PTPRZ1 deletion on mTORC1 activation and protein synthesis, suggesting that mTORC1 activation is downstream of cMet. The mTORC1 inhibitor rapamycin abolishes the stimulatory effect of PTN or PTPRZ1 deletion on endothelial cell migration, suggesting that mTORC1 is involved in the PTN/PTPRZ1-dependent cell migration. The αvβ3 integrin blocking antibody LM609 and the peptide PTN112-136, both known to bind to ανβ3 and inhibit PTN-induced endothelial cell migration, increase cMet phosphorylation and activate mTORC1, suggesting that cMet and mTORC1 activation are required but are not sufficient to stimulate cell migration. Overall, our data highlight novel aspects of the signaling pathway downstream of the PTN/PTPRZ1 axis that regulates endothelial cell functions.

Silmitasertib in Combination With Cabozantinib Impairs Liver Cancer Cell Cycle Progression, Induces Apoptosis, and Delays Tumor Growth in a Preclinical Model.

The rising incidence of hepatocellular carcinoma (HCC) is a global problem. Several approved treatments, including immune therapy and multi-tyrosine kinase inhibitors, are used for treatment, although the results are not optimum. There is an unmet need to develop highly effective chemotherapies for HCC. Targeting multiple pathways to attack cancer cells is beneficial. Cabozantinib is an orally available bioactive multikinase inhibitor and has a modest effect on HCC treatment. Silmitasertib is an orally bioavailable, potent CK2 inhibitor with a direct role in DNA damage repair and is in clinical trials for other cancers. In this study, we planned to repurpose these existing drugs on HCC treatment. We observed a stronger antiproliferative effect of these two combined drugs on HCC cells generated from different etiologies as compared to the single treatment. Global RNA-seq analyses revealed a decrease in the expression of G2/M cell cycle transition genes in HCC cells following combination treatment, suggesting G2 phase cell arrest. We observed G2/M cell cycle phase arrest in HCC cells upon combination treatment compared to the single-treated or vehicle-treated control cells. The downregulation of CCNA2 and CDC25C following combination therapy further supported the observation. Subsequent analyses demonstrated that combination treatment inhibited 70 kDa ribosomal protein S6 kinase (p70S6K) phosphorylation, and increased Bim expression. Apoptosis of HCC cells were accompanied by increased poly (ADP-ribose) polymerase cleavage and caspase-9 activation. Next, we observed that a combination therapy significantly delayed the progression of HCC xenograft growth as compared to vehicle control. Together, our results suggested combining cabozantinib and silmitasertib would be a promising treatment option for HCC.

Punicalagin as a novel selective aryl hydrocarbon receptor (AhR) modulator upregulates AhR expression through the PDK1/p90RSK/AP-1 pathway to promote the anti-inflammatory response and bactericidal activity of macrophages

Aryl hydrocarbon receptor (AhR) plays an important role in inflammation and immunity as a new therapeutic target for infectious disease and sepsis. Punicalagin (PUN) is a Chinese herbal monomer extract of pomegranate peel that has beneficial anti-inflammatory, antioxidant and anti-infective effects. However, whether PUN is a ligand of AhR, its effect on AhR expression, and its signaling pathway remain poorly understood. In this study, we found that PUN was a unique polyphenolic compound that upregulated AhR expression at the transcriptional level, and regulated the AhR nongenomic pathway. AhR expression in lipopolysaccharide-induced macrophages was upregulated by PUN in vitro and in vivo in a time- and dose-dependent manner. Using specific inhibitors and siRNA, induction of AhR by PUN depended on sequential phosphorylation of 90-kDa ribosomal S6 kinase (p90RSK), which was activated by the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-dependent protein kinase (PDK)1 pathways. PUN promoted p90RSK-mediated activator protein-1 (AP-1) activation. AhR knockout or inhibitors reversed suppression of interleukin (IL)-6 and IL-1β expression by PUN. PUN decreased Listeria load and increased macrophage survival via AhR upregulation. In conclusion, we identified PUN as a novel selective AhR modulator involved in AhR expression via the MEK/ERK and PDK1 pathways targeting p90RSK/AP-1 in inflammatory macrophages, which inhibited macrophage inflammation and promoted bactericidal activity.

Dysregulating mTORC1-4E-BP2 signaling in GABAergic interneurons impairs hippocampus-dependent learning and memory.

Memory formation is contingent on molecular and structural changes in neurons in response to learning stimuli-a process known as neuronal plasticity. The initiation step of mRNA translation is a gatekeeper of long-term memory by controlling the production of plasticity-related proteins in the brain. The mechanistic target of rapamycin complex 1 (mTORC1) controls mRNA translation, mainly through phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and ribosomal protein S6 kinases (S6Ks). mTORC1 signaling decreases throughout brain development, starting from the early postnatal period. Here, we discovered that in mice, the age-dependent decrease in mTORC1 signaling occurs selectively in excitatory but not inhibitory neurons. Using a gene conditional knockout (cKO) strategy, we demonstrate that either up- or downregulating the mTORC1-4E-BP2 axis in GAD65 inhibitory interneurons, but not excitatory neurons, results in long-term object recognition and object location memory deficits. Our data indicate that the mTORC1 pathway in inhibitory but not excitatory neurons plays a key role in memory formation.

RPS6KA1 is a histone acetylation-related oncoprotein in acute myeloid leukemia which is targeted by afzelin

BackgroundHistone acetylation plays a critical role in the progression of acute myeloid leukemia (AML). This study aimed to explore the prognostic significance and biological implications of histone acetylation-related genes in AML and to identify potential oncoproteins and therapeutic compounds.MethodsGenes associated with AML and histone acetylation were identified using the TCGA-LAML and IMEx Interactome databases. A histone acetylation-related risk model was developed using the least absolute shrinkage and selection operator method. The prognostic value of the model was evaluated through Kaplan-Meier survival analysis, time-dependent receiver operating characteristic curve, univariate and multivariate Cox regression, and nomogram calibration. Key genes were identified using random forest, support vector machine, and multivariate Cox analysis. Molecular docking was employed to assess the binding affinity between ribosomal protein S6 kinase A1 (RPS6KA1) and potential compounds. Furthermore, the effects of RPS6KA1 and afzelin on the malignant behaviors and downstream pathways of AML cells were validated through in vitro experiments.ResultsA risk model composed of 6 genes, including HDAC6, CREB3, KLF13, GOLGA2, RPS6KA1 and ZMIZ2, was established, demonstrating strong prognostic predictive capability. Among these, RPS6KA1 emerged as a key risk factor linked to histone acetylation status in AML. Elevated RPS6KA1 expression was observed in AML samples and was associated with poor prognosis. RPS6KA1 knockdown suppressed AML cell proliferation, migration, and invasion, induced G0/G1 phase arrest, and promoted apoptosis. Additionally, RPS6KA1 was identified as a potential target for afzelin, which exhibited anti-AML activity by inactivating RPS6KA1.ConclusionHistone acetylation status is closely associated with AML patient prognosis. RPS6KA1 acts as an oncoprotein in AML, facilitating disease progression. Afzelin may represent a novel therapeutic agent for AML by targeting RPS6KA1, which requires validation by clinical trials.

Abstract C111: Characterization of kisspeptin receptor signaling pathways in cervical cancer: Unveiling novel mechanisms and therapeutic potentials

Abstract Introduction: This study delves into the complex signaling mechanisms of the kisspeptin system in cervical cancer, aiming to unravel the intricate interactions between kisspeptin and its receptor, as well as their roles in modulating cancer progression. Given the emerging significance of the kisspeptin system in tumor biology, our research seeks to illuminate how kisspeptin signaling influences cellular behaviors critical to cancer development, such as proliferation and migration. By exploring the nuanced effects of kisspeptin receptor activation, this study contributes to a deeper understanding of its therapeutic potential and underscores the importance of kisspeptin pathways in the broader context of cancer research. Methods: Was employed Bioluminiscence Resonance Energy Transfer (BRET) assays to explore the activation of the kisspeptin receptor in cervical cancer cell lines, using synthesized analogs of kisspeptin-10 for detailed signaling analysis. Functional effects were assessed through proliferation and migration assays. Additionally, we evaluated the activation or reduction in phosphorylation of specific kinases involved in tumoral processes, providing a nuanced understanding of the signaling mechanisms at play. Results: Our data demonstrate that activation of the kisspeptin receptor substantially modulates cellular proliferation and motility in cervical cancer cell lines. We uncovered novel activation of the Gz protein by kisspeptin-10, which is integral to the kisspeptin receptor function, and identified an upregulation of signaling pathways critical for cell survival and metastasis. Importantly, we observed activation of kinases such as Chk2, c-Jun, p70 S6 kinase, and RSK 1/2/3, as well as members of the STAT family, following kisspeptin-10 stimulation. This kinase activation underscores the multifaceted role of kisspeptin in tumor progression, suggesting its involvement in both pro-oncogenic and tumor-suppressive pathways, and provides direct insights into the mechanistic underpinnings of kisspeptin effect on cancer cell behavior. Conclusion: In conclusion, our study elucidates the complex role of kisspeptin-10 in cervical cancer, demonstrating its ability to modulate cellular proliferation and motility through activation of the kisspeptin receptor. The novel activation of the Gz protein and subsequent upregulation of signaling pathways associated with cell survival and metastasis highlight the intricate involvement of kisspeptin in cancer progression. Furthermore, the activation of key kinases such as Chk2, c-Jun, p70 S6 kinase, RSK 1/2/3, and STAT family members upon kisspeptin-10 stimulation reveals a dual-faceted influence on tumor dynamics, promoting both pro-oncogenic and tumor-suppressive mechanisms. These findings provide significant insights into the molecular actions of kisspeptin in cervical cancer, offering potential targets for therapeutic intervention and a deeper understanding of its role in tumor biology. Citation Format: Deisy Y. Rodríguez-Sarmiento, Pedro H. Scarpelli-Pereira, Michel Bouvier. Characterization of kisspeptin receptor signaling pathways in cervical cancer: Unveiling novel mechanisms and therapeutic potentials [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C111.

Antidiabetic and Antihyperlipidemic Activities and Molecular Mechanisms of Phyllanthus emblica L. Extract in Mice on a High-Fat Diet.

We planned to explore the protective activities of extract of Phyllanthus emblica L. (EPE) on insulin resistance and metabolic disorders including hyperlipidemia, visceral obesity, and renal dysfunction in high-fat diet (HFD)-progressed T2DM mice. Mice treatments included 7 weeks of HFD induction followed by EPE, fenofibrate (Feno), or metformin (Metf) treatment daily for another 4-week HFD in HFD-fed mice. Finally, we harvested blood to analyze some tests on circulating glycemia and blood lipid levels. Western blotting analysis was performed on target gene expressions in peripheral tissues. The present findings indicated that EPE treatment reversed the HFD-induced increases in blood glucose, glycosylated HbA1C, and insulin levels. Our findings proved that treatment with EPE in HFD mice effectively controls hyperglycemia and hyperinsulinemia. Our results showed that EPE reduced blood lipid levels, including a reduction in blood triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA); moreover, EPE reduced blood leptin levels and enhanced adiponectin concentrations. EPE treatment in HFD mice reduced BUN and creatinine in both blood and urine and lowered albumin levels in urine; moreover, EPE decreased circulating concentrations of inflammatory NLR family pyrin domain containing 3 (NLRP3) and kidney injury molecule-1 (KIM-1). These results indicated that EPE displayed antihyperglycemic and antihyperlipidemic activities but alleviated renal dysfunction in HFD mice. The histology examinations indicated that EPE treatment decreased adipose hypertrophy and hepatic ballooning, thus contributing to amelioration of lipid accumulation. EPE treatment decreased visceral fat amounts and led to improved systemic insulin resistance. For target gene expression levels, EPE enhanced AMP-activated protein kinase (AMPK) phosphorylation expressions both in livers and skeletal muscles and elevated the muscular membrane glucose transporter 4 (GLUT4) expressions. Treatment with EPE reduced hepatic glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) expressions to suppress glucose production in the livers and decreased phosphorylation of glycogen synthase kinase 3β (GSK3β) expressions to affect hepatic glycogen synthesis, thus convergently contributing to an antidiabetic effect and improving insulin resistance. The mechanism of the antihyperlipidemic activity of EPE involved a decrease in the hepatic phosphorylation of mammalian target of rapamycin complex C1 (mTORC1) and p70 S6 kinase 1 (S6K1) expressions to improve insulin resistance but also a reduction in hepatic sterol regulatory element binding protein (SREBP)-1c expressions, and suppression of ACC activity, thus resulting in the decreased fatty acid synthesis but elevated hepatic peroxisome proliferator-activated receptor (PPAR) α and SREBP-2 expressions, resulting in lowering TG and TC concentrations. Our results demonstrated that EPE improves insulin resistance and ameliorates hyperlipidemia in HFD mice.

Targeting RSK2 in Cancer Therapy: A Review of Natural Products.

P90 ribosomal S6 kinase 2 (RSK2) is an important member of the RSK family, functioning as a kinase enzyme that targets serine and threonine residues and contributes to regulating cell growth. RSK2 comprises two major functional domains: the N-terminal kinase domain (NTKD) and the C-terminal kinase domain (CTKD). RSK2 is situated at the lower end of the Mitogen-activated protein kinases (MAPK) signaling pathway and is phosphorylated by the direct regulation of Extracellular signal-regulating kinase (ERK). RSK2 has been found to play a pivotal role in regulating cell proliferation, apoptosis, metastasis, and invasion in various cancer cells, including breast cancer and melanoma. Consequently, RSK2 has emerged as a potential target for the development of anti-cancer drugs. Presently, several inhibitors are undergoing clinical trials, such as SL0101. Current inhibitors of RSK2 mainly bind to its NTK or CTK domains and inhibit their activity. Natural products serve as an important resource for drug development and screening and with the potential to identify RSK2 inhibitors. This article discusses how RSK2 influences tumor cell proliferation, prevents apoptosis, arrests the cell cycle process, and promotes cancer metastasis through its regulation of downstream pathways or interaction with other biological molecules. Additionally, the paper also covers recent research progress on RSK2 inhibitors and the mechanisms of action of natural RSK2 inhibitors on tumors. This review emphasizes the significance of RSK2 as a potential therapeutic target in cancer and offers a theoretical basis for the clinical application of RSK2 inhibitors.

Scorpiones, Scolopendra and Gekko Inhibit Lung Cancer Growth and Metastasis by Ameliorating Hypoxic Tumor Microenvironment via PI3K/AKT/mTOR/HIF-1α Signaling Pathway.

To investigate whether Buthus martensii karsch (Scorpiones), Scolopendra subspinipes mutilans L. Koch (Scolopendra) and Gekko gecko Linnaeus (Gekko) could ameliorate the hypoxic tumor microenvironment and inhibit lung cancer growth and metastasis by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor-1α (PI3K/AKT/mTOR/HIF-1α) signaling pathway. Male C57BL/6J mice were inoculated with luciferase labeled LL/2-luc-M38 cell suspension to develop lung cancer models, with rapamycin and cyclophosphamide as positive controls. Carboxy methyl cellulose solutions of Scorpiones, Scolopendra and Gekko were administered intragastrically as 0.33, 0.33, and 0.83 g/kg, respectively once daily for 21 days. Fluorescent expression were detected every 7 days after inoculation, and tumor growth curves were plotted. Immunohistochemistry was performed to determine CD31 and HIF-1α expressions in tumor tissue and microvessel density (MVD) was analyzed. Western blot was performed to detect the expression of PI3K/AKT/mTOR/HIF-1α signaling pathway-related proteins. Enzyme-linked immunosorbent assay was performed to detect serum basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in mice. Scorpiones, Scolopendra and Gekko prolonged the survival time and inhibited lung cancer metastasis and expression of HIF-1α (all P<0.01). Moreover, Scorpiones, Scolopendra and Gekko inhibited the phosphorylation of AKT and ribosomal protein S6 kinase (p70S6K) (P<0.05 or P<0.01). In addition, they also decreased the expression of CD31, MVD, bFGF, TGF-β1 and VEGF compared with the model group (P<0.05 or P<0.01). Scorpiones, Scolopendra and Gekko all showed beneficial effects on lung cancer by ameliorating the hypoxic tumor microenvironment via PI3K/AKT/mTOR/HIF-1α signaling pathway.