S6 Kinase Research Articles

Abstract 5963: VGLL1-derived peptides demonstrated anticancer effect by inhibiting VGLL1-TEAD4 interaction

Abstract YAP/TAZ, cofactors of TEA domain-containing transcription factor TEADs (TEAD1-4), play a key role in the HIPPO pathway, which is involved in various function including embryonic development, organ size, cell proliferation, tissue regeneration, and the development and progression of several cancers. YAP/TAZ is therefore considered as master regulators of various cancers. However, we are interested in VGLL1, a cofactor of TEAD4, because it is mainly involved in the proliferation and metastasis of various cancers, including gastric, breast, pancreatic, and ovarian cancers. In gastric cancer, VGLL1 expression is regulated by the PI3K/AKT/β-catenin pathway. VGLL1 is then phosphorylated at S84 by TGF-β/ribosomal S6 kinase 2 (RSK2) and interacts with TEAD4 to express target genes such as MMP9, cyclin D1, IGFBP3, IGFBP5 and ITGαV. which are involved in cancer malignancy. We designed S84-containing VGLL1-derived peptides (VGLL1S84 peptides, SCVP), which consist of 7 to 15 amino acids linked to CPP, a cell-penetration peptide. We evaluated SCVP to inhibit the VGLL1-TEAD4 interaction and their therapeutic potential as anticancer agents. SCVP suppressed the growth of gastric and breast cancer cells expressing high levels of VGLL1, EGFR, and/or HER2. In the RNA-seq analysis of NUGC3 to elucidate the molecular mechanisms of SCVP induced cell death, SCVP-treated cells showed gene expression changes in metabolic pathway, RAS signaling, PI3K-AKT signaling, Wnt signaling, TNF signaling, and mTOR signaling. However, RNA-seq analysis of breast cancer cells treated with SCVP showed changes in the expression of genes involved in cell cycle, DNA replication, DNA repair, and apoptosis. Then, we performed CRISPR/Cas9 screening using SCVP and sgRNA library to explore companion biomarkers and genetic interactors of VGLL1 for targeted cancer therapy. As a result, we identified several candidate genes, including ZNF292 and PLK1, as synthetic lethal genes of VGLL1. As expected, HCC70 cells with ZNF292 mutation showed increased sensitivity to SCVP. We also found that SCVP in combination and the PLK inhibitor volasertib resulted in a synergistic effect on the growth inhibition of breast cancer cells. In conclusion, we demonstrated VGLL1 as a novel target for anticancer drug development, and elucidated the molecular mechanism and therapeutic potential of the SCVP in gastric and breast cancers. Citation Format: Sangmi Ock, Changsub Lee, Wongi Jung, Ina Whang, Kyung-Soo Kim, Jongmin Han, Sujin Yim, Bo-Kyung Kim, Hong-Sub Lee, Kwan-Young Chang, Misun Won. VGLL1-derived peptides demonstrated anticancer effect by inhibiting VGLL1-TEAD4 interaction [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 5963.

Modulation of mGlu5 reduces rewarding associative properties of nicotine via changes in mesolimbic plasticity: Relevance to comorbid cigarette smoking in psychosis

RationaleAntipsychotic medications that are used to treat psychosis are often limited in their efficacy by high rates of severe side effects. Treatment success in schizophrenia is further complicated by high rates of comorbid nicotine use. Dopamine D2 heteroreceptor complexes have recently emerged as targets for the development of more efficacious pharmaceutical treatments for schizophrenia. ObjectiveThe current study sought to explore the use of the positive allosteric modulator of the mGlu5 receptor 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) as a treatment to reduce symptoms related to psychosis and comorbid nicotine use. MethodsNeonatal treatment of animals with the dopamine D2-like receptor agonist quinpirole (NQ) from postnatal day (P)1–21 produces a lifelong increase in D2 receptor sensitivity, showing relevance to psychosis and comorbid tobacco use disorder. Following an 8-day conditioning paradigm, brain tissue in the mesolimbic pathway was analyzed for several plasticity markers, including brain derived neurotrophic factor (BDNF), phosphorylated p70 ribosomal S6 kinase (phospho-p70S6K), and cadherin-13 (Cdh13). ResultsPretreatment with CDPPB was effective to block enhanced nicotine conditioned place preference observed in NQ-treated animals. Pretreatment was additionally effective to block the nicotine-induced increase in BDNF and sex-dependent increases in cadherin-13 in the ventral tegmental area (VTA), as well as increased phospho-p70S6K in the nucleus accumbens (NAcc) shell found in NQ-treated animals. ConclusionIn conjunction with prior work, the current study suggests positive allosteric modulation of the mGlu5 receptor, an emerging target for schizophrenia therapeutics, may be effective for the treatment of comorbid nicotine abuse in psychosis.

Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression

BackgroundBecause obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (–)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PurposeThis study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. MethodsPreadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. ResultsHere, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. ConclusionsThese findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

Novel 4th-generation phytase improves broiler growth performance and reduces woody breast severity through modulation of muscle glucose uptake and metabolism.

The objective of the present study was to determine the effect of a novel (4th generation) phytase supplementation as well as its mode of action on growth, meat quality, and incidence of muscle myopathies. One-day old male broilers (n = 720) were weighed and randomly allocated to 30 floor pens (24 birds/pen) with 10 replicate pens per treatment. Three diets were fed from hatch to 56- days-old: a 3-phase corn-soy based diet as a positive control (PC); a negative control (NC) formulated to be isocaloric and isonitrogenous to the PC and with a reduction in Ca and available P, respectively; and the NC supplemented with 2,000 phytase units per kg of diet (NC + P). At the conclusion of the experiment, birds fed with NC + P diet were significantly heavier and had 2.1- and 4.2-points better feed conversion ratio (FCR) compared to birds offered NC and PC diets, respectively. Processing data showed that phytase supplementation increased live weight, hot carcass without giblets, wings, tender, and skin-on drum and thigh compared to both NC and PC diets. Macroscopic scoring showed that birds fed the NC + P diet had lower woody breast (WB) severity compared to those fed the PC and NC diets, however there was no effect on white striping (WS) incidence and meat quality parameters (pH, drip loss, meat color). To delineate its mode of action, iSTAT showed that blood glucose concentrations were significantly lower in birds fed NC + P diet compared to those offered PC and NC diets, suggesting a better glucose uptake. In support, molecular analyses demonstrated that the breast muscle expression (mRNA and protein) of glucose transporter 1 (GLUT1) and glucokinase (GK) was significantly upregulated in birds fed NC + P diet compared to those fed the NC and PC diets. The expression of mitochondrial ATP synthase F0 subunit 8 (MT-ATP8) was significantly upregulated in NC + P compared to other groups, indicating intracellular ATP abundance for anabolic pathways. This was confirmed by the reduced level of phosphorylated-AMP-activated protein kinase (AMPKα1/2) at Thr172 site, upregulation of glycogen synthase (GYS1) gene and activation of mechanistic target of rapamycin and ribosomal protein S6 kinase (mTOR-P70S6K) pathway. In conclusion, this is the first report showing that in-feed supplementation of the novel phytase improves growth performance and reduces WB severity in broilers potentially through enhancement of glucose uptake, glycolysis, and intracellular ATP production, which used for muscle glycogenesis and protein synthesis.

Computer-assisted discovery and evaluation of potential ribosomal protein S6 kinase beta 2 inhibitors

S6K2 is an important protein in mTOR signaling pathway and cancer. To identify potential S6K2 inhibitors for mTOR pathway treatment, a virtual screening of 1,575,957 active molecules was performed using PLANET, AutoDock GPU, and AutoDock Vina, with their classification abilities compared. The MM/PB(GB)SA method was used to identify four compounds with the strongest binding energies. These compounds were further investigated using molecular dynamics (MD) simulations to understand the properties of the S6K2/ligand complex. Due to a lack of available 3D structures of S6K2, OmegaFold served as a reliable 3D predictive model with higher evaluation scores in SAVES v6.0 than AlphaFold, AlphaFold2, and RoseTTAFold2. The 150 ns MD simulation revealed that the S6K2 structure in aqueous solvation experienced compression during conformational relaxation and encountered potential energy traps of about 19.6 kJ mol−1. The virtual screening results indicated that Lys75 and Lys99 in S6K2 are key binding sites in the binding cavity. Additionally, MD simulations revealed that the ligands remained attached to the activation cavity of S6K2. Among the compounds, compound 1 induced restrictive dissociation of S6K2 in the presence of a flexible region, compound 8 achieved strong stability through hydrogen bonding with Lys99, compound 9 caused S6K2 tightening, and the binding of compound 16 was heavily influenced by hydrophobic interactions. This study suggests that these four potential inhibitors with different mechanisms of action could provide potential therapeutic options.

Inhibition of S6K lowers age-related inflammation and increases lifespan through the endolysosomal system

Suppression of target of rapamycin complex 1 (TORC1) by rapamycin ameliorates aging in diverse species. S6 kinase (S6K) is an essential mediator, but the mechanisms involved are unclear. Here we show that activation of S6K specifically in Drosophila fat-body blocked extension of lifespan by rapamycin, induced accumulation of multilamellar lysosomes and blocked age-associated hyperactivation of the NF-κB-like immune deficiency (IMD) pathway, indicative of reduced inflammaging. Syntaxin 13 mediated the effects of TORC1–S6K signaling on lysosome morphology and inflammaging, suggesting they may be linked. Inflammaging depended on the IMD receptor regulatory isoform PGRP-LC, and repression of the IMD pathway from midlife extended lifespan. Age-related inflammaging was higher in females than in males and was not lowered in males by rapamycin treatment or lowered S6K. Rapamycin treatment also elevated Syntaxin 12/13 levels in mouse liver and prevented age-related increase in noncanonical NF-κB signaling, suggesting that the effect of TORC1 on inflammaging is conserved from flies to mammals.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

Effects of lactic acid bacteria on protein metabolism of Sunit lamb

To investigate the effect of adding lactic acid bacteria (LAB) to Sunit lambs’ diet on their protein metabolism, 18 3-month-old Sunit sheep were randomly divided into two groups (Group C (non-feeding of LAB) and Group L (feeding of LAB)) for a 90 d feeding trial. After the animals were slaughtered, we measured several key factors, including basal indices, amino acid composition, protein metabolism enzyme activity, and protein metabolism-related gene expression. There were nine replications of each group. The data show that LAB had a significant effect on the intramuscular fat (IMF) content (P<0.001). The composition of amino acids in the serum and mutton was improved by the addition of LAB. The addition of LAB led to a significant increase in the expression of genes related to AKT serine/threonine kinase 1 (Akt1), mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K1), and eukaryotic initiation factor 4E (eIF4E) protein synthesis (P<0.05), while the expression of all genes related to protein catabolism was reduced. Thus, dietary LAB can increase IMF content, improve amino acid composition, regulate protein metabolism in lambs, and provide a new way and theoretical basis for dietary management to improve meat quality in housed sheep.

Identification of RSK substrates using an analog-sensitive kinase approach

The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase (MAPK) pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest non-redundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells, and validated RanBP3, PDCD4, IRS2 and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissue-specific expression pattern.

Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway.

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G1 phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G1 phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G1 phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

Biochemical interaction of twenty steroid derivatives with ribosomal protein kinase 4 S6 (RSK-4) surface using a theoretical model

Several genetic expressions have been involved in the development of cancer such as the expression of a ribosomal kinase S6 P90 (RSK-4). It is important to mention that some compounds such as LJH685, 2073047-06-8, and SL0101 can act as RSK-4 inhibitors; however, its interaction with the surface of RSK-4 is very confusing. The aim of this research was to evaluate the interaction of twenty-nine steroid derivatives (1 to 29) with of RSK-4 surface using 6rv2 protein, LJH685, 2073047-06-8 and SL0101 as theoretical tools in the Dockingserver program. The results showed differences in the aminoacid residues involved in the interaction of steroid derivatives with 6rv2 protein surface compared with LJH685, 2073047-06-8 and SL0101. Besides, the inhibition constant for steroid derivatives 1, 12, 14, 19 and 22 was lower compared to 2073047-06-8 drug. In conclusion, the steroid derivatives 1, 12, 14, 19 and 22 could be a good alternative as RSK-4 inhibitors to decrease cancer cells growth.

RSK/GSK3-mediated phosphorylation of FilGAP regulates chemotactic cancer invasion.

Cell migration plays a crucial role in various biological processes, such as gastrulation, immune response, and cancer metastasis. In response to chemoattractant-like growth factors, cells form protrusions and migrate toward the source of the signal. Rho family small GTPase Rac is a key regulator of cell migration by stimulating actin polymerization to generate lamellipodia, flat membrane protrusions at the leading edge of migrating cells. FilGAP (ARHGAP24), a Rac-specific GTPase-activating protein (GAP), suppresses lamellipodia formation, and controls tumor cell migration. In this study, we found that FilGAP is phosphorylated downstream of epidermal growth factor (EGF) signaling. Upon EGF stimulation, FilGAP is phosphorylated at Ser625 by p90 ribosomal S6 kinase (RSK) and then at Ser621 by glycogen synthase kinase 3 (GSK3). Phosphorylation of FilGAP induces its dissociation from actin filaments. We identified a novel actin-localization domain of FilGAP that is essential for stabilizing cell adhesion. Additionally, we found that phosphorylation of FilGAP inhibits its lamellipodia suppression activity. Finally, we showed the expression of nonphosphorylatable FilGAP mutant, but not wild-type FilGAP, reduced cell migration speed and persistence toward the EGF gradient. Taken together, our results suggest that phosphorylation of FilGAP downstream of EGF-signaling plays a critical role in regulating chemotactic tumor cell migration by controlling cell-matrix adhesion and protrusion formation.

Establishing a carcinoembryonic antigen-associated competitive endogenous RNA network and forecasting an important regulatory axis in colon adenocarcinoma patients.

Colorectal cancer is one of the top five malignant tumors in the world in terms of morbidity and mortality. Numerous long non-coding RNAs (lncRNAs) are specifically expressed in tumours and can affect various types of human cancer by participating in competitive endogenous RNA (ceRNA) regulatory networks. However, the specific mechanisms and complex networks of ceRNA regulatory patterns in colon adenocarcinoma (COAD) remain unclear. Using The Cancer Genome Atlas (TCGA) database, we identified the differentially expressed lncRNA, microRNA (miRNA), and messenger RNA (mRNA) between colon cancer and normal tissues, as well as between groups with high and low CEACAM5 expression. Then, we constructed CEACAM5-related ceRNA networks, established the key lncRNA-miRNA-mRNA regulatory axis, and explored the biological mechanisms of this axis and its clinical significance in colon cancer from multiomic aspects. We constructed a ceRNA network of 18 lncRNAs, 177 miRNAs, and 25 mRNAs associated with CEACAM5 and finally established the key LCMT1-AS2/miR-454-3p/ribosomal protein S6 kinase A5 (RPS6KA5) axis associated with overall survival. Subsequent investigations have indicated that this regulatory axis could potentially participate in the progression of COAD and exert influence on the therapeutic outcomes of chemotherapy and immunotherapy. It may be involved in the PI3K-Akt signaling pathway and may modify the tumor immune microenvironment and influence the course of COAD. Additionally, it may be related to ferroptosis, N6-methyladenosine (m6A) methylation, and tumor stemness and interfere with the sensitivity of tumor cells to 5-fluorouracil and immunotherapy. The LCMT1-AS2/RPS6KA5 axis may be instrumental in tumor progression, potentially acting as a prognostic biomarker and therapeutic target.

Effect of Erchen Decoction on liver mitochondrial function by inhibiting mTORC1/SREBP1/CAV1 pathway in mice with high-fat diet

This study aims to investigate the effect of Erchen Decoction(ECD) on liver mitochondrial function in mice with a high-fat diet and its possible mechanism. A total of sixty C57BL/6J mice were randomly divided into a normal group, high-fat group, ECD group, mTORC1 activator(MHY) group, ECD+MHY group, and polyene phosphatidyl choline(PPC) group, with 10 rats in each group. The normal group was given a normal diet, and the other groups were fed a high-fat diet for 20 weeks. At the 17th week, the ECD group and ECD+MHY group were given ECD(8.7 g·kg~(-1)) daily, and the PPC group was given PPC(0.18 g·kg~(-1)) daily, while the remaining groups were given normal saline(0.01 mL·g~(-1)) daily for four weeks. In the 19th week, the MHY group and ECD+MHY group were injected intraperitoneally with MHY(5 mg·kg~(-1)) every other day for two weeks. During the experiment, the general conditions of the mice were observed. The contents of triglyceride(TG) and total cholesterol(TC) in serum were measured. Morphological changes in liver tissue were examined through HE and oil red O staining. The content of adenosine triphosphate(ATP) was determined using chemiluminescence, and mitochondrial membrane potential was assessed using a fluorescence probe(JC-1). Western blot was performed to detect the expression of rapamycin target protein complex 1(mTOR1), ribosomal protein S6 kinase B1(S6K), sterol regulatory element binding protein 1(SREBP1), and caveolin 1(CAV1). RESULTS:: revealed that compared with the normal group, the mice in the high-fat group exhibited significant increases in body weight and abdominal circumference(P&lt;0.01). Additionally, there were significant increases in TG and TC levels(P&lt;0.01). HE and oil red O staining showed that the boundaries of hepatic lobules were unclear; hepatocytes were enlarged, round, and irregularly arranged, with obvious lipid droplet deposition and inflammatory cell infiltration. The liver ATP content and mitochondrial membrane potential decreased significantly(P&lt;0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 increased significantly(P&lt;0.01), while the expression of CAV1 decreased significantly(P&lt;0.01). Compared with the high-fat group, the body weight and TG content of mice in the ECD group and PPC group decreased significantly(P&lt;0.05). Improvements were observed in hepatocyte morphology, lipid deposition, and inflammatory cell infiltration. Furthermore, there were significant increases in ATP content and mitochondrial membrane potential(P&lt;0.05 or P&lt;0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly in the ECD group(P&lt;0.01), while CAV1 expression increased significantly(P&lt;0.01). However, the indices mentioned above did not show improvement in the MHY group. When the ECD+MHY group was compared with the MHY group, there were significant reductions in body weight and TG contents(P&lt;0.05). The morphological changes of hepatocytes, lipid deposition, and inflammatory cell infiltration were recovered. Moreover, there were significant increases in liver ATP content and mitochondrial membrane potential(P&lt;0.05 or P&lt;0.05). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly(P&lt;0.01), while CAV1 expression increased significantly(P&lt;0.01). In conclusion, ECD can improve mitochondrial function by regulating the mTORC1/SREBP1/CAV1 pathway. This mechanism may be involved in the resolution of phlegm syndrome and the regulation of lipid metabolism.

Enhanced Antipediatric Sarcoma Effect of Everolimus with Secukinumab by Targeting IL17A.

In this study, we explored the therapeutic potential of everolimus, an mTOR inhibitor, in a patient-derived xenograft (PDX) of rhabdomyosarcoma, the most prevalent malignant pediatric sarcoma. In addition, rhabdoid tumor cell line A-204 and Ewings sarcoma cell line A-673 were cultured to assess the in vitro effect of everolimus. Furthermore, the cell-derived xenograft (CDX) of A-673 was established and treated with everolimus in vivo. IHC and Western blotting were performed to detect the expressions of pertinent proteins. Results showed that everolimus intervention had limited inhibitory effect on PDX tumor growth compared with cyclophosphamide. Nevertheless, everolimus treatment significantly influenced the phosphorylation levels of S6 kinase beta 1 (S6K1) and eIF4E-binding protein 1 (p-4E-BP1), resulting in the inhibition of angiogenesis in vitro and in vivo. Interestingly, everolimus led to an upregulation in the level of IL17A in sarcoma cells. Notably, when secukinumab, a mAb of IL17A, was combined with everolimus, it synergistically enhanced the inhibitory effect of everolimus on sarcoma cell proliferation in vitro and on the growth of PDX or CDX xenograft tumors in vivo. Importantly, this combination therapy did not affect the mTOR signaling. These results indicate that everolimus exerts an antipediatric sarcoma effect by inhibiting mTOR signal. However, everolimus induces sarcoma cells to produce IL17A, which promotes tumor cell survival and counteracts its antipediatric sarcoma effect. The combination of secukinumab effectively eliminates the effects of IL17A, thereby improving the therapeutic efficacy of everolimus in the context of pediatric sarcomas.

Ribosomal protein S6 kinase 2 (RPS6KB2) is a potential immunotherapeutic target for cancer that upregulates proinflammatory cytokines.

Cancer is still a leading cause of mortality. Over the years, cancer therapy has undergone significant advances driven by advancements in science and technology. A promising area of drug discovery in this field involves the development of therapeutic targets for cancer treatment. The urgent need to identify new pharmacological targets arises from the impact of tumor resistance on the effectiveness of current medications. Specifically, the RPS6KB2 gene on chromosome 11 has been implicated in cell cycle regulation and exhibits higher expression levels in tumor tissue. Given this association, there is a potential for this gene to serve as a target for cancer treatment. We conducted an analysis using the GTEx, TCGA, and CCLE databases to explore the relationship between RPS6KB2 and immune infiltration, the tumor microenvironment (TME), microsatellite instability (MSI), and more. Cell proliferation was assessed using EDU detection, while cell invasion and migration were evaluated via wound healing and Transwell assays. Additionally, western blot analysis was employed to measure expression of Bax, Bcl-2, MMP2, MMP9, PCNA, and proinflammatory factors. Through data analysis and molecular biology methods, our study carefully examined the potential role of RPS6KB2 in cancer therapy. The data revealed that RPS6KB2 is aberrantly expressed in most cancers and is associated with poor prognosis. Further analysis indicated its involvement in cancer cell apoptosis and migration, as well as its role in cancer immune processes. We validated the significance of RPS6KB2 in hepatocellular carcinoma (HCC), highlighting its capacity to upregulate proinflammatory cytokines. Our research indicates that RPS6KB2 is a prognostic biomarker associated with immune infiltration in cancer that can affect antitumor immunity by increasing secretion of proinflammatory factors, providing a potential drug target for cancer treatment.

Ilex paraguariensis A.St.-Hil. improves lipid metabolism in high-fat diet-fed obese rats and suppresses intracellular lipid accumulation in 3T3-L1 adipocytes via the AMPK-dependent and insulin signaling pathways.

Obesity is closely associated with several chronic diseases, and adipose tissue plays a major role in modulating energy metabolism. This study aimed to determine whether Mate, derived from I. paraguariensis A.St.-Hil., ameliorates lipid metabolism in 3T3-L1 adipocytes and high-fat diet (HFD)-fed obese Sprague-Dawley (SD) rats. 3T3-L1 adipocytes were cultured for 7 days, following which intracellular lipid accumulation and expression levels of lipid metabolism-related factors were examined. Dorsomorphin was used to investigate the potential pathways involved, particularly the adenosine monophosphate-activated protein kinase (AMPK)- dependent pathway. Mate was administered to rat HFD-fed obese SD models for 8 consecutive weeks. The expression of lipid metabolism-related factors in the organs and tissues collected from dissected SD rats was evaluated. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes, increased the protein and gene expression levels of AMPK, hormone sensitive lipase (HSL), calmodulin kinase kinase (CaMKK), liver kinase B1 (LKB1), protein kinase A (PKA), CCAAT/enhancer binding protein β (C/EBPβ), insulin receptor b (IRβ), and insulin receptor substrate 1 (IRS1) (Tyr465), and decreased those of sterol regulatory element binding protein 1C (Srebp1c), fatty acid synthase (FAS), peroxisome-activated receptor γ (PPARγ), and IRS1 (Ser1101). Furthermore, an AMPK inhibitor abolished the effects exerted by Mate on intracellular lipid accumulation and HSL and FAS expression levels. Mate treatment suppressed body weight gain and improved serum cholesterol levels in HFD-fed obese SD rats. Treatment with Mate increased the protein and gene expression levels of AMPK, PKA, Erk1/Erk2 (p44/p42), and uncoupling protein 1 and reduced those of mammalian target of rapamycin, S6 kinase, Srebp1c, ap2, FAS, Il6, Adiponectin, Leptin, and Fabp4 in rat HFD-fed obese SD models. Mate suppressed intracellular lipid accumulation in 3T3-L1 adipocytes and improved lipid metabolism in the epididymal adipose tissue of HFD-fed obese SD rats via the activation of AMPK-dependent and insulin signaling pathways.

Abstract B078: A complex of NF1 and SPRED2 non-canonically behave as tumor promoters in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with poor prognosis due to late detection and lack of effective therapeutic options. &amp;gt;90% of PDAC tumors are driven by activating mutations in the KRAS oncogene, making it an appealing subject for further investigation into understanding PDAC biology and offering a potential genetic vulnerability to target in patients. Our laboratory has previously reported a mutant-KRAS-proximal complex at the plasma membrane that consists of tumor-suppressor Neurofibromatosis type 1 (NF1), Sprouty Related EVH1 Domain Containing 2 (SPRED2), and Ribosomal S6 Kinase 1 (RSK1). This complex mechanistically suppresses WT KRAS signaling via RSK1, a suppression which is then relieved upon mutant KRAS inhibition, ultimately promoting adaptive resistance for PDAC cells under these conditions. We find that both knockout of either NF1 or SPRED2 in PDAC organoids induces a substantial growth defect in vivo. Our preliminary data suggests that NF1 and SPRED2 support KRAS activity in mouse pancreatic cancer in vivo. Moreover, both SPRED2 and NF1 KO show decreased proliferation, invasion, and attachment on collagen family members in vitro. Given NF1's well-established role as a tumor-suppressor, our observations of decreased tumorigenic properties upon NF1 deletion are unexpected. We hypothesize that NF1 and SPRED2 promote tumorigenesis via recruitment of RSK1 to the plasma membrane, inducing the phosphorylation of putative substrates to stimulate a pro-migratory cellular behavior. Our work suggests the existence of a unique RSK1 program localized at the plasma membrane by mutant KRAS, providing a new facet of oncogenesis to investigate for fundamental understanding and potential therapeutic opportunities. Citation Format: Sun Kim, Hsiu-Chi Ting, Astrid Deschenes, Jonathan Kastan, Youngkyu Park, David Tuveson. A complex of NF1 and SPRED2 non-canonically behave as tumor promoters in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B078.

In-silico investigation of RPS6KB1 associated cancer inhibitor: a drug repurposing study

The ribosomal protein S6 kinase beta-1 (RPS6KB1), also known as p70S6 kinase, plays a crucial role in various disease-related conditions such as diabetes, obesity, and cancer. Its activity is regulated by phosphorylation events, including phosphorylation of Threonine 389 in the hydrophobic motif by the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of Threonine 229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1). However, other phenomena connected to RPS6KB1 remain unknown. In this study, we employed virtual screening and molecular docking techniques on the molecules in the ZINC library to identify potential inhibitors. Molecular dynamics (MD) simulations and MMGBSA calculations were carried out on promising compounds to determine their binding affinity and stability. We also evaluated the drug-likeness properties of the selected compounds. A comparative study between the native RPS6KB1 structure, co-crystal ligands, and the shortlisted molecules from the ZINC dataset was carried out. The identified molecules possess significant potential for future in vitro and in vivo studies, paving the way for developing effective cancer treatments. Communicated by Ramaswamy H. Sarma

Lactate-induced autophagy activation: unraveling the therapeutic impact of high-intensity interval training on insulin resistance in type 2 diabetic rats

Impaired autophagy is a hallmark of diabetes. The current study proposed to investigate if high intensity interval training (HIIT) induced lactate accumulation could stimulate autophagy in type 2 diabetic male rats. 28 male Wistar rats were randomly assigned into four groups: Healthy Control (CO), Diabetes Control (T2D), Exercise (EX), and Diabetes + Exercise (T2D + EX). Diabetes was induced by feeding high-fat diet and administrating single dose of streptozotocin (35 mg/kg). After becoming diabetic, the animals in the exercise groups (EX and T2D + EX) performed an eight-week HIIT (4–10 interval, 80–100% Vmax, 5 days per week). Serum levels of lactate, glucose and insulin as well as the levels of lactate, pyruvate, lactate transporter monocarboxylate transporter 1 (MCT1), phosphorylated mitogen-activated protein kinases (p-MAP 1 and 2), phosphorylated extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1 and 2), mammalian target of rapamycin (p-mTOR), ribosomal protein S6 kinase beta-1 (p-70S6k), p90 ribosomal S6 kinases (p-90RSK), autophagy related 7 (ATG7), Beclin-1, microtubule-associated protein 1A/1B, and 2A/2B -light chain 3 levels (LC3-I), (LC3- II), (LC3I/LC3II) in soleus muscle were measured. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and serum glucose was lower in T2D + EX compared to T2D group (P < 0.0001). While serum and soleus muscle levels of lactate was not different between T2D and T2D + Ex, the levels of Pyruvate (P < 0.01), MCT1, p-ERK1/2, p-mTOR, p70S6k, P-90RSK, ATG7, LC3-II, and LC3-II/LC3I ratios were higher in T2D + EX compared to T2D group (P < 0.0001). We concluded that eight weeks of high-intensity interval training could activated ERK/P90SRK while inhibiting mTOR/P70S6K signaling pathway in lactate dependent manner. It means increased autophagy which resulted in improve insulin resistance (IR) and reduce blood glucose.