P70 Ribosomal S6 Kinase Research Articles

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.

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.

Phytochemical composition, antioxidant activity, and cytotoxicity of the aqueous extracts of Dracaena arborea and Bridelia ferruginea: In vitro and in silico studies

Dracaena arborea (DA) and Bridelia ferruginea (BF) are medicinal plants used in the treatment of various ailments such as diabetes, infertility, and sexual dysfunctions. The aim of this work was to characterize these plants chemically and investigate their antioxidant and cytotoxicity in vitro and in silico. The cytotoxicity was determined in PC3, NIH3T3 and BT474 cells, and through hemolysis using erythrocytes. Furthermore, selected compounds from DA and BF were docked against some oxidative stress (Keap1: Kelch-like ECH-associated protein 1 and GST: glutathione S-transferase) and cytotoxicity (PARP10: poly(ADP-ribose) polymerase family member 10 and p90 RSK: p90 ribosomal S6 kinases) proteins to predict the ligand-protein interaction. We found that the total phenolic and flavonoid content in BF was significantly (p < 0.001) higher than that of DA. 16 and 32 phytocompounds were identified in DA and BF, respectively. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) free radical scavenging activity, and cupric and ferric reducing power of BF were significantly (p < 0.05–0.001) higher than those of DA. Moreover, the ability of BF to inhibit lipid peroxidation was higher than that of DA. DA dose-dependently decreased the viability of PC3, NIH3T3, and BT474 cells, while BF tended to feed the cells. Drug-Likeness and toxicity prediction of selected compounds found in DA and BF were within the acceptable standards, according to Lipinski's rule. The majority of compounds found in DA and BF exhibited a strong binding affinity with Keap1, GST, PARP10 and p90 RSK, which supports the in vitro results. For instance, Cis-cyclohexane-1,3-diamine from DA showed a binding affinity of -6.73 kcal/mol and interacted with Keap1 through five conventional hydrogen bonds, while Nrf2 (a Keap1 inhibitor) exhibited a binding affinity of -11.82 kcal/mol and interacted with Keap1 via two conventional hydrogen bonds. 1,3-propanediamine, n-methyl identified in BF exhibited a high stability with Keap1 (-5.95 kcal/mol) due to the presence of 6 conventional hydrogen bonds. Among the compounds identified in DA, the best stable ligand interacting with PARP10 was 2-deoxy-alpha-d-ribopyranose (-7.62 kcal/mol), as it was well-matched in the PARP10 pocket through five conventional hydrogen bonds. In parallel, among the compounds present in BF, 2-[(methylamino)methyl]cyclohexanol showed the best binding affinity with PARP10 (-9.80 kcal/mol), which was higher than that of veliparib (-8.26 kcal/mol), used as a reference inhibitor. In conclusion, BF has the possibility to be exploited in the prevention/treatment of oxidative stress-related diseases, while DA could be a potential anticancer agent. This could justify their ethnomedicinal uses.

Insulin restores retinal ganglion cell functional connectivity and promotes visual recovery in glaucoma.

Dendrite pathology and synaptic loss result in neural circuit dysfunction, a common feature of neurodegenerative diseases. There is a lack of strategies that target dendritic and synaptic regeneration to promote neurorecovery. We show that daily human recombinant insulin eye drops stimulate retinal ganglion cell (RGC) dendrite and synapse regeneration during ocular hypertension, a risk factor to develop glaucoma. We demonstrate that the ribosomal protein p70S6 kinase (S6K) is essential for insulin-dependent dendritic regrowth. Furthermore, S6K phosphorylation of the stress-activated protein kinase-interacting protein 1 (SIN1), a link between the mammalian target of rapamycin complexes 1 and 2 (mTORC1/2), is required for insulin-induced dendritic regeneration. Using two-photon microscopy live retinal imaging, we show that insulin rescues single-RGC light-evoked calcium (Ca2+) dynamics. We further demonstrate that insulin enhances neuronal survival and retina-brain connectivity leading to improved optomotor reflex-elicited behaviors. Our data support that insulin is a compelling pro-regenerative strategy with potential clinical implications for the treatment and management of glaucoma.

Impaired Mitochondrial Energy Metabolism Regulated by p70S6K: A Putative Pathological Feature in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease. Mitochondrial energy metabolism and p70 ribosomal protein S6 kinase (p70S6K) play significant roles in AD pathology. However, the potential relationship between them is unclear. In this study, bioinformatics methods were initially applied to analyze the transcriptomic data in the CA1 and the primary visual cortex of patients with AD and Aβ42-treated SH-SY5Y cells. By applying secreted Aβ42 and p70S6K gene silencing in cells, we explored disorders in mitochondrial function and the regulatory roles of p70S6K by flow cytometry, laser scanning confocal microscopy, high-performance liquid chromatography, Western blotting, and quantitative reverse transcription PCR. The study reveals that impaired mitochondrial energy metabolism is a potential pathological feature of AD and that p70S6K gene silencing reversed most of the changes induced by Aβ42, such as the activities of the electron transport chain complexes I and III, as well as ATP synthase, ATP production, generation of reactive oxygen species, mitochondrial membrane potential, and phosphorylation of AMPK, PINK1, and Parkin, all of which are required for mitochondria to function properly in the cell.

2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis

Sirtuin 3 (SIRT3) is well known as a conserved nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase located in the mitochondria that may regulate oxidative stress, catabolism and ATP production. Accumulating evidence has recently revealed that SIRT3 plays its critical roles in cardiac fibrosis, myocardial fibrosis and even heart failure (HF), through its deacetylation modifications. Accordingly, discovery of SIRT3 activators and elucidating their underlying mechanisms of HF should be urgently needed. Herein, we identified a new small-molecule activator of SIRT3 (named 2-APQC) by the structure-based drug designing strategy. 2-APQC was shown to alleviate isoproterenol (ISO)-induced cardiac hypertrophy and myocardial fibrosis in vitro and in vivo rat models. Importantly, in SIRT3 knockout mice, 2-APQC could not relieve HF, suggesting that 2-APQC is dependent on SIRT3 for its protective role. Mechanically, 2-APQC was found to inhibit the mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K), c-jun N-terminal kinase (JNK) and transforming growth factor-β (TGF-β)/ small mother against decapentaplegic 3 (Smad3) pathways to improve ISO-induced cardiac hypertrophy and myocardial fibrosis. Based upon RNA-seq analyses, we demonstrated that SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) axis was closely assoiated with HF. By activating PYCR1, 2-APQC was shown to enhance mitochondrial proline metabolism, inhibited reactive oxygen species (ROS)-p38 mitogen activated protein kinase (p38MAPK) pathway and thereby protecting against ISO-induced mitochondrialoxidative damage. Moreover, activation of SIRT3 by 2-APQC could facilitate AMP-activated protein kinase (AMPK)-Parkin axis to inhibit ISO-induced necrosis. Together, our results demonstrate that 2-APQC is a targeted SIRT3 activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis, which may provide a new clue on exploiting a promising drug candidate for the future HF therapeutics.

Abstract PO3-29-04: Patient selection for high RSK2 expression is key for achieving improved PFS in metastatic breast cancer in the PMD-026 Phase 1/1b study

Abstract Background: PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK and PDK-1 pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Both pathways are implicated in HR+ and triple negative breast cancer (TNBC). RSK2 can modulate the growth of breast cancer (BC) by promoting cell cycle progression through G2/M, specifically through pYB-1S102 which nucleates spindle pole formation in M-phase of cancer cells. This mechanism of action (MOA) for PMD-026 can be monitored by measuring the loss of pYB-1S102 and pCDK2Y15, a marker for G2/M arrest. Therefore, PMD-026 presents a novel mechanism to inhibit resistance at G1/S which is common for CDK4/6 inhibitors as well as ADCs such as those that contain Topoisomerase 1 conjugates. Here in, we show for the first time that PMD-026 inhibits P-YB-1S102 and pCDK2y15 in a dose-dependent manner in TNBC cells and address whether this could be translated as a pharmacodynamic marker of PMD-026 activity in patients. To guide patient selection, we are co-developing a unique and simple IHC based companion diagnostic (CDx) with Roche based on the principle that activated RSK2 translocates from the cytoplasm into the nucleus. In mBC, RSK2 was found to be highly expressed in ~70% of patients using a staining algorithm. An H-score was developed and ultimately H-score &amp;gt;180 (75% nuclear staining with staining intensity of 2+ or 3+ by IHC) was used as the cut-off in our Phase 1/1b trial retrospective study. This IHC assay will be used to select patients in the upcoming Phase II, as PFS in the PMD-026 Phase I/1b study showed remarkable differences when comparing patients with high and low RSK2. Results: In this Phase 1/1b study, PMD-026 was evaluated in an ethnically diverse population of women across all breast cancer subtypes. Patients had a median of 5 prior lines of therapy and PMD-026 achieved stable disease in 44% (11/25) of the subjects. Consistent with the MOA, PMD-026 inhibited pCDK2Y15 in PBMCs from patients at C1D15. Corresponding plasma concentrations were ~ 1µM, which correlates with the IC90 of PMD-026 in preclinical models. To guide patient selection in the future, the RSK2 IHC with an H:Score &amp;gt;180 was used to evaluate patient outcomes. Using this criterion, PFS was significantly longer in RSK2 high vs low patients (3.6 months vs 1.3 month, HR = 0.27, p = 0.031, N=20 patients representing de novo TNBC and HR+/CDK4/6 refractory subtypes). Upon further analysis, patients who had 5 lines of therapy or less benefited the most. PFS in these less heavily pretreated RSK2 high patients was 4.8 months vs 1.3 months for RSK2 low patients (HR = 0.07, p = 0.001 N=14 representing the same subtypes above). It was clear that the time on study was longer for patients with high RSK2 vs low, and this was specifically noted in de novo TNBC and HR+ patients that previously failed CDK4/6 inhibitors as well as endocrine therapies. A key observation in this study was that the PFS benefit was based on the RSK2 levels and not on any pharmacokinetic differences between patients, as the AUC (p=0.53), Cmax (p=0.73) and plasma levels (p=0.76) did not differ between RSK2 high and RSK2 low groups. These data strongly support the activity of this first in class RSK inhibitor, PMD-026, in RSK2 high breast cancers. Our integrated IHC based CDx strategy plans to prospectively enroll RSK2 high patients for our Phase 2 trials will be described. Conclusions/Translational Significance: CDx guided breast cancer therapeutics are often lacking for the treatment of TNBC, as well as HR+ breast cancers that have failed CDK4/6 inhibitors. The ability to identify patients who could potentially benefit most from RSK-targeted agents such as PMD-026 is an important step forward in delivering practice changing therapies for patients. Citation Format: Sandra Dunn, Judy Wang, Hyo Han, Robert Wesolowski, Amita Patnaik, Shakeela Bahadur, Nicolaos Palaskas, My-my Huynh, Aarthi Jayanthan, Gerrit Los, Andrew Dorr. Patient selection for high RSK2 expression is key for achieving improved PFS in metastatic breast cancer in the PMD-026 Phase 1/1b study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-29-04.

Anabolic Signaling After Withdrawl of AMPK-Activating Drugs in C2C12 Cells

Adenosine monophosphate-activated protein kinase (AMPK) is a master metabolic regulator that is activated under low-energy conditions, which generally promotes catabolic processes, including an increase of ATP synthesis and inhibiting anabolic processes that consume ATP. Indeed, acute AMPK activation potently inhibits anabolic pathways, including Akt/mTORC1 signaling. However, recent research reported that withdrawal of the AMPK-activating drug AICAR after a short treatment period results in enhanced anabolic signaling through Akt/mTORC1. This work aims to extend those findings using a more specific AMPK activator since AICAR is well-known to have numerous AMPK-independent effects. To do so, we treated differentiated C2C12 cells with either 2 mM AICAR, 5uM MK8722 (a pan-AMPK activator), or vehicle control (DMSO) for 60 minutes. After 60 minutes of incubation with the drugs, the treatment media was removed, the cells were rinsed and lysed immediately or incubated with media without drug treatment for 30, 60, 120, or 180 minutes, and then lysed. Phosphorylation of ACC (as an indicator of AMPK activity) and anabolic markers Akt, p70 ribosomal protein S6 kinase (S6k), and eIF4E-binding protein (4EBP1) were measured by western blotting. ACC phosphorylation (reflective of AMPK activity) remained elevated through 180 minutes after drug withdrawal for both AICAR and MK8722. Both AKT and S6k phosphorylation remained suppressed for at least 60 minutes after withdrawal of both AICAR and MK8722 and were not elevated at any time point up to 3 hours after withdrawal of the drugs. In contrast to previously reported data, we conclude that anabolic signaling remains inhibited in skeletal muscle myotubes for up to 3 hours after 1 hour of pharmacological activation of AMPK. This work was funded by a BYU Gerontology Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of mTOR and HIF-1α in LMP2A-mediated increase in ATP production

Abstract Numerous studies indicate that latency proteins from Epstein-Barr virus contribute not only to the development, but also the survival of B cell lymphomas. Our laboratory has demonstrated that LMP2A promotes B cell lymphoma survival by inducing the activation of STAT3. More recently, pathways that link STAT3 activation with increased cellular metabolism suggested that LMP2A may increase B cell lymphoma ATP production via the mTOR-dependent activation of STAT3. Using LMP2A-negative and -positive B cell lymphoma cell lines, our data suggest that LMP2A increases ATP production in an mTOR-dependent pathway. However, the addition of the STAT3 inhibitor, STATTIC, did not impact the LMP2A-mediated increase in ATP production. Further investigation indicated that LMP2A-mediated activation of mTOR results in increases in HIF-1α protein, but not RNA, that are required for the enhancements in ATP production. Finally, our data demonstrate that LMP2A requires the activation of phosphorylated p70 ribosomal s6 kinase and 4E-BP1 to increase protein levels of HIF-1α that are required to increase ATP levels in LMP2A-expressing cell lines. Taken together, our data suggest that LMP2A gives lymphoma cells a competitive advantage to produce more ATP through increases in HIF-1α that are mTOR and p70s6K/4E-BP-1 dependent.

MiR-29 and MiR-140 regulate TRAIL-induced drug tolerance in lung cancer

ABSTRACT Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has chemotherapeutic potential as a regulator of an extrinsic apoptotic ligand, but its effect as a drug is limited by innate and acquired resistance. Recent findings suggest that an intermediate drug tolerance could mediate acquired resistance, which has made the main obstacle for limited utility of TRAIL as an anti-cancer therapeutics. We propose miRNA-dependent epigenetic modification drives the drug tolerant state in TRAIL-induced drug tolerant (TDT). Transcriptomic analysis revealed miR-29 target gene activation in TDT cells, showing oncogenic signature in lung cancer. Also, the restored TRAIL-sensitivity was associated with miR-29ac and 140-5p expressions, which is known as tumor suppressor by suppressing oncogenic protein RSK2 (p90 ribosomal S6 kinase), further confirmed in patient samples. Moreover, we extended this finding into 119 lung cancer cell lines from public data set, suggesting a significant correlation between TRAIL-sensitivity and RSK2 mRNA expression. Finally, we found that increased RSK2 mRNA is responsible for NF-κB activation, which we previously showed as a key determinant in both innate and acquired TRAIL-resistance. Our findings support further investigation of miR-29ac and -140-5p inhibition to maintain TRAIL-sensitivity and improve the durability of response to TRAIL in lung cancer.

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4’s transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

The involvement of Akt, mTOR, and S6K in the in vivo effect of IGF-1 on the regulation of rat cardiac Na+/K+-ATPase.

We previously demonstrated that insulin-like growth factor-1 (IGF-1) regulates sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) in vascular smooth muscle cells (VSMC) via phosphatidylinositol-3 kinase (PI3K). Taking into account that others' work show that IGF-1 activates the PI3K/protein kinase B (Akt) signaling pathway in many different cells, we here further questioned if the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein p70 S6 kinase (S6K) pathway stimulates Na+/K+-ATPase, an essential protein for maintaining normal heart function. There were 14 adult male Wistar rats, half of whom received bolus injections of IGF-1 (50μg/kg) for 24h. We evaluated cardiac Na+/K+-ATPase expression, activity, and serum IGF-1 levels. Additionally, we examined the phosphorylated forms of the following proteins: insulin receptor substrate (IRS), phosphoinositide-dependent kinase-1 (PDK-1), Akt, mTOR, S6K, and α subunit of Na+/K+-ATPase. Additionally, the mRNA expression of the Na+/K+-ATPase α1 subunit was evaluated. Treatment with IGF-1 increases levels of serum IGF-1 and stimulates Na+/K+-ATPase activity, phosphorylation of α subunit of Na+/K+-ATPase on Ser23, and protein expression of α2 subunit. Furthermore, IGF-1 treatment increased phosphorylation of IRS-1 on Tyr1222, Akt on Ser473, PDK-1 on Ser241, mTOR on Ser2481 and Ser2448, and S6K on Thr421/Ser424. The concentration of IGF-1 in serum positively correlates with Na+/K+-ATPase activity and the phosphorylated form of mTOR (Ser2448), while Na+/K+-ATPase activity positively correlates with the phosphorylated form of IRS-1 (Tyr1222) and mTOR (Ser2448). These results indicate that the Akt/mTOR/S6K signalling pathway may be involved in the IGF-1 regulating cardiac Na+/K+-ATPase expression and activity.

The Therapeutic Efficacy and Mechanism of Action of Gnetin C, a Natural Compound from the Melinjo Plant, in a Preclinical Mouse Model of Advanced Prostate Cancer.

The metastasis-associated protein 1/protein kinase B (MTA1/AKT) signaling pathway has been shown to cooperate in promoting prostate tumor growth. Targeted interception strategies by plant-based polyphenols, specifically stilbenes, have shown great promise against MTA1-mediated prostate cancer progression. In this study, we employed a prostate-specific transgenic mouse model with MTA1 overexpression on the background of phosphatase and tensin homolog (Pten) null (R26MTA1; Ptenf/f) and PC3M prostate cancer cells which recapitulate altered molecular pathways in advanced prostate cancer. Mechanistically, the MTA1 knockdown or pharmacological inhibition of MTA1 by gnetin C (dimer resveratrol) in cultured PC3M cells resulted in the marked inactivation of mammalian target of rapamycin (mTOR) signaling. In vivo, mice tolerated a daily intraperitoneal treatment of gnetin C (7 mg/kg bw) for 12 weeks without any sign of toxicity. Treatment with gnetin C markedly reduced cell proliferation and angiogenesis and promoted apoptosis in mice with advanced prostate cancer. Further, in addition to decreasing MTA1 levels in prostate epithelial cells, gnetin C significantly reduced mTOR signaling activity in prostate tissues, including the activity of mTOR-target proteins: p70 ribosomal protein S6 kinase (S6K) and eukaryotic translational initiation factor 4E (elF4E)-binding protein 1 (4EBP1). Collectively, these findings established gnetin C as a new natural compound with anticancer properties against MTA1/AKT/mTOR-activated prostate cancer, with potential as monotherapy and as a possible adjunct to clinically approved mTOR pathway inhibitors in the future.

Empagliflozin mitigates cardiac hypertrophy through cardiac RSK/NHE-1 inhibition

BackgroundSGLT2i reduce cardiac hypertrophy, but underlying mechanisms remain unknown. Here we explore a role for serine/threonine kinases (STK) and sodium hydrogen exchanger 1(NHE1) activities in SGLT2i effects on cardiac hypertrophy. MethodsIsolated hearts from db/db mice were perfused with 1 µM EMPA, and STK phosphorylation sites were examined using unbiased multiplex analysis to detect the most affected STKs by EMPA. Subsequently, hypertrophy was induced in H9c2 cells with 50 µM phenylephrine (PE), and the role of the most affected STK (p90 ribosomal S6 kinase (RSK)) and NHE1 activity in hypertrophy and the protection by EMPA was evaluated. ResultsIn db/db mice hearts, EMPA most markedly reduced STK phosphorylation sites regulated by RSKL1, a member of the RSK family, and by Aurora A and B kinases. GO and KEGG analysis suggested that EMPA inhibits hypertrophy, cell cycle, cell senescence and FOXO pathways, illustrating inhibition of growth pathways. EMPA prevented PE-induced hypertrophy as evaluated by BNP and cell surface area in H9c2 cells. EMPA blocked PE-induced activation of NHE1. The specific NHE1 inhibitor Cariporide also prevented PE-induced hypertrophy without added effect of EMPA. EMPA blocked PE-induced RSK phosphorylation. The RSK inhibitor BIX02565 also suppressed PE-induced hypertrophy without added effect of EMPA. Cariporide mimicked EMPA’s effects on PE-treated RSK phosphorylation. BIX02565 decreased PE-induced NHE1 activity, with no further decrease by EMPA. ConclusionsRSK inhibition by EMPA appears as a novel direct cardiac target of SGLT2i. Direct cardiac effects of EMPA exert their anti-hypertrophic effect through NHE-inhibition and subsequent RSK pathway inhibition.

Abstract 567: Fisetin sensitizes triple negative breast cancer cells to radiation

Abstract Triple negative breast cancer (TNBC) represents a highly aggressive subtype, constituting approximately 20% of all breast cancer cases. Y-box binding protein-1 (YB-1) is often overexpressed in various tumor types and has been implicated in conferring resistance to cell death induced by radiotherapy. Fisetin, a flavonoid compound with demonstrated anti-cancer properties, operates in part through inhibiting phosphorylation of YB-1 by p90 ribosomal S6 kinase (RSK). In this study, we investigated the potential synergistic effects of combining fisetin with radiotherapy in TNBC. The activation status of the RSK signaling pathway was assessed in both total cell lysate and subcellular fractions using Western blotting. A standard clonogenic assay was employed to evaluate post-irradiation cell survival. To investigate the frequency of double-strand breaks (DSBs) and chromosomal aberrations, γH2AX foci assay and 3-color fluorescence in situ hybridization analyses were conducted, respectively. The DSB repair pathways targeted by fisetin were examined in cells expressing genomically integrated reporter constructs by quantifying green fluorescence protein expression through flow cytometry. Apoptosis and autophagy were measured using flow cytometric quantification of sub-G1 cells and the assessment of LC3-II protein expression, respectively. Kinase array and phosphoproteomics analyses were carried out to study the potential impact of fisetin on the signaling involved in the DNA damage response (DDR) after irradiation. We demonstrated that fisetin mimics the effects of RSK pharmacological inhibitors in terms of effect on YB-1 phosphorylation. Treatment with fisetin increased frequency of DSB in non-irradiated cells and impaired repair of DSB after irradiation. Fisetin attenuated DSB repair by suppressing the classical non-homologous end-joining and homologous recombination repair pathways. The frequency of chromosomal aberration was enhanced by fisetin treatment. The effect of fisetin on inhibiting DSB repair was partially YB-1 dependent. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. Fisetin neither inhibited DSB repair nor radiosensitized normal human fibroblast HSF7 cells. The radiosensitizing effect of fisetin was not attributed to enhancing apoptosis or modulating autophagy. In summary, combining fisetin with radiotherapy may enhance TNBC radiotherapy outcomes. -This research was supported by a grant from the German Research Council (DFG,TO 685/2-3). Citation Format: Shayan Khozooei, Soundaram Veerappan, Mahmoud Toulany. Fisetin sensitizes triple negative breast cancer cells to radiation [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 567.

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.

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.

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.