Cytochrome c levels affect the TOR pathway to regulate growth and metabolism under energy-deficient conditions.

Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.

Growth promotion in Arabidopsis thaliana by bacterial cyclodipeptides involves the TOR/S6K pathway activation.

Cyclodipeptides (CDPs) are the smallest peptidic molecules that can be produced by diverse organisms such as bacteria, fungi, and animals. They have multiple biological effects. In this paper, we examined the CDPs produced by the bacteria Pseudomonas aeruginosa PAO1, which are known as opportunistic pathogens of humans and plants on TARGET OF RAPAMYCIN (TOR) signaling pathways, and regulation of root system architecture. This bacterium produces the bioactive CDPs: cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In a previous report, these molecules were found to modulate basic cellular programs not only via auxin mechanisms but also by promoting the phosphorylation of the S6 ribosomal protein kinase (S6K), a downstream substrate of the TOR kinase. In the present work, we found that the inoculation of Arabidopsis plants with P. aeruginosa PAO1, the non-pathogenic P. aeruginosa ΔlasI/Δrhll strain (JM2), or by direct exposure of plants to CDPs influenced growth and promoted root branching depending upon the treatment imposed, while genetic evidence using Arabidopsis lines with enhanced or decreased TOR levels indicated a critical role of this pathway in the bacterial phytostimulation.

Increased mTOR activation in idiopathic multicentric Castleman disease.

Idiopathic multicentric Castleman disease (iMCD) is a rare and poorly understood hematologic disorder characterized by lymphadenopathy, systemic inflammation, cytopenias, and life-threatening multiorgan dysfunction. Interleukin-6 (IL-6) inhibition effectively treats approximately one-third of patients. Limited options exist for nonresponders, because the etiology, dysregulated cell types, and signaling pathways are unknown. We previously reported 3 anti-IL-6 nonresponders with increased mTOR activation who responded to mTOR inhibition with sirolimus. We investigated mTOR signaling in tissue and serum proteomes from iMCD patients and controls. mTOR activation was increased in the interfollicular space of iMCD lymph nodes (N = 26) compared with control lymph nodes by immunohistochemistry (IHC) for pS6, p4EBP1, and p70S6K, known effectors and readouts of mTORC1 activation. IHC for pS6 also revealed increased mTOR activation in iMCD compared with Hodgkin lymphoma, systemic lupus erythematosus, and reactive lymph nodes, suggesting that the mTOR activation in iMCD is not just a product of lymphoproliferation/inflammatory lymphadenopathy. Further, the degree of mTOR activation in iMCD was comparable to autoimmune lymphoproliferative syndrome, a disease driven by mTOR hyperactivation that responds to sirolimus treatment. Gene set enrichment analysis of serum proteomic data from iMCD patients (n = 88) and controls (n = 42) showed significantly enriched mTORC1 signaling. Finally, functional studies revealed increased baseline mTOR pathway activation in peripheral monocytes and T cells from iMCD remission samples compared with healthy controls. IL-6 stimulation augmented mTOR activation in iMCD patients, which was abrogated with JAK1/2 inhibition. These findings support mTOR activation as a novel therapeutic target for iMCD, which is being investigated through a trial of sirolimus (NCT03933904).

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

O papel de RSK na fosforilação de alvos compartilhados com S6K e no controle da tradução de 5'top mRNAS em glioblastomas / The role of RSK in phosphorylation of targets shared with S6K and in translational control of 5'TOP mRNAs in glioblastomas

O glioblastoma (GBM) é o tumor cerebral mais comum e agressivo, com sobrevida média de 12 a 15 meses após o diagnóstico. Os pacientes são tratados com ressecção cirúrgica, seguida por quimiorradioterapia, porém o prognóstico é desfavorável devido à alta propensão para recorrência. Algumas classificações para o GBM baseadas em dados de genômica e de transcriptômica foram propostas pelo The Cancer Genome Atlas (TCGA), mas nenhum avanço clínico ocorreu de fato. Como os estudos de transcriptômica incluem todos os mRNAs presentes em uma célula, inclusive aqueles que não estão sendo traduzidos, eles não refletem adequadamente os níveis de proteínas, que são o produto funcional de praticamente todos os genes. Por outro lado, o translatoma corresponde apenas aos mRNAs sendo ativamente traduzidos e, portanto, representa uma melhor abordagem para entender como o proteoma da célula está sendo regulado. Duas importantes vias reguladoras da tradução são RTK/PI3K/Akt/mTORC1 e RTK/Ras/ERK, que apresentam alterações em 90% dos casos de GBM. De forma interessante, há uma família de proteínas chamada 90 kDa ribosomal protein S6 kinase (RSK), que medeia um cross-talk entre essas duas vias. Há quatro isoformas de RSK (RSK1-4), que foram relacionadas a diversos processos celulares em outros tipos tumorais, tais como crescimento, proliferação, sobrevivência, motilidade e tradução. Em GBMs, o papel das RSKs ainda é pouco conhecido. Resultados anteriores do nosso grupo sugeriram a participação da isoforma RSK1 na regulação da fosforilação do fator de início da tradução eIF4B quando mTORC1 está inativo. Neste trabalho, avaliamos não apenas a fosforilação de eIF4B, mas também de rpS6 e eEF2K, todos alvos compartilhados pelas vias PI3K/Akt/mTORC1/S6K e Ras/ERK/RSK. Para investigar o papel das RSKs na fosforilação dessas proteínas, foram utilizadas linhagens celulares selvagens de GBM com diferentes níveis de RSK1. Os experimentos foram conduzidos também em células LN-229 nocautes para RSK1, RSK2 e nocaute duplos para RSK1 e RSK2 (DKO) geradas neste trabalho pela técnica de CRISPR/Cas9. Nossos resultados confirmam que a fosforilação de eIF4B é mais dependente da via Ras/ERK/RSK e indicam que a fosforilação de rpS6 é mais dependente de PI3K/Akt/mTORC1/S6K. Além disso, RSK1 é a principal isoforma envolvida na fosforilação de eIF4B. Outro ponto a ser investigado era o papel da isoforma RSK1 na tradução de mRNAs com sequência 5'TOP, como observado em resultados anteriores de translatômica, que buscaram identificar famílias de mRNAs reguladas pelas RSKs. Através de experimentos de perfil polissomal e avaliação da expressão dos mRNAs 5'TOP por RT-qPCR, neste estudo foi demonstrado que a isoforma RSK1 é capaz de manter a tradução de mRNAs com sequência 5'TOP quando mTORC1 está inibido. Nossos resultados revelam funções exercidas por RSK1 em GBMs e possíveis mecanismos de resistência aos inibidores de mTORC1.

Glioblastoma (GBM) is a very aggressive type of brain tumor, with average survival of 12 to 15 months after diagnosis. Patients are treated with surgical resection followed by chemoradiotherapy, but most of them suffer recurrence. Classifications for GBM using genomic and transcriptomic data were proposed by TCGA, but without clinically relevant benefits. Transcriptome comprises all mRNAs in a cell, whereas the translatome comprises only mRNAs being actively translated to produce proteins. Thus, translatome provides information that is closer to the tumor proteome. RTK/ PI3K/Akt/mTORC1 and RTK/Ras/ERK regulates translation and both of them present with alterations in 90% of GBMs. Importantly, a family of proteins called 90 kDa ribosomal protein S6 kinase (RSK), a substrate for ERK1/2, mediates a crosstalk between these two pathways. RSK family of proteins comprises four isoforms (RSK1-4), related to several cellular processes in other types of cancer, such as cell growth, proliferation, survival, motility and translation. However, the role of RSKs in GBMs is poorly understood. Previous results from our group suggested that RSK controls phosphorylation of translation initiation factor eIF4B when mTORC1 is inactive. In this work, we evaluated not only eIF4B phosphorylation but also rpS6 and eEF2K because all of them are substrates of both PI3K/Akt/mTORC1/S6K and Ras/ERK/RSK. To this, we used GBM cell lines expressing different levels of RSK1 and also LN-229 knockout cells for RSK1, RSK2 and RSK1/2 (DKO) generated by CRISPR/Cas9 system. Our results confirm that eIF4B phosphorylation is more dependent on Ras/ERK/RSK pathway, whereas rpS6 phosphorylation is more dependent on PI3K/Akt/mTORC1/S6K pathway. In addition, RSK1 is the main isoform involved in eIF4B phosphorylation. Another point investigated here was the role of RSK1 in translation of 5'TOP mRNAs, as suggested by previous identification of mRNAs regulated by this isoform using translatomics. Polysome profiling was performed followed by evaluation of 5'TOP mRNAs expression through RT-qPCR. Our results validate that RSK1 isoform can sustain translation of 5'TOP mRNAs when mTORC1 is inactive. In conclusion, this study reveals roles for RSK1 in GBMs and possible mechanisms of resistance to mTORC1 inhibitors.

PKB is a central molecule in the modulation of Na+-ATPase activity by albumin in renal proximal tubule cells.

Evidence points to a possible role of tubular sodium reabsorption in worsening renal injury. Proximal tubule (PT) albumin overload is a critical process in the development of tubule-interstitial injury (TII), and consequently in progression of renal disease. We studied the possible correlation between changes in albumin concentration in the lumen of PT with modification of Na+-ATPase activity. An albumin overload animal model and LLC-PK1 cells as a model of PT cells were used. Albumin overload was induced by intraperitoneal injection of BSA in 14-week-old male Wistar rats. An increase in sodium clearance, fractional excretion of sodium, proteinuria, ratio between urinary protein and creatinine, and albuminuria were observed. These observations indicate that there could be a correlation between an increase in albumin in the lumen of PTs and renal sodium excretion. We observed that the activity of both Na+-ATPase and (Na++K+)ATPase decreased in the renal cortex of an albumin overload animal model. Using LLC-PK1 cells as a model of PT cells, inhibition of Na+-ATPase activity was observed with higher albumin concentrations, similar to that observed in the animal model. The inhibition of protein kinase B by higher albumin concentration was found to be a critical step in the inhibition of Na+-ATPase activity. Interestingly, activation of the ERK1/2/mTORC1/S6K pathway was required for protein kinase B inhibition. This mechanism leads to a decrease in protein kinase C activity and, consequently to inhibition of Na+-ATPase activity. Taken together, our results indicate that the molecular mechanism underlying the modulation of PT Na+-ATPase activity by albumin overload involves activation of the ERK1/2/mTORC1/S6K pathway, which leads to inhibition of the mTORC2/PKB/PKC pathway. Our findings contribute to better understanding regarding handing of renal Na+ induced by albumin overload in the lumen of PTs and, consequently, in the progression of renal disease.

The polyproline-motif of S6K2: eIF5A translational dependence and importance for protein-protein interactions.

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Efeitos da regulação da tradução via RSKs em glioblastomas / Effects of translation regulation via RSKs in glioblastoma

O Glioblastoma (GBM) é o tumor cerebral mais comum e maligno, caracterizado por sua alta agressividade e respostas ineficazes aos tratamentos disponíveis. O diagnóstico impõe uma elevada taxa de mortalidade aos pacientes acometidos, que apresentam um tempo de sobrevida médio menor do que dois anos. A nível molecular, GBMs apresentam alterações em componentes chave das vias de sinalização de receptores tirosina quinase (RTKs), Ras/ERK e PI3K/AKT/mTORC1. A família das "ribosomal protein S6 kinases" (RSKs), ativada pela via Ras/ERK, foi proposta como um regulador da via de mTORC1, convergindo na regulação da síntese de proteínas. Em humanos, foram descritas quatro isoformas de RSKs (RSK1 - 4), que apresentam alta homologia e regulam diferentes funções celulares, sendo a desregulação das RSKs responsável por diferentes processos oncogênicos em diversos tipos tumorais. Contudo, a função das RSKs na regulação da síntese proteica global e de mRNAs específicos em GBMs ainda não foi descrita. Com a finalidade de se estabelecer um modelo robusto para o estudo dos efeitos das RSKs, especialmente do papel específico das isoformas no processo de tradução, foram geradas células nocaute para RSK1, RSK2 e duplo nocautes para RSK1 e RSK2 na linhagem celular de GBM LN-18, através da técnica CRISPR/Cas9. Os estudos com os clones nocautes validou o alvo P(S1798)-TSC2 para avaliação dos efeitos das RSKs. Além disso, detectamos que o alvo de RSKs, P(S422)-eIF4B, pode ser preferencialmente regulado pelas RSKs e não por mTORC1. Importantemente, realizamos a translatômica das células nocaute e analisamos os mRNAs diferencialmente traduzidos na presença ou ausência do inibidor de mTOR, Torin1, para estabelecer um modelo para o estudo da tradução de mRNAs dependentes de RSKs. A análise por meio de microarranjos mostrou que as isoformas RSK1 e RSK2 regulam diferentes conjuntos de mRNAs. A família de mRNAs contendo sequências 5'TOP são um dos principais alvos de mTORC1. De maneira surpreendente, observamos que a maioria dos 5'TOP mRNAs tem a tradução dependente de RSK1, em um mecanismo visualizado somente quando mTORC1 está inativado. Essa observação indica fortemente que RSK1, mas não RSK2 estaria mediando um mecanismo redundante e de resistência contra a inativação de mTORC1. Além disso, propomos um mecanismo inédito de controle da tradução de 5'TOP mRNAs. Desse modo, descrevemos um importante modelo para o entendimento das funções biológicas da família das RSKs em GBMs, que contribuirá com o desenho de alvos terapêuticos mais eficientes (AU)

Glioblastoma (GBM) is the most frequent and malignant brain tumor, characterized by its aggressiveness and poor response to the available treatments. Once diagnosed, GBM patients are inflicted with high mortality rates, and a mean survival lower than two years. From a molecular point of view, GBMs display alterations in key components of receptor tyrosine kinases (RTK), Ras/ERK and/or PI3K/AKT/mTORC1 pathways. The p90 ribosomal S6 kinase family (RSK) is directly regulated by the Ras/ERK pathway, and is thought to regulate mTORC1 pathway, converging on regulation of protein synthesis. Human cells display four RSK isoforms (RSK 1 - 4) that share high levels of sequence homology and regulate several cellular functions. The deregulation of RSKs seems to be responsible for different oncogenic outcomes in several types of tumors. Nevertheless, the function of RSKs in regulation of global protein synthesis and in translation of specific subsets of mRNA in GBM was not described so far. In order to establish a robust model for studying the effects of RSKs focusing on isoform-specific roles in the control of translation, we generated knockout cells using CRISPR/Cas9 system technology for RSK1, RSK2 and double-knockout cells for RSK1 and RSK2 in LN-18 cell line. The studies with knockout cells validated the target P(S1798)-TSC2 to evaluate the effects of RSKs. Also, we found that the RSK target P(S422)-eIF4B might be preferably regulated by RSKs, rather than mTORC1 in LN-18 cells. Importantly, we performed the translatomics on knockout cells and analyzed differentially translated mRNAs in the presence or absence of the mTOR inhibitor, Torin1, in order to determine a model for studying the translation of RSK-dependent mRNAs. Microarray data analysis revealed that RSK1 and RSK2 regulate different sets of mRNAs. The family of mRNAs containing a 5'TOP motif is the main target of mTORC1. Surprisingly, we observed that the majority of 5'TOP mRNAs have its translation dependent on RSK1, in a mechanism that emerges only when mTORC1 is inactivated. This observation strongly suggests that RSK1, but not RSK2, is mediating a mechanism of resistance against mTORC1 inactivation. Moreover, we propose an unprecedented mechanism for translation control of 5'TOP mRNAs. With this study, we were able to describe an important model for understanding the biological functions of RSK in GBMs that will contribute with development of more efficient therapeutic targets (AU)

Agmatine potentiates neuroprotective effects of subthreshold concentrations of ketamine via mTOR/S6 kinase signaling pathway.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is one of the most robust neurobiological findings in the pathophysiology of major depressive disorder (MDD) over the last 40 years. The persistent increase in glucocorticoids levels induces morphological and anatomical changes in the brain, especially in the hippocampus. Ketamine represents a major advance for the treatment of MDD, however the psychotomimetic effects of this compound limit its widespread use. Agmatine is a neuromodulator that has been shown to be a putative novel and well-tolerated antidepressant/augmenter drug. In this study, the exposure of HT22 hippocampal neuronal cell line to corticosterone (50 µM) induced a significant neuronal cell death. Interestingly, the incubation of HT22 cells with the fast-acting antidepressant drug ketamine (1 µM) prevented the corticosterone-induced toxicity. Similarly, agmatine caused a significant cytoprotection at the concentration of 0.1 µM against corticosterone (50 µM) cell damage. Notably, the incubation with a subthreshold concentration of ketamine (0.01 µM) in combination with a subthreshold concentration of agmatine (0.001 µM) prevented the neuronal damage elicited by corticosterone (50 µM). A 24 h co-incubation with subthreshold concentrations of ketamine (0.01 µM) and agmatine (0.001 µM) was able to cause a significant increase in the phosphorylation levels of Akt (Ser473) and p70S6 kinase (Thr389) as well as PSD95 immunocontent. Neither glycogen synthase kinase-3ß (Ser9) phosphorylation nor ß catenin immunocontent were altered by a 24 h co-incubation period. Finally, the co-incubation of cells for 30 min did not produce any effect in the phosphorylation or immunocontent of any protein investigated. Taken together, our results support the notion that the combination of subthreshold concentrations of ketamine and agmatine has cytoprotective effects against corticosterone-induced cell death. This effect is accompanied by its ability to activate Akt and mTOR/S6 kinase signaling pathway, and increase the expression of synaptic proteins.

Overexpression of mTOR and p(240-244)S6 in IDH1 Wild-Type Human Glioblastomas Is Predictive of Low Survival.

PI3K/Akt/mTOR pathway activation is a hallmark of high-grade gliomas, which prompted clinical trials for the use of PI3K and mTOR inhibitors. However, the poor results in the original trials suggested that better patient profiling was needed for such drugs. Thus, accurate and reproducible monitoring of mTOR complexes can lead to improved therapeutic strategies. In this work, we evaluated the expression and phosphorylation of mTOR, RAPTOR, and rpS6 in 195 human astrocytomas and 30 normal brain tissue samples. The expression of mTOR increased in glioblastomas, whereas mTOR phosphorylation, expression of RAPTOR, and expression and phosphorylation of rpS6 were similar between grades. Interestingly, the overexpression of total and phosphorylated mTOR as well as phosphorylated rpS6 (residues 240-244) were associated with wild-type IDH1 only glioblastomas. The expression and phosphorylation of mTOR and phosphorylation of rpS6 at residues 240-244 were associated with a worse prognosis in glioblastomas. Our results suggest that mTOR and rpS6 could be used as markers of overactivation of the PI3K-mTOR pathway and are predictive factors for overall survival in glioblastomas. Our study thus suggests that patients who harbor IDH1 wild-type glioblastomas might have increased benefit from targeted therapy against mTOR.

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60-250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

Guaraná, a Highly Caffeinated Food, Presents in vitro Antitumor Activity in Colorectal and Breast Cancer Cell Lines by Inhibiting AKT/mTOR/S6K and MAPKs Pathways.

The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinases (MAPKs) pathways are frequently upregulated in cancer. Some authors have reported that some antioxidant molecules could be potential inhibitors of these pathways. Therefore, we investigated the in vitro antitumor effect of guaraná by inhibiting the AKT/mTOR/S6K and MAPKs pathways. Colorectal and breast cancer cell lineages, HT-29 and MCF-7 cells, respectively, were exposed to different guaraná concentrations (0.1, 1, 10, and 100 µg/mL) as well as its main bioactive molecule, caffeine, in proportional concentrations to those found in the extract. Western blot, clonogenic assay, and growth curve were performed. Moreover, we investigated the potential cytotoxic effect of guaraná in normal cells. The results revealed that guaraná and caffeine inhibited some MAPKs proteins (p-p38 and p-HSP27) in MCF-7 cells. However, they did not affect this pathway in HT-29 cells. Furthermore, guaraná inhibited mTORC1 (p-S6K) and mTORC2 (p-AKT) in MCF-7 cells, but only mTORC1 in HT-29 cells. Caffeine only inhibited the mTOR pathway in MCF-7 cells. Guaraná decreased the colony formation and cell growth in MCF-7 and HT-29 cells. Guaraná did not affect normal cells. In conclusion, guaraná could be an important agent in antitumor pharmacologic therapies by inhibiting the mTOR and MAPKs pathways.

S6Ks isoforms contribute to viability, migration, docetaxel resistance and tumor formation of prostate cancer cells.

BACKGROUND: The S6 Kinase (S6K) proteins are some of the main downstream effectors of the mammalian Target Of Rapamycin (mTOR) and act as key regulators of protein synthesis and cell growth. S6K is overexpressed in a variety of human tumors and is correlated to poor prognosis in prostate cancer. Due to the current urgency to identify factors involved in prostate cancer progression, we aimed to reveal the cellular functions of three S6K isoforms-p70-S6K1, p85-S6K1 and p54-S6K2-in prostate cancer, as well as their potential as therapeutic targets. METHODS: In this study we performed S6K knockdown and overexpression and investigated its role in prostate cancer cell proliferation, colony formation, viability, migration and resistance to docetaxel treatment. In addition, we measured tumor growth in Nude mice injected with PC3 cells overexpressing S6K isoforms and tested the efficacy of a new available S6K1 inhibitor in vitro. RESULTS: S6Ks overexpression enhanced PC3-luc cell line viability, migration, resistance to docetaxel and tumor formation in Nude mice. Only S6K2 knockdown rendered prostate cancer cells more sensitive to docetaxel. S6K1 inhibitor PF-4708671 was particularly effective for reducing migration and proliferation of PC3 cell line. CONCLUSIONS: These findings demonstrate that S6Ks play an important role in prostate cancer progression, enhancing cell viability, migration and chemotherapy resistance, and place both S6K1 and S6K2 as a potential targets in advanced prostate cancer. We also provide evidence that S6K1 inhibitor PF-4708671 may be considered as a potential drug for prostate cancer treatment.

Estudo do papel de RSK na sinalização celular em glioblastoma / Study of the role of RSK in cell signaling in glioblastoma

Os astrocitomas são neoplasias primárias do Sistema Nervoso Central, graduadas de I a IV com base em critérios clínicos e histológicos. O astrocitoma de grau IV, também denominado glioblastoma, é o tipo mais comum e agressivo dos tumores gliais e apresenta baixa resposta a agentes quimioterápicos. Em glioblastomas, mutações e superexpressão dos receptores de fatores de crescimento podem levar à ativação desregulada da via das proteínas cinases ativadas por mitógenos (MAPK). A via de MAPK é ativadora direta da cinase RSK (do inglês, p90 ribosomal S6 kinase), que está envolvida com diversos processos celulares. Apesar dessa proteína ter sido estudada em diferentes tipos tumorais, sua participação em glioblastomas nunca foi avaliada. Dessa maneira, neste trabalho tivemos como objetivo analisar o envolvimento de RSK na tumorigênese em glioblastoma, e observar a participação dessa proteína em processos como proliferação e senescência celular. Observamos que a regulação dessa proteína ocorre principalmente a nível traducional em linhagens celulares de glioblastoma. Utilizando células nocaute para RSK1 e/ou 2 obtidas pela metodologia CRISPR/Cas9, pudemos constatar o envolvimento dessa proteína na proliferação celular. Além disso, a relação entre RSK e a senescência celular induzida pela perda de PTEN foi demonstrada em células não transformadas, utilizando siRNA e inibidores químicos de RSK. Pudemos também descrever que inibidores de RSK comumente utilizados na literatura possuem importantes efeitos inespecíficos que podem levar a interpretações errôneas sobre as funções de RSK. Adicionalmente, determinamos que o melhor alvo para acessar a atividade de RSK é a proteína TSC2 (Ser1798).

Astrocytomas are primary Central Nervous System tumors graded from I to IV based on histological and clinical criteria. The grade IV astrocytoma, also known as glioblastoma, is the most common and aggressive of glial tumors and presents low response to chemotherapeutic agents. In glioblastomas, mutations and overexpression of growth factor receptors can lead to the upregulated activation of the mitogen-activated protein kinases pathways (MAPK). MAPK pathway is the major activator of the p90 ribosomal S6 kinase (RSK), which is involved in many cellular processes. RSK involvement has been performed in several tumor types; however it has never been studied in glioblastomas. Thus, this work aimed to analyze the involvement of RSK in glioblastoma. Firstily, it was observed that the regulation of this protein occurs mainly at translational level or by pos-tranlstional control in glioblastoma cell lines. Using RSK1 and/or RSK2 knockout cells obtained by the CRISPR/Cas9 methodology, it was found the involvement of this protein on cell proliferation. Furthermore, the possible relationship between RSK and cellular senescence induced by PTEN loss has been demonstrated in untransformed cells by using siRNA and chemical RSK inhibitors. Additionally, it also was observed that RKS inhibitors commonly used in the literature have unspecific effects that can lead to wrongful conclusions about the true functions of RSKs. It was also demonstrated that the best target to access the RSK activity is the TSC2 protein (Ser1798).

Silencing FKBP38 gene by siRNA induces activation of mTOR signaling in goat fetal fibroblasts.

FKBP38 (also known as FKBP8) is a unique member of the FK506-binding protein (FKBP) family, and its role is controversial because it acts as an upstream regulator of the mTOR signaling pathway, which controls cell growth, proliferation, and differentiation. This study aimed to explore the role of FKBP38 in the activation of mTOR signaling in Cashmere goat (Capra hircus) fetal fibroblasts. To construct a Cashmere goat FKBP38 siRNA eukaryotic expression vector that targets FKBP38 mRNA, we designed shRNA based on the gene sequence deposited in GenBank (accession No. JF714970) and synthesized a DNA fragment encoding the shRNA. The DNA fragment was inserted into the pRNAT-U6.1/Neo vector to construct an expression vector of shRNA, which was labeled pRNAT-FKBP38-shRNA. The recombinant plasmid was used to transfect Cashmere goat fetal fibroblasts (GFb) using lipofectamine™2000. We found that cells were successfully transfected with pRNAT-U6.1/Neo-FKBP38-shRNA. Green fluorescence could be observed in cells following 48-h transfection. Proteins were then isolated from GFbs transfected with pRNAT-FKBP38-shRNA and from control cells, and protein expression was analyzed by western blot. Expression of FKBP38 decreased and mTOR signaling was activated, which induced the phosphorylation of mTOR, S6, and 4EBP1. Thus, FKBP38 gene-silencing activates mTOR signaling in goat cells.

Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Testosterone signals through mTOR and androgen receptor to induce muscle hypertrophy.

PURPOSE: The anabolic hormone testosterone induces muscle hypertrophy, but the intracellular mechanisms involved are poorly known. We addressed the question whether signal transduction pathways other than the androgen receptor (AR) are necessary to elicit hypertrophy in skeletal muscle myotubes. METHODS: Cultured rat skeletal muscle myotubes were preincubated with inhibitors for ERK1/2 (PD98059), PI3K/Akt (LY294002 and Akt inhibitor VIII) or mTOR/S6K1 (rapamycin), and then stimulated with 100 nM testosterone. The expression of α-actin and the phosphorylation levels of ERK1/2, Akt and S6K1 (a downstream target for mTOR) were measured by Western blot. mRNA levels were evaluated by real time RT-PCR. Myotube size and sarcomerization were determined by confocal microscopy. Inhibition of AR was assessed by bicalutamide. RESULTS: Testosterone-induced myotube hypertrophy was assessed as increased myotube cross-sectional area (CSA) and increased α-actin mRNA and α-actin protein levels, with no changes in mRNA expression of atrogenes (MAFbx and MuRF-1). Morphological development of myotube sarcomeres was evident in testosterone-stimulated myotubes. Known hypertrophy signaling pathways were studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after testosterone stimulus at 5 and 15 min, respectively. S6K1 was phosphorylated at 60 min. This response was abolished by PI3K/Akt and mTOR inhibition but not by ERK1/2 inhibition. Similarly, the CSA increase at 12 h was abolished by inhibitors of the PI3K/Akt pathway as well as by AR inhibition. CONCLUSIONS: These results suggest a crosstalk between pathways involving fast intracellular signaling and the AR to explain testosterone-induced skeletal muscle hypertrophy.

TOR pathway activation in Zea mays L. tissues: conserved function between animal and plant kingdoms.

In most non-photosynthetic eukaryotes it has been demonstrated a conserved signal transduction pathway, namely TOR-S6K, that coordinates growth and cell proliferation. This pathway targets the translational apparatus to induce selective translation of ribosomal mRNAs as well as stimulate the cell cycle transition through the G1/S phase. Thus, by activation of this pathway through environmental signals, nutrients, stress, or specific growth factors, such as insulin or insulin-like growth factors (IGF), this pathway allows organisms to regulate growth and cell division. In plants, evidence has shown that TOR protein has been highly conserved through evolution, being involved in growth and cell proliferation control as well. Particularly in maize, a peptide named ZmIGF has been found in actively growing tissues. It targets the maize TOR pathway at the same extent as insulin and, by doing so it induces growth, as well as ribosomal proteins and DNA synthesis. Thus, higher metazoans and plants seem to conserve similar biochemical paths to regulate cell growth through equivalent targets that conduce to activation of the TOR-S6K pathway. Recent research shows evidence that supports this proposal by uncovering the ZmIGF receptor in maize, providing further means for analyzing the role of the conserved TOR signaling pathway in this plant.

Mammalian target of rapamycin complex 1 is involved in differentiation of regenerating myofibers in vivo.

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.

FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of the mTOR complex.

AIMS: Cardiac fibroblasts are activated by mechanical stress, but the underlying mechanisms involved remain poorly understood. In this study, we investigated whether focal adhesion kinase (FAK) plays a role in the activation of cardiac fibroblasts in response to cyclic stretch. METHODS AND RESULTS: Neonatal (NF-P3/80--third passage, 80% confluence) and adult (AF-P1/80--first passage, 80% confluence) rat cardiac fibroblasts were exposed to cyclic stretch (biaxial, 1 Hz), which enhanced FAK phosphorylation at Tyr397. Proliferation (anti-5-bromo-2'-deoxyuridine and anti-Ki67 nuclear labelling), differentiation into myofibroblasts (expression of alpha-smooth muscle actin--alpha-SMA), and the activity of matrix metalloproteinase-2 were equally enhanced in stretched NF-P3/80 and AF-P1/80. Treatment with the integrin inhibitor RGD peptide impaired FAK phosphorylation and increased apoptosis (TUNEL) in non-stretched and stretched NF-P3/80, whereas FAK silencing induced by small interfering RNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF-P3/80 invoked by cyclic stretch. In addition, NF-P3/80 depleted of FAK were defective in AKT Ser473, TSC-2 Thr1462, and S6 kinase Thr389 phosphorylation induced by cyclic stretch. The activation of NF-P3/80 invoked by cyclic stretch was prevented by pre-treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin, whereas supplementation with the amino acid, leucine, activated S6K and rescued the stretch-induced activation of NF-P3/80 depleted of FAK. CONCLUSIONS: These findings demonstrate a critical role for the mTOR complex, downstream from FAK, in mediating the activation of cardiac fibroblasts in response to mechanical stress.