Isadora Pavan

The S6K protein family in health and disease.

The S6K proteins are mTOR pathway effectors and accumulative evidence suggest that mTOR/S6K signaling contributes to several pathological conditions, such as diabetes, cancer and obesity. The activation of the mTOR/S6K axis stimulates protein synthesis and cell growth. S6K1 has two well-known isoforms, p70-S6K1 and p85-S6K1, generated by alternative translation initiation sites. A third isoform, named p31-S6K1, has been characterized as a truncated type of the protein due to alternative splicing, and reports have shown its important role in cancer. Studies involving S6K2 are scarce. This article aims to review what is new in the literature about these kinases and establish differences regarding their interacting proteins, activation and function, connecting their roles in the homeostasis of the cell and in pathological conditions.

Different interactomes for p70‐S6K1 and p54‐S6K2 revealed by proteomic analysis

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70‐S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG‐tagged p70‐S6K1 and p54‐S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high‐scoring interactions. By a comparative approach, p70‐S6K1 interacting proteins were predominantly related to “cytoskeleton” and “stress response,” whereas p54‐S6K2 interactome was more associated to “transcription,” “splicing,” and “ribosome biogenesis.” Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.

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

BackgroundThe 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.MethodsIn 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.ResultsS6Ks 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.ConclusionsThese 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.

p53 and metabolism: from mechanism to therapeutics

The tumor cell changes itself and its microenvironment to adapt to different situations, including action of drugs and other agents targeting tumor control. Therefore, metabolism plays an important role in the activation of survival mechanisms to keep the cell proliferative potential. The Warburg effect directs the cellular metabolism towards an aerobic glycolytic pathway, despite the fact that it generates less adenosine triphosphate than oxidative phosphorylation; because it creates the building blocks necessary for cell proliferation. The transcription factor p53 is the master tumor suppressor; it binds to more than 4,000 sites in the genome and regulates the expression of more than 500 genes. Among these genes are important regulators of metabolism, affecting glucose, lipids and amino acids metabolism, oxidative phosphorylation, reactive oxygen species (ROS) generation and growth factors signaling. Wild-type and mutant p53 may have opposing effects in the expression of these metabolic genes. Therefore, depending on the p53 status of the cell, drugs that target metabolism may have different outcomes and metabolism may modulate drug resistance. Conversely, induction of p53 expression may regulate differently the tumor cell metabolism, inducing senescence, autophagy and apoptosis, which are dependent on the regulation of the PI3K/AKT/mTOR pathway and/or ROS induction. The interplay between p53 and metabolism is essential in the decision of cell fate and for cancer therapeutics.

Correction: p53 and metabolism: from mechanism to therapeutics

[This corrects the article DOI: 10.18632/oncotarget.25267.].

Interactome analysis of the human Cap-specific mRNA (nucleoside-2′-O-)-methyltransferase 1 (hMTr1) protein: SIMABUCO et al.

In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap‐specific messenger RNA (mRNA) (nucleoside‐2′‐O‐)‐methyltransferase 1 (hMTr1), which catalyzes methylation of the 2′‐O ‐ribose of the first nucleotide of capped mRNAs. Pre‐mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5′‐end of the whole cellular pre‐mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre‐mRNA maturation was identified, including splicing factors, spliceosome‐associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA‐binding proteins and proteins involved in mRNA 5′‐ and 3′‐end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre‐mRNA synthesis.

ANÁLISE DO PROTEOMA DE CÉLULAS A549 DE CÂNCER DE PULMÃO SUBMETIDAS A TRATAMENTO COM CISPLATINA

Resumo A combinação terapêutica de cisplatina e metformina tem se mostrado bastante efetiva no tratamento do câncer de pulmão. A administração de metformina mostrou ao longo dos anos relevante redução da incidência e da mortalidade por câncer e, por isso, passou a ser estudada como um possível tratamento contra a doença. Diversos estudos demonstram a efetividade da metformina em reverter a resistência adquirida ao tratamento com cisplatina em pacientes com câncer de pulmão, fator limitante neste tipo de tratamento. Entretanto, os mecanismos moleculares que envolvem essa efetividade ainda não estão esclarecidos. Por este motivo, o presente trabalho teve como objetivo realizar uma análise proteômica de células de câncer de pulmão A549 submetidas primeiramente ao tratamento com cisplatina, identificando proteínas que têm sua expressão modificada através da técnica SILAC (Stable Isotope Labeling by Amino acids in Cell culture).

Characterization of the different S6Ks isoforms interacting partners

mTOR signaling pathway has been related to several human diseases and disorders, including obesity, diabetes and many types of cancer. In the cell, mTOR signaling is responsible to induce cell growth and metabolism upon activation by signals from nutrients processing, such as insulin, ATP and amino acids. S6Ks, which phosphorylates ribosomal S6 protein, are effectors of the mTOR pathway. S6K family is composed of two genes (encoding S6K1 and S6K2) and different isoforms from alternative translation initiation (p70-S6K1, p85-S6K1, p54-S6K2, p56S6K2) and alternative splicing (p31-S6K1). The main function of S6Ks, upon activation, is to lead increased protein synthesis. Nevertheless, one of the main questions in this field is to address the specific roles of the different S6Ks isoforms. In this study, we overexpressed p70-S6K1, p85-S6K1 and p54-S6K2 in human cells and analyzed by immunoprecipitation, followed by mass spectrometry identification, the different interacting partners of those S6Ks isoforms. The different roles of S6Ks isoforms were also evaluated in an adipocyte model and in cancer cell lines. Our study provide new possible functions to the different S6Ks isoforms which may help the understanding of their roles in the mTOR signaling pathway, metabolism and cancer.