mTOR: from growth signal integration to cancer, diabetes and ageing

In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.National Institutes of Health (U.S.)Howard Hughes Medical InstituteWhitehead Institute for Biomedical ResearchJane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship)Human Frontier Science Program (Strasbourg, France

TOR and PKA Pathways Synergize at the Level of the Ste11 Transcription Factor to Prevent Mating and Meiosis in Fission Yeast

[Background]: In the fission yeast Schizosaccharomyces pombe, the TOR (target of rapamycin) and PKA (protein kinase A) signaling transduction pathways regulate the expression of genes required for cell growth and sexual differentiation in response to the nutritional environment. Inhibition of Tor2 signaling results in the induction of genes involved in sexual differentiation, and the cells undergo mating and meiosis, even under good nutritional conditions. The same phenotype is observed in mutants in which the PKA pathway is inactive. By contrast, Tor2 overexpression or mutations that hyperactivate PKA signaling impair sexual differentiation, even under poor nutritional conditions. Accordingly, a very important question is to understand the molecular mechanism by which these two pathways coordinately regulate gene expression in response to nutrients. [Methodology/Principal Findings]: Here we demonstrate that TOR and PKA pathways operate coordinately to negatively regulate sexual differentiation by inhibiting the nuclear accumulation of the Ste11 transcription factor. However, the Tor2 pathway is unable to block the nuclear localization of Ste11 under good nutritional conditions when the PKA pathway is inactive. Using microarray analyses, we found that both pathways inhibit sexual differentiation by blocking ste11-dependent gene expression. [Conclusions/Significance]: We conclude that both the PKA and the TOR pathways inhibit Ste11 nuclear accumulation to repress Ste11-dependent gene expression. However, the PKA pathway plays a quantitatively more important role than the TOR pathway in this process.N.V. is supported by a postdoctoral grant from the Carlos III Institute, Ministerio de Sanidad. Our group is supported by grants from la Junta de Castilla y Leon (Grupo de Excelencia grant GR265) and the Spanish Ministry of Science and Innovation (BFU2008-01808 and Consolider Ingenio CSD2007-00015).Peer reviewe

Preoperative external beam radiotherapy and reduced dose brachytherapy for carcinoma of the cervix: survival and pathological response

PURPOSE: To evaluate the pathologic response of cervical carcinoma to external beam radiotherapy (EBRT) and high dose rate brachytherapy (HDRB) and outcome. MATERIALS AND METHODS: Between 1992 and 2001, 67 patients with cervical carcinoma were submitted to preoperative radiotherapy. Sixty-five patients were stage IIb. Preoperative treatment included 45 Gy EBRT and 12 Gy HDRB. Patients were submitted to surgery after a mean time of 82 days. Lymphadenectomy was performed in 81% of patients. Eleven patients with residual cervix residual disease on pathological specimen were submitted to 2 additional insertions of HDRB. RESULTS: median follow up was 72 months. Five-year cause specific survival was 75%, overall survival 65%, local control 95%. Complete pelvic pathological response was seen in 40%. Surgery performed later than 80 days was associated with pathological response. Pelvic nodal involvement was found in 12%. Complete pelvic pathological response and negative lymphnodes were associated with better outcome (p = .03 and p = .005). Late grade 3 and 4 urinary and intestinal adverse effects were seen in 12 and 2% of patients. CONCLUSION: Time allowed between RT and surgery correlated with pathological response. Pelvic pathological response was associated with improved outcome. Postoperative additional HDRB did not improve therapeutic results. Treatment was well tolerated

Genome-Wide Identification of Transcription Start Sites, Promoters and Transcription Factor Binding Sites in E. coli

Despite almost 40 years of molecular genetics research in Escherichia coli a major fraction of its Transcription Start Sites (TSSs) are still unknown, limiting therefore our understanding of the regulatory circuits that control gene expression in this model organism. RegulonDB (http://regulondb.ccg.unam.mx/) is aimed at integrating the genetic regulatory network of E. coli K12 as an entirely bioinformatic project up till now. In this work, we extended its aims by generating experimental data at a genome scale on TSSs, promoters and regulatory regions. We implemented a modified 5′ RACE protocol and an unbiased High Throughput Pyrosequencing Strategy (HTPS) that allowed us to map more than 1700 TSSs with high precision. From this collection, about 230 corresponded to previously reported TSSs, which helped us to benchmark both our methodologies and the accuracy of the previous mapping experiments. The other ca 1500 TSSs mapped belong to about 1000 different genes, many of them with no assigned function. We identified promoter sequences and type of σ factors that control the expression of about 80% of these genes. As expected, the housekeeping σ70 was the most common type of promoter, followed by σ38. The majority of the putative TSSs were located between 20 to 40 nucleotides from the translational start site. Putative regulatory binding sites for transcription factors were detected upstream of many TSSs. For a few transcripts, riboswitches and small RNAs were found. Several genes also had additional TSSs within the coding region. Unexpectedly, the HTPS experiments revealed extensive antisense transcription, probably for regulatory functions. The new information in RegulonDB, now with more than 2400 experimentally determined TSSs, strengthens the accuracy of promoter prediction, operon structure, and regulatory networks and provides valuable new information that will facilitate the understanding from a global perspective the complex and intricate regulatory network that operates in E. coli

Diet and Energy-Sensing Inputs Affect TorC1-Mediated Axon Misrouting but Not TorC2-Directed Synapse Growth in a Drosophila Model of Tuberous Sclerosis

The Target of Rapamycin (TOR) growth regulatory system is influenced by a number of different inputs, including growth factor signaling, nutrient availability, and cellular energy levels. While the effects of TOR on cell and organismal growth have been well characterized, this pathway also has profound effects on neural development and behavior. Hyperactivation of the TOR pathway by mutations in the upstream TOR inhibitors TSC1 (tuberous sclerosis complex 1) or TSC2 promotes benign tumors and neurological and behavioral deficits, a syndrome known as tuberous sclerosis (TS). In Drosophila, neuron-specific overexpression of Rheb, the direct downstream target inhibited by Tsc1/Tsc2, produced significant synapse overgrowth, axon misrouting, and phototaxis deficits. To understand how misregulation of Tor signaling affects neural and behavioral development, we examined the influence of growth factor, nutrient, and energy sensing inputs on these neurodevelopmental phenotypes. Neural expression of Pi3K, a principal mediator of growth factor inputs to Tor, caused synapse overgrowth similar to Rheb, but did not disrupt axon guidance or phototaxis. Dietary restriction rescued Rheb-mediated behavioral and axon guidance deficits, as did overexpression of AMPK, a component of the cellular energy sensing pathway, but neither was able to rescue synapse overgrowth. While axon guidance and behavioral phenotypes were affected by altering the function of a Tor complex 1 (TorC1) component, Raptor, or a TORC1 downstream element (S6k), synapse overgrowth was only suppressed by reducing the function of Tor complex 2 (TorC2) components (Rictor, Sin1). These findings demonstrate that different inputs to Tor signaling have distinct activities in nervous system development, and that Tor provides an important connection between nutrient-energy sensing systems and patterning of the nervous system

Defining the Cellular Environment in the Organ of Corti following Extensive Hair Cell Loss: A Basis for Future Sensory Cell Replacement in the Cochlea

Background: Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss. Methodology/Principal Findings: Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca’s, suggesting genetic background influences the rate of re-organisation

Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer

Human telomerase activity regulation

Telomerase has been recognized as a relevant factor distinguishing cancer cells from normal cells. Thus, it has become a very promising target for anticancer therapy. The cell proliferative potential can be limited by replication end problem, due to telomeres shortening, which is overcome in cancer cells by telomerase activity or by alternative telomeres lengthening (ALT) mechanism. However, this multisubunit enzymatic complex can be regulated at various levels, including expression control but also other factors contributing to the enzyme phosphorylation status, assembling or complex subunits transport. Thus, we show that the telomerase expression targeting cannot be the only possibility to shorten telomeres and induce cell apoptosis. It is important especially since the transcription expression is not always correlated with the enzyme activity which might result in transcription modulation failure or a possibility for the gene therapy to be overcome. This review summarizes the current state of knowledge of numerous telomerase regulation mechanisms that take place after telomerase subunits coding genes transcription. Thus we show the possible mechanisms of telomerase activity regulation which might become attractive anticancer therapy targets

Cancer: evolutionary, genetic and epigenetic aspects

There exist two paradigms about the nature of cancer. According to the generally accepted one, cancer is a by-product of design limitations of a multi-cellular organism (Greaves, Nat Rev Cancer 7:213–221, 2007). The essence of the second resides in the question “Does cancer kill the individual and save the species?” (Sommer, Hum Mutat 3:166–169, 1994). Recent data on genetic and epigenetic mechanisms of cell transformation summarized in this review support the latter point of view, namely that carcinogenesis is an evolutionary conserved phenomenon—a programmed death of an organism. It is assumed that cancer possesses an important function of altruistic nature: as a mediator of negative selection, it serves to preserve integrity of species gene pool and to mediate its evolutionary adjustment. Cancer fulfills its task due apparently to specific killer function, understanding mechanism of which may suggest new therapeutic strategy