Aric N. Rogers

With TOR, Less Is More: A Key Role for the Conserved Nutrient-Sensing TOR Pathway in Aging

Target of rapamycin (TOR) is an evolutionarily conserved nutrient-sensing protein kinase that regulates growth and metabolism in all eukaryotic cells. Studies in flies, worms, yeast, and mice support the notion that the TOR signaling network modulates aging. TOR is also emerging as a robust mediator of the protective effects of various forms of dietary restriction (DR), which can extend life span and slow the onset of certain age-related diseases across species. Here we discuss how modulating TOR signaling slows aging through downstream processes including mRNA translation, autophagy, endoplasmic reticulum (ER) stress signaling, stress responses, and metabolism. Identifying the mechanisms by which the TOR signaling network works as a pacemaker of aging is a major challenge and may help identify potential drug targets for age-related diseases, thereby facilitating healthful life span extension in humans.

Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans

Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the cap‐binding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg‐1, the worm homologue of eIF4G, which is a scaffold protein in the cap‐binding complex; and rsks‐1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg‐1 or rsks‐1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg‐1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans.

Insulin-like Signaling Determines Survival during Stress via Posttranscriptional Mechanisms in C. elegans

The insulin-like signaling (ILS) pathway regulates metabolism and is known to modulate adult life span in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF-16 and HSF-1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stress-induced transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are posttranscriptionally regulated in response to altered ILS during heat shock in a DAF-16-dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered ILS results in metabolic and physiological changes. These DAF-16-induced changes precondition a translational response under acute stress to modulate survival.

mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms

Resistance of breast cancers to targeted hormone receptor (HR) or human epidermal growth factor receptor 2 (HER2) inhibitors often occurs through dysregulation of the phosphoinositide 3-kinase, protein kinase B/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Presently, no targeted therapies exist for breast cancers lacking HR and HER2 overexpression, many of which also exhibit PI3K/AKT/mTOR hyper-activation. Resistance of breast cancers to current therapeutics also results, in part, from aberrant epigenetic modifications including protein acetylation regulated by histone deacetylases (HDACs). We show that the investigational drug MLN0128, which inhibits both complexes of mTOR (mTORC1 and mTORC2), and the hydroxamic acid pan-HDAC inhibitor TSA synergistically inhibit the viability of a phenotypically diverse panel of five breast cancer cell lines (HR−/+, HER2−/+). The combination of MLN0128 and TSA induces apoptosis in most breast cancer cell lines tested, but not in the non-malignant MCF-10A mammary epithelial cells. In parallel, the MLN0128/TSA combination reduces phosphorylation of AKT at S473 more than single agents alone and more so in the 5 malignant breast cancer cell lines than in the non-malignant mammary epithelial cells. Examining polysome profiles from one of the most sensitive breast cancer cell lines (SKBR3), we demonstrate that this MLN0128/TSA treatment combination synergistically impairs polysome assembly in conjunction with enhanced inhibition of 4eBP1 phosphorylation at S65. Taken together, these data indicate that the synergistic growth inhibiting consequence of combining a mTORC1/C2 inhibitor like MLN0128 with a pan-HDAC inhibitor like TSA results from their mechanistic convergence onto the PI3K/AKT/mTOR pathway, profoundly inhibiting both AKT S473 and 4eBP1 S65 phosphorylation, reducing polysome formation and cancer cell viability.

Role of Translation Initiation Factor 4G in Lifespan Regulation and Age-related Health

Inhibiting expression of eukaryotic translation initiation factor 4G (eIF4G) arrests normal development but extends lifespan when suppressed during adulthood. In addition to reducing overall translation, inhibition alters the stoichiometry of mRNA translation in favor of genes important for responding to stress and against those associated with growth and reproduction in C. elegans. In humans, aberrant expression of eIF4G is associated with certain forms of cancer and neurodegeneration. Here we review what is known about the roles of eIF4G in molecular, cellular, and organismal contexts. Also discussed are the gaps in understanding of this factor, particularly with regard to the roles of specific forms of expression in individual tissues and the importance of understanding eIF4G for development of potential therapeutic applications.

Comparative biology of tissue repair, regeneration and aging

The Symposium on the Comparative Biology of Tissue Repair, Regeneration and Aging, held 26 June to 28 June 2015 at the MDI Biological Laboratory in Salisbury Cove, Maine, brought together a diverse group of biologists with a common interest in understanding why regenerative capacity varies among animal species, why it is progressively lost in senescence, and how answers obtained from studies that address those questions might be applied in regenerative medicine.

Reducing translation through eIF4G/IFG-1 improves survival under ER stress that depends on heat shock factor HSF-1 in Caenorhabditis elegans.

Although certain methods of lowering and/or altering mRNA translation are associated with increased lifespan, the mechanisms underlying this effect remain largely unknown. We previously showed that the increased lifespan conferred by reducing expression of eukaryotic translation initiation factor 4G (eIF4G/IFG‐1) enhances survival under starvation conditions while shifting protein expression toward factors involved with maintaining ER‐dependent protein and lipid balance. In this study, we investigated changes in ER homeostasis and found that lower eIF4G/IFG‐1 increased survival under conditions of ER stress. Enhanced survival required the ER stress sensor gene ire‐1 and the ER calcium ATPase gene sca‐1 and corresponded with increased translation of chaperones that mediate the ER unfolded protein response (UPRER). Surprisingly, the heat‐shock transcription factor gene hsf‐1 was also required for enhanced survival, despite having little or no influence on the ability of wild‐type animals to survive ER stress. The requirement for hsf‐1 led us to re‐evaluate the role of eIF4G/IFG‐1 on thermotolerance. Results show that lowering expression of this translation factor enhanced thermotolerance, but only after prolonged attenuation, the timing of which corresponded to increased transcription of heat‐shock factor transcriptional targets. Results indicate that restricting overall translation through eIF4G/IFG‐1 enhances ER and cytoplasmic proteostasis through a mechanism that relies heavily on hsf‐1.

Translational Control of Longevity

There is accumulating evidence to suggest that that certain methods of limiting translation, while slowing growth and development, enhance somatic maintenance and lifespan. Much of this work has been based on studies in the model organism C. elegans. With its abundant genetic toolbox, rapid lifecycle, and transparency with diverse yet tractable tissues, many physiological responses to translation modulation were first characterized utilizing C. elegans. Translational regulation is complex and governed by hundreds of factors and noncoding RNAs, far too many to discuss in any one chapter. Instead, we explore the basic concepts of translational responsiveness to environment changes and inputs from signalling pathways associated with longevity regulation. We also discuss how studies aimed at diagnosing translation in C. elegans are enhancing efforts to understand mechanisms underlying the pro-longevity effects of attenuating translation.

Abstract P2-05-04: Differential potency of TORC1/C2 (INK/MLN-128) and pan-PI3K (GDC0941) inhibitors on breast cancer polysome composition and phosphoprotein response biomarkers

The PI3K (phosphoinositide-3 kinase)/AKT (protein kinase B) pathway is dysregulated in over 70% of breast cancers, including more than 40% of non-basal breast cancers that possess activating PIK3CA mutations and 15% of basal breast cancers with functional loss of PTEN (n = 357 TCGA samples), suggesting that inhibitors of this signaling pathway may be clinically beneficial. Moreover, the recently reported BOLERO-2 trial (N Engl J Med, 2012) demonstrated the potential clinical benefit of using a TORC1 (target of rapamycin complex I) inhibitor, Everolimus, to treat patients with advanced non-basal, ER-positive breast cancers presumeably enriched with PIK3CA mutations. Among the new PI3K/AKT/TOR pathway-targeted therapeutics now under clinical development are the dual TORC1/C2 inhibitor, INK/MLN-128, and the pan-PI3K inhibitor, GDC0941. Despite having comparable nanomolar target affinities, it is unclear whether similar subsets of breast cancers will respond similarly to these agents, or if targeting this PI3K/AKT/TOR pathway either upstream (e.g. by GDC0941) or downstream (e.g. by INK/MLN-128) will yield comparable antitumor effects. Many of the downstream cellular responses regulated by this pathway, including tumor cell survival, growth, metabolism, invasiveness and angiogenic potential, are mediated by phosphorylation of polysome effector proteins, including the known TORC1 regulated eukaryotic translation initiation factor 4 binding protein (4eBP1) and 40S ribosomal protein S6. Therefore, we compared the effects of INK/MLN-128 and GDC0941 on cell growth and polysome protein phosphorylation profiles using a model breast cancer cell line (SKBr3) with amplified upstream receptor (HER2) activation of the PI3K/AKT/TOR pathway. Continuous dosing of these two agents against SKBr3 cultures (0–625 nM x5 days) revealed a 50-fold greater anti-proliferative efficacy for INK/MLN-128, with IC 50 = 9nM compared to the GDC0941 IC 50 = 479nM. This difference in antiproliferative activity correlated with their 50-fold different doses required to inhibit polysome formation (relative to free 40S, 60S, and 80S ribosomes) and polysomal 40S Rack1 and 60S RPL24 content. Likewise, INK/MLN-128 more potently inhibited the phosphorylation of two polysome-associated proteins of 32 KD and 60 KD size, recognized by an antibody toward a motif phosphorylated by AKT and related kinases (RXXS*/T*); the 32 KD substrate has been identified as S6. With equipotent dosing (0.1 mM INK/MLN-128 and 5 mM GDC0941), these two agents showed comparable inhibitory effects on polysome formation and protein phosphorylation. In summary, in a breast cancer model bearing upstream receptor activation of the PI3K/AKT/TOR pathway, distal pathway inhibition of TORC1/C2 by INK/MLN-128 appeared more potent and effective than more proximal inhibition of pan-PI3K by GDC0941. Furthermore, polysome inhibitory effects following INK/MLN-128 exposure were apparent within 8 h of treatment, and the most sensitive polysome response biomarker for this agent appeared to be phospho(T389)-S6 kinase, providing rationale for the use of this response biomarker in future clinical trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-04.

Abstract 3602: Differential effects of mTOR and PI3K inhibition on the phosphorylation of polysome-associated proteins

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The PI3K (phosphoinositide-3 kinase) pathway is upregulated in many malignancies, including a significant proportion of human breast cancers. PI3K dysregulation activates downstream targets, such as the serine-threonine kinase AKT (also known as protein kinase B) and the mammalian target of rapamycin (mTOR) complexes, TORC1 and TORC2, serving to enhance cell survival, growth, invasiveness, and angiogenic potential as well as altering cellular metabolism. Many of these downstream cellular responses are induced by PI3K/AKT/mTOR control of mRNA translation initiation and elongation via phosphorylation of polysome-associated effectors, such as eukaryotic translation initiation factor 4 components, its inhibitory binding protein (eIF4BP), and the 40S ribosomal protein S6. Due to the importance of the PI3K pathway in tumorigenesis, several different PI3K/AKT/mTOR pathway inhibitors are under clinical development, including dual TORC1/C2 inhibitors (e.g. INK128; Intellikine) and pan-inhibitors of all four isoforms of class 1 PI3K (e.g. GDC0941; Genentech). Despite the specificity of such mTOR and PI3K inhibitors, it remains unclear which subsets of breast cancer will respond to these agents and which biomarkers are best used to monitor and distinguish their target-specific antitumor responses. Furthermore, known translation-regulating phosphoproteins downstream of mTORC1 (e.g. eIF4B, S6) can also be activated by alternative kinase pathways (e.g. PDK1) downstream of PI3K. Therefore, we sought to discover translation-associated biomarkers capable of distinguishing the responsiveness of breast cancer cells to INK128 and GDC0941, focusing on post-translationally modified proteins contained within the polysome-enriched cell compartment. We used an antibody specific for phospho-AKT substrates to probe 1-D and 2-D gel separated polysome-enriched fractions from the HER2-positive breast cancer cell line, SKBR3, following control or growth inhibiting doses of INK128 or GDC0941. The 5-day SKBR3 IC50 doses (inhibiting cell proliferation by 50%) for INK128 and GDC0941 were found to be 16 nM and 565 nM, respectively. Following 24 h exposure to identical doses of these two drugs, INK128 significantly reduced the phosphorylation of a small subset of polysome-associated proteins (including ∼30 kD and ∼50 kD species) while GDC0941 had no apparent effect on these proteins, whose identification is pending. These findings suggest that phosphorylation of specific polysome-associated proteins likely mediates translational control exerted by some downstream effectors of the PI3K/AKT/mTOR pathway. Furthermore these specific polysome-associated targets may serve as biomarkers differentiating cell responses to TORC1/C2 from pan-PI3K inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3602. doi:1538-7445.AM2012-3602