Rapamycin Addition Research Articles

Structural insights into rapamycin-induced oligomerization of a FRB-FKBP fusion protein.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.

Rapamycin enhanced the production of 2-phenylethanol during whole-cell bioconversion by yeast.

2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, has been widely utilized in food, perfume, and beverages. Saccharomyces cerevisiae is one of the most promising microorganisms for the biosynthesis of natural 2-PE. However, the growth of S. cerevisiae is generally inhibited by 2-PE, which makes its production in yeast cell factories challenging. Here, the whole-cell bioconversion was used to avert growth inhibition, leading to an increase in the concentration and productivity of 2-PE. Moreover, rapamycin (Rap) addition further improved the efficiency of 2-PE synthesis. The concentration of 2-PE (2.20g/L) was 1.68-fold higher than that in the absence of Rap during the whole-cell bioconversion by S. cerevisiae BY4741. RT-qPCR results showed that Rap addition increased the transcription of ARO9, ARO10, ADH2, GAP1, ARO80, GLN3, and GDH2. When the GLN3 was knocked out, the transcriptional levels of the genes were dramatically decreased, and the concentration of 2-PE significantly decreased to 0.21g/L. The results indicated that Rap enhanced the flux of the Ehrlich pathway, and Gln3 exerted a central role in the regulation of Rap. Furthermore, commercial yeast (S. cerevisiae FY202001) was selected to verify the applicability of Rap. In the presence of Rap, 3.67g/L 2-PE was obtained by whole-cell bioconversion in flask, which was increased by 9% than that in the absence of Rap. Finally, the 2-PE titer reached 4.93g/L by whole-cell bioconversion in a 5 L bioreactor, with a yield of 84mol% from L-phenylalanine and a productivity of 0.103g/L h, which was far higher than that of the currently reported in S. cerevisiae. These findings provided a new idea for the efficient synthesis of 2-PE. KEY POINTS: • Whole-cell bioconversion was used to produce 2-PE. • The regulation of the Ehrlich pathway by Rap provides a theoretical basis for developing an effective yeast cell factory to produce 2-PE. • The 2-PE productivity of 0.103g/L h is far higher than that of the currently reported in S. cerevisiae .

Insulin growth factor-1 enhances proliferation and inhibits apoptosis of neural progenitor cells by phosphorylation of Akt/mTOR/p70S6K molecules and triggering intrinsic apoptosis signaling pathway

Neural progenitor cells (NPCs) transplantation is known as a potential strategy for treating spinal cord injury (SCI). This study aimed to investigate effects of insulin growth factor-1 (IGF-I) on NPCs proliferation and clarify associated mechanisms. NPCs isolated from T8-T10 segmental spinal cord tissues of rats were cultured and identification. Then, lentivirus packing plasmids containing IGF-I was constructed and used for NPCs infection. Cell proliferation was evaluated by detecting 5-Bromodeoxyuridine (BrdU) expression in NPCs, cell differentiation was detected using double-labeling immunofluorescence staining while cell apoptosis was detected using TUNEL assay. In addition, the signal expression of Akt/mTOR/p70S6K in NPCs cells were investigated using immunofluorescence staining and western blot assay. The experimental group was defined as pCMV-IGF-I group, while the negative control group was defined as pCMV-LacZ group. Cells infected with pCMV-IGF-I lentivirus followed by addition of 100mg/ml rapamycin were defined as pCMV-IGF-I + Rapa group. NPCs were successfully isolated, identified and cultured. IGF-I overexpression significantly inhibited cell apoptosis and enhanced cell migration. Akt/mTOR/ p70S6K signaling cascade was proved to be present in NPCs, IGF-I overexpression significantly activated Akt/mTOR/p70S6K signaling cascade, while rapamycin addition inhibited its expression. Also, the activated Akt/mTOR/p70S6K signal cascade induced by IGF-I significantly enhanced BrdU expression and inhibited cell apoptosis, and promoted the differentiation of NPC into the neuronal system. However, the rapamycin addition inhibited the cell response induced by IGF-I overexpression. IGF-I overexpression could enhance cell proliferation, inhibit cell apoptosis and promote their differentiation into neuronal systems by activating Akt/mTOR/p70S6K signaling cascade in vitro, indicating that the Akt/mTOR/p70S6K signaling cascade may be the potentially mechanism for the endogenous repair and remodeling of spinal cord after injury.

Fine-tuning of AMPK\u2013ULK1\u2013mTORC1 regulatory triangle is crucial for autophagy oscillation

Autophagy is an intracellular digestive process, which has a crucial role in maintaining cellular homeostasis by self-eating the unnecessary and/or damaged components of the cell at various stress events. ULK1, one of the key elements of autophagy activator complex, together with the two sensors of nutrient and energy conditions, called mTORC1 and AMPK kinases, guarantee the precise function of cell response mechanism. We claim that the feedback loops of AMPK–mTORC1–ULK1 regulatory triangle determine an accurate dynamical characteristic of autophagic process upon cellular stress. By using both molecular and theoretical biological techniques, here we reveal that a delayed negative feedback loop between active AMPK and ULK1 is essential to manage a proper cellular answer after prolonged starvation or rapamycin addition. AMPK kinase quickly gets induced followed by AMPK-P-dependent ULK1 activation, whereas active ULK1 has a rapid negative effect on AMPK-P resulting in a delayed inhibition of ULK1. The AMPK-P → ULK1 ˧ AMPK-P negative feedback loop results in a periodic repeat of their activation and inactivation and an oscillatory activation of autophagy, as well. We demonstrate that the periodic induction of self-cannibalism is necessary for the proper dynamical behaviour of the control network when mTORC1 is inhibited with respect to various stress events. By computational simulations we also suggest various scenario to introduce “delay” on AMPK-P-dependent ULK1 activation (i.e. extra regulatory element in the wiring diagram or multi-phosphorylation of ULK1).

Mmi1, the Yeast Ortholog of Mammalian Translationally Controlled Tumor Protein (TCTP), Negatively Affects Rapamycin-Induced Autophagy in Post-Diauxic Growth Phase.

Translationally controlled tumor protein (TCTP) is a multifunctional and highly conserved protein from yeast to humans. Recently, its role in non-selective autophagy has been reported with controversial results in mammalian and human cells. Herein we examine the effect of Mmi1, the yeast ortholog of TCTP, on non-selective autophagy in budding yeast Saccharomyces cerevisiae, a well-established model system to monitor autophagy. We induced autophagy by nitrogen starvation or rapamycin addition and measured autophagy by using the Pho8Δ60 and GFP-Atg8 processing assays in WT, mmi1Δ, and in autophagy-deficient strains atg8Δ or atg1Δ. Our results demonstrate that Mmi1 does not affect basal or nitrogen starvation-induced autophagy. However, an increased rapamycin-induced autophagy is detected in mmi1Δ strain when the cells enter the post-diauxic growth phase, and this phenotype can be rescued by inserted wild-type MMI1 gene. Further, the mmi1Δ cells exhibit significantly lower amounts of reactive oxygen species (ROS) in the post-diauxic growth phase compared to WT cells. In summary, our study suggests that Mmi1 negatively affects rapamycin-induced autophagy in the post-diauxic growth phase and supports the role of Mmi1/TCTP as a negative autophagy regulator in eukaryotic cells.

Inhibiting expression of Cxcl9 promotes angiogenesis in MSCs-HUVECs co-culture

The insufficient vascularization is a major challenge in bone tissue engineering, leading to partial necrosis of the implant. Pre-vascularization is a promising way via in vitro cells co-culture strategies using osteogenic cells and vasculogenic cells, and the cross-talk of cells is essential. In the present study, the effect of rat bone-marrow derived mesenchymal stem cells (BMSCs) on angiogenic capability of human umbilical vein endothelial cells (HUVECs) in growth medium (GM) and osteogenic induction medium (OIM) was investigated. It was demonstrated that cells co-cultured in OIM showed high efficiency in osteogenesis but failed to form capillary-like structure while the results of co-culture in GM were the opposite. By comparing the angiogenic capacity of co-cultures under GM and OIM, chemokine (C-X-C motif) ligand 9 (Cxcl9), secreted by BMSCs in OIM, was identified to be an angiostatic factor to counter-regulate vascular endothelial growth factor (VEGF) and prevent its binding to HUVECs, which abrogated angiogenesis of MSCs-ECs co-culture. Moreover, Cxcl9 was proved to suppress the osteogenic differentiation of BMSCs monoculture. The molecular mechanism of Cxcl9 activation in BMSCs involved mTOR/STAT1 signaling pathway. Therefore, blocking this signaling pathway via rapamycin addition resulted in the inhibition of Cxcl9 and improvement of osteogenic differentiation and angiogenic capacity of co-culture in OIM. These results reveal that Cxcl9 is a negative modulator of angiogenesis and osteogenesis, and its inhibition could promote pre-vascularization of bone tissue engineering.

Rapamycin treatment increases hippocampal cell viability in an mTOR-independent manner during exposure to hypoxia mimetic, cobalt chloride

BackgroundCobalt chloride (CoCl2) induces chemical hypoxia through activation of hypoxia-inducible factor-1 alpha (HIF-1α). Mammalian target of rapamycin (mTOR) is a multifaceted protein capable of regulating cell growth, angiogenesis, metabolism, proliferation, and survival. In this study, we tested the efficacy of a well-known mTOR inhibitor, rapamycin, in reducing oxidative damage and increasing cell viability in the mouse hippocampal cell line, HT22, during a CoCl2-simulated hypoxic insult.ResultsCoCl2 caused cell death in a dose-dependent manner and increased protein levels of cleaved caspase-9 and caspase-3. Rapamycin increased viability of HT22 cells exposed to CoCl2 and reduced activation of caspases-9 and -3. Cells exposed to CoCl2 displayed increased reactive oxygen species (ROS) production and hyperpolarization of the mitochondrial membrane, both of which rapamycin successfully blocked. mTOR protein itself, along with its downstream signaling target, phospho-S6 ribosomal protein (pS6), were significantly inhibited with CoCl2 and rapamycin addition did not significantly lower expression further. Rapamycin promoted protein expression of Beclin-1 and increased conversion of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, suggesting an increase in autophagy. Pro-apoptotic protein, Bcl-2 associated × (Bax), exhibited a slight, but significant decrease with rapamycin treatment, while its anti-apoptotic counterpart, B cell lymphoma-2 (Bcl-2), was to a similar degree upregulated. Finally, the protein expression ratio of phosphorylated mitogen-activated protein kinase (pMAPK) to its unphosphorylated form (MAPK) was dramatically increased in rapamycin and CoCl2 co-treated cells.ConclusionsOur results indicate that rapamycin confers protection against CoCl2-simulated hypoxic insults to neuronal cells. This occurs, as suggested by our results, independent of mTOR modification, and rather through stabilization of the mitochondrial membrane with concomitant decreases in ROS production. Additionally, inhibition of caspase-9 and -3 activation and stimulation of protective autophagy reduces cell death, while a decrease in the Bax/Bcl-2 ratio and an increase in pMAPK promotes cell survival during CoCl2 exposure. Together these results demonstrate the therapeutic potential of rapamycin against hypoxic injury and highlight potential pathways mediating the protective effects of rapamycin treatment.

Translocatable voltage-gated Ca2+ channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B-β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

Induced prodrug activation by conditional protein degradation

Enzyme prodrug therapies hold potential as a targeted treatment option for cancer patients. However, off-target effects can be detrimental to patient health and represent a safety concern. This concern can be alleviated by including a failsafe mechanism that can abort the therapy in healthy cells. This feature can be included in enzyme prodrug therapies by use of conditional degradation tags, which degrade the protein unless stabilized. We call this process Degradation-Directed Enzyme Prodrug Therapy (DDEPT). Herein, we use traceless shielding (TShld), a mechanism that degrades a protein of interest unless it is rescued by the addition of rapamycin, to test this concept. We demonstrated that TShld rapidly yielded only native protein products within 1h after rapamycin addition. The rapid protection phenotype of TShld was further adapted to rescue yeast cytosine deaminase, a prodrug converting enzyme. As expected, cell viability was adversely affected only in the presence of both 5-fluorocytosine (5-FC) and rapamycin. We believe that the DDEPT system can be easily combined with other targeting strategies to further increase the safety of prodrug therapies.

PLC‐Dependent Hydrolysis of Phosphatidylinositol 4‐Phosphate is Responsible for the Majority of Total Inositol Phosphate Production and PKC/D Activation Downstream of Multiple GPCRs

Receptor activation of phospholipase C (PLC) is important for many physiological processes, including neurotransmission and cell proliferation. PLC enzymes are 6 families of proteins that hydrolyze the plasma membrane lipid, phosphatidylinositol 4,5‐bisphosphate (PIP2) into inositol trisphosphate and diacylglycerol (DAG). Our group identified a novel pathway in cardiac cells, where G protein coupled receptor (GPCR) stimulation leads to PLC‐dependent hydrolysis of the PIP2‐precursor, phosphatidylinositol 4‐phosphate (PI4P) on intracellular Golgi membranes. We proposed that this localized DAG production was essential for nuclear processes involved in cardiac hypertrophy.Our hypothesis is that receptor‐stimulated PI4P hydrolysis expands beyond cardiac cells as a mechanism for DAG production without calcium signaling. We measured PI4P in various cell types using PI4P specific binding domains fused to GFP and found that PI4P was depleted both at the Golgi and plasma membrane (PM) in response to GPCR‐stimulation. Since PI4P is the precursor of PIP2, one could argue that depletion of PI4P happens to replenish PIP2 used by PLC and not as an actual substrate. To test this, we depleted PIP2 prior to GPCR stimulation. We used the PM‐targeted FRB/FKBP‐5‐phosphatase rapamycin‐induced dimerization system. After rapamycin addition, the PI‐5‐phosphatase is recruited to the PM where it removes the 5 phosphate from the head group of PIP2. As predicted, depletion of PIP2 did not inhibit GPCR‐stimulated PM or Golgi PI4P depletion, total IP production or PKD activation. In contrast, pharmacological inhibition of PI4‐kinases to deplete PI4P specifically at the PM, without affecting PIP2 levels, completely blocked GPCR‐dependent total IP production and PKD activation. These data strongly suggest that PI4P hydrolysis is the major reaction catalyzed by PLCs in terms of phosphoinositide mass and is a key source of DAG for regulation of protein kinases.While we have identified PI4P hydrolysis in various cell types, it is not clear which PLC isoforms catalyze this reaction. PLC epsilon performs this function at the Golgi‐Nuclear Envelope interface, but all PLC isoforms hydrolyze PI4P in vitro and may participate in PI4P hydrolysis in different cell types. To determine the identity of PLC isoforms responsible for PI4P hydrolysis, we used CRISPR technology to knockout individual PLC isoforms and will assess their roles in total IP production and PKD activation. Ultimately, finding which PLC isoforms are involved may provide new and more specific therapeutic targets for diseases.Support or Funding InformationSupport from NIH, RO1 grants GM053536 and GM111735 (A.V.S.) and NRSA predoctoral grant F31 GM116557 (R.G.).

GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy.

The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress.

Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis.

Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi-cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor-mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain-containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions.

Capsule growth in Cryptococcus neoformans is coordinated with cell cycle progression.

ABSTRACTThe fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans.

Rapamycin‐responsiveness and Gln3‐Tor1 interaction are associated with different target sites on the Gln3 protein (609.13)

Nitrogen‐sensitive control is imposed by TorC1‐dependent and ‐independent regulatory pathways directing localization of the transcription activator, Gln3. In nitrogen excess Gln3 is cytoplasmic and Gln3‐mediated transcription is minimal. During nitrogen limitation, or after rapamycin (Rap, a specificTorC1 inhibitor) addition, Gln3 relocates to the nucleus and activates transcription. Alteration of a Gln3 α‐helix656‐662 abolishes Gln3‐Tor1 interaction, Rap‐responsiveness and diminishes cytoplasmic Gln3 sequestration in nitrogen excess. This prompts the question, are these characteristics inextricably linked to the Tor1 interacting site on Gln3? We demonstrate that they are not. Amino acid substitutions throughout Gln3510‐589, remote from the Gln3‐Tor1 interaction site, completely abolish Rap‐responsiveness, but leave cytoplasmic Gln3 sequestration, Gln3‐Tor1 interaction and nitrogen‐responsiveness of Gln3 localization untouched. These data identify a specific Gln3 region required for Rap‐responsiveness and suggest that events triggered by Rap inhibition of TorC1 resulting in nuclear Gln3 localization occur outside of the Gln3 region interacting with Tor1 or responding to nitrogen availability.Grant Funding Source: Supported by NIH GM‐35642

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc(13) localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc(13) localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.

Abstract 1081: Rapamycin is a chemopreventive and chemotherapeutic agent for ras-driven epidermal squamous cell carcinoma: evidence from mouse models.

Abstract Braf inhibitors are clinically important agents for the treatment of advanced melanoma however secondary cutaneous tumors are a common side effect; non-melanoma skin cancers (NMSCs) arise in 15-30% of patients on Braf inhibitor therapy and 60% of these harbor ras mutations. NMSC also occurs in 40% of organ transplant patients receiving immunosuppressive therapy, and switching from a calcineurin inhibitor-containing regimen to rapamycin reduces the incidence. To examine the effect of rapamycin on ras-driven epidermal squamous cell tumors, we treated mutant K-RasLA2 mice with rapamycin or vehicle by intraperitoneal injection and found that rapamycin prevented the development of squamous skin tumors (0/21 vs 6/24; p=0.04), and also rapidly reduced the tumor size. To explore this effect we employed a syngeneic orthotopic grafting model using H-Ras mutant primary murine keratinocytes to determine if rapamycin could hamper the ability of a Braf inhibitor to enhance squamous skin tumor growth. Similar to results in the K-RasLA2 model, rapamycin alone diminished the growth of tumors (size difference; 334.5 vs 22.3mm3; p=0.03), and decreased the volume even more in the presence of a Braf inhibitor (size difference; 699.6 vs 17.6 mm3; p=0.02). Treatment of established tumors with rapamycin resulted in significant tumor shrinkage even in the continuous presence of a Braf inhibitor. Size reduction was 78.7% and 66.7% in Braf inhibitor untreated and treated groups respectively. In vitro, Braf inhibition enhanced mutant H-Ras-induced activation of the Raf-ERK and mTOR pathways in keratinocytes, while rapamycin addition blocked the activation of signaling pathways and decreased cell proliferation. Taken together, rapamycin prevents murine skin tumor development arising from oncogenic mutations in two distinct types of ras gene alleles, and reduced the tumor size by inhibiting downstream oncogenic pathways. Rapamycin may have clinical application as a chemopreventive and therapeutic agent for patients at high risk to develop ras-driven NMSC, including those receiving Braf inhibitors. Citation Format: Hiroshi Kitagawa, Christophe Cataisson, Alessandra Handisurya, Patricia M. Day, Stuart H. Yuspa, Phillip A. Dennis. Rapamycin is a chemopreventive and chemotherapeutic agent for ras-driven epidermal squamous cell carcinoma: evidence from mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1081. doi:10.1158/1538-7445.AM2013-1081

Gln3 Mutations Dissociate Responses to Nitrogen Catabolite Repression and Rapamycin Inhibition of TorC1

The GATA‐family transcription activator, Gln3 responds to the cell's nitrogen requirements and environmental resources. In nitrogen excess (YNB‐Gln medium), Gln3 is sequestered in the cytoplasm and the transcription it mediates is minimal. In contrast, during nitrogen‐limitation (YNB‐Pro), Gln3 relocates to the nucleus and activates transcription of genes required to scavenge poor nitrogen sources; these responses are designated nitrogen catabolite repression (NCR). Since rapamycin (Rap) addition also elicits nuclear Gln3 localization [Rap is an inhibitor of the global regulator TorC1], TorC1 has been thought to be responsible for NCR‐sensitive Gln3 regulation. Accumulating evidence, however, suggests that GATA factor regulation occurs by two separate pathways, one TorC1‐dependent and the other NCR‐sensitive. Here, we identify Gln3 alterations that dissect the individual contributions of these two pathways to overall Gln3 regulation. Gln3 C‐terminal truncations or three Gln3 amino acid substitutions on one face of a putative 11 residue α‐helix completely abolish the response of Gln3 localization to Rap addition, but have no affect on its response to nitrogen limitation. This suggests that the two regulatory systems can be effectively separated. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. NIH GM‐35642.

Nucleotide degradation and ribose salvage in yeast

Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose-derived carbon accumulates as sedoheptulose-7-phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde-3-phosphate. Oxidative stress increases glyceraldehyde-3-phosphate, resulting in rapid consumption of sedoheptulose-7-phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.

The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis inSchizosaccharomyces pombe

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin–separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2–M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1—securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.

Mammalian Target of Rapamycin (mTOR) Regulates Cellular Proliferation and Tumor Growth in Urothelial Carcinoma

Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth.