Rapamycin Resistant Research Articles

Biological Effects of Abdominal Irradiation on Intestinal Barrier Breakdown Identified By Second-Generation Probiotic, LR-IL-22

Toxic effects of total body irradiation and whole abdomen irradiation include breakdown of the intestinal barrier function and bacterial translocation into the abdominal lymphatic and systemic vascular circulations. Therapeutic effects of countermeasures against intestinal radiation damage have relied upon serum assays for bacterial endotoxin and citrulline, biomarkers of relatively late effects in barrier dysfunction. Intestinal barrier breakdown effects on mesenteric lymph nodes and spleen have been incompletely documented. In the present experiments, we gavaged C57BL/6NTac female mice one hour before irradiation (19.75 Gy whole abdomen) with 100 microliters containing 109 second-generation probiotic, Lactobacillus reuteri-Interleukin-22 (LR-IL-22) containing transgenes for both mammalian IL-22 and Rapamycin resistance (Alexander LM, et. al., Applied and Environmental Microbiology 85(10): e02335-18, 2019), LR alone, or sham-gavage. Twenty-four hours later, Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood were collected at serial times after gavage and assayed for growth of Rapamycin-resistant bacteria, and for relative compositions of immunocytes in mesenteric lymph nodes spleen and blood including: CD3, CD19, CD8, CD45, NK1.1, CD25, Foxp3, CD103, and PD-1+ lymphocytes. Compared to non-irradiated, non-gavaged mice, animals receiving LR or LR-IL-22 showed no significant changes in weight or length of the spleen or lymph nodes 24 hr after gavage. There was an increase in PD-1 expression in the spleen of the mice gavaged with LR-IL-22. There were no other significant changes in the composition of immunocytes in abdominal lymph nodes, or spleen and detectable second-generation probiotic growth in these organs. At 24 hr after irradiation, abdominal irradiated mice demonstrated a significant loss of weight and length of the spleen (p < 0.05) as well as decreased lymphocytes in the blood (p < 0.05). Mice gavaged with LR-IL-22 and irradiated to 19.75 Gy had a significant increase in lymphocytes and red blood cells compared to irradiation only (p < 0.05). There was no significant change in the size or weight of the lymph nodes. In the lymph nodes there was an increase in CD8+ cells after irradiation while the LR-IL-22 gavaged mice had an increase in PD-1 expression. There was no detectable growth of Rapamycin resistant bacteria in non-gavaged animals. Mice gavaged with LR-IL-22, but not LR showed decreased biomarkers of intestinal barrier breakdown. Thus, gavage of genetically labeled second-generation probiotics represents a sensitive assay system for irradiation changes in the intestinal barrier function, translocation of bacteria into the abdominal lymphatic system, and therapeutic effect of enteric delivered radiation countermeasures. Supported by U19-AI1068021 Grant. Disclosures No relevant conflicts of interest to declare.

Inhibition of MDM2 Re-Sensitizes Rapamycin Resistant Renal Cancer Cells via the Activation of p53

Background/Aims: Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. Methods: Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. Results: Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. Conclusion: Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs.

Abstract 1496: Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines.

Abstract Activation of the mTOR signaling pathway is common in multiple cancers. Rapamycin has inhibitory effects on solid tumors through inhibition of the mTOR kinase and alteration of downstream signaling. Though rapamycin and its analogs have yielded promising results from in vitro studies in sensitive cell lines, they have generated only a modest effect in clinical application. Epithelial to mesenchymal transition (EMT) in cancer cells involves not only the loss of cell-cell adhesion and acquisition of migratory and invasive properties but also resistance to cell death, senescence, and chemotherapy. We hypothesized that the EMT status of cancer cells regulates their sensitivity to rapamycin. We classified a panel of immortalized cancer cell lines as resistant or sensitive to rapamycin based on their IC50 as determined by Sulforhodamine B (SRB) assay. Using reverse phase proteomic array, we found that EMT markers were differentially expressed in sensitive compared to resistant immortalized cell lines. Rapamycin sensitive (RS) cell lines had increased expression of E-cadherin and N-cadherin while rapamycin resistant (RR) cell lines had increased expression of Smad3 (p<0.02). To validate these findings, we performed western blotting for EMT markers in selected RR and RS cell lines. E-cadherin expression was higher in RS cell lines than RR cell lines; in turn, RR cell lines expressed increased levels of the mesenchymal marker Vimentin. To determine if inducing EMT decreases rapamycin sensitivity, we assessed E-cadherin and Vimentin expression by western blotting in the epithelial, immortalized breast cancer cell line, MCF7, which was transfected with a constitutively-active mutant of Snail, a known EMT driver. Constitutively-active Snail mutant MCF7 demonstrated loss of E-cadherin and gain of Vimentin expression compared to cells transfected with nonfunctional mutant Snail or Wild-type Snail. Furthermore, we found decreased growth inhibition with rapamycin by SRB assay in constitutively-active Snail mutant MCF7 compared to controls (p<0.05). To determine if modulating EMT increases rapamycin sensitivity, we induced MET using ZEB1 siRNA knockdown and miR200b/c transfection in two mesenchymal cancer cell lines_ACHN and MDA-MB-231. Although miR200b/c transfection induced E-cadherin expression by immunoblotting in both cell lines, there was no significant increase in growth inhibition by SRB assay. In contrast, ZEB1 siRNA knockdown partially reversed EMT in these cells, which exhibited increased sensitivity to rapamycin by SRB assay. Most but not all mechanisms of EMT modulation altered rapamycin sensitivity in immortalized cancer cell lines. Future studies to determine the mechanism of EMT modulation that results in increased sensitivity to rapamycin could have important clinical applications for combination therapies utilizing rapamycin and its analogs. Citation Format: Ashley M. Holder, Farrell Adkins, Argun Akcakanat, Huiqin Chen, Kim-Anh Do, Mien-Chie Hung, Funda Meric-Bernstam. Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines. [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 1496. doi:10.1158/1538-7445.AM2013-1496

Abstract 2448: mTOR inhibitor MLN0128 Has antitumor efficacy in cell lines with intrinsic and acquired rapamycin resistance in vitro and in vivo.

Abstract Introduction: The mammalian target of rapamycin (mTOR) pathway plays a central role in cancer biology. mTOR forms two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin and analogues are allosteric mTOR inhibitors that predominantly inhibit mTORC1. Rapamycin analogues have been FDA approved for several cancer types and are in clinical trials for treatment of many others. MLN0128 is a mTOR kinase inhibitor, that inhibit s both mTORC1 and mTORC2. We hypothesized that MLN0128 may have antitumor efficacy in cancer cell lines with intrinsic or acquired rapamycin-resistance. Methods: Twenty immortalized human cancer cell lines representing various cancer types and a range of rapamycin sensitivity were treated with Rapamycin, MLN0128 or vehicle. Sulforhodamine B assay was used to determine rapamycin and MLN0128 half maximal inhibitory concentrations (IC50), which were later confirmed with colony formation assay (CFA). Acquired resistance models were created in BT-474 and MCF-7 cell lines through culturing in progressively increasing doses of rapamycin until sustained growth at supratherapeutic (10uM) doses was achieved. Western blotting was used to characterize PI3K/mTOR signaling pathway expression. Flow cytometry and cell sorting (FACS) was used to detect and quantify apoptosis of Annexin-V-FLUOS stained cancer cell lines. Xenograft models of nu/nu mice were treated with MLN0128 and rapamycin or everolimus for 28 days. Results: Cell lines that were intrinsically rapamycin sensitive as well as those that were intrinsically rapamycin resistant were sensitive to MLN0128. MLN0128 induced cell cycle arrest; Induction of apoptosis or autophagy with MLN0128 was not detected with western blotting and annexin labeling in most cell lines tested. MLN0128 demonstrated greater inhibition of mTORC1 signaling, especially with a decrease in p4E-BP1 across all cell lines. In addition, MLN0128 demonstrated inhibition of mTORC2, with decline in pAKT473 and pAKT450. However a feedback-dependent phosphorylation of AKT S308 was observed. In cell lines with acquired rapamycin resistance, treatment with MLN0128 produced a significantly higher growth inhibition compared to rapamycin. Thus far, we have demonstrated that MLN0128 treatment resulted in increased tumor growth inhibition compared to rapamycin in xenograft models of BT474 cells with acquired rapamycin resistance. Conclusion: Cancer cell lines with intrinsic as well as acquired resistance to rapamycin are responsive to mTOR kinase inhibitor MLN0128. Further investigations are needed to study the efficacy of MLN0128 in clinical trials and to evaluate its potential as a treatment option in cases of resistance to rapalogs. Citation Format: Burhan Hassan, Argun Akcakanat, Takafumi Sangai, Farrell Adkins, Kurt Evans, Gordon B. Mills, Funda Meric-Bernstam. mTOR inhibitor MLN0128 Has antitumor efficacy in cell lines with intrinsic and acquired rapamycin resistance in vitro and in vivo. [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 2448. doi:10.1158/1538-7445.AM2013-2448

Abstract B58: The ATP-competitive mammalian target of rapamycin (mTOR) inhibitors suppress proliferation and induce apoptosis in the acquired rapamycin resistant breast cancer cells

Abstract The mammalian target of rapamycin (mTOR) is the major regulator of cell growth, metabolism and survival. The anti-cancer effect of allosteric mTOR-inhibitors, rapamycin and its analogs (rapalogs), has been tested extensively in different types of cancer cells. However, there is only limited clinical efficacy observed by treatment of these traditional mTOR inhibitors, partly due to the occurring of acquired rapamycin resistance (ARR). Several new mTOR inhibitors have been developed which specifically target to the catalytic sites of mTOR by competing with ATP binding, and are suggested to be more effective in inhibiting mTOR signaling and cell growth. However, their effect and mechanisms in ARR cells are not clear yet. In the present study, we used an in vitro model of acquired rapamycin-resistant (ARR) breast cancer cells to evaluate the efficacy of two major classes of ATP-competitive mTOR inhibitors, PI3K/mTOR dual and mTORC1/mTORC2 inhibitors. We found that ATP-competitive mTOR inhibitors acutely suppress the proliferation and prominently inhibit PI3K/mTOR signaling of ARR breast cancer cells. In contrast to the cytostatic effect of rapamycin, the ATP-competitive mTOR inhibitors induced apoptosis in ARR breast cancer cells. Unexpectedly, one of PI3K/mTOR dual inhibitors we tested, PI-103, only transiently inhibits its proposed signaling targets and the anti-proliferation effects are possibly mediated through the off-target mechanism. Our results support the rationale to use ATP-competitive mTOR inhibitors to treat rapamycin-resistant breast cancer cells and highlight the putative off-target effect of the new generation of mTOR inhibitor on cancer cells.

Abstract 5607: TLE4 is a key modulator of resistance to mTOR pathway specific inhibition in glioblastoma

Abstract Rapamycin, a selective inhibitor of TOR (target of rapamycin), has been used in clinical trials for the treatment of glioma (Cloughesy et al., 2008). However, even though some patients respond temporarily to rapamycin treatment, virtually all recur. The mechanisms underlying this resistance to therapy are unknown. Here, we utilized patient-derived glioblastoma (GBM) neurosphere cultures to examine these mechanisms. GBM cell cultures exposed to chronic rapamycin yield a population of rapamycin resistant cells with an increased malignant phenotype upon removal from treatment, enhanced sphere formation in vitro and enhanced tumorigenesis. We used an unbiased, proteomics-based approach to discover phosphopeptides that were upregulated during rapamycin treatment in vitro. Amongst the peptides that we uncovered was Tle4/Groucho, a global co-repressor and known modulator of several key signaling pathways, including the Wnt and Notch pathways. In an expression microarray, Tle4 was found to be significantly upregulated within chronic rapa treated GBM cells. We show that TLE4 is a key modulator of resistance to mammalian target of rapamycin (mTOR) pathway specific inhibition. While TLE4 knockdown has no clear effect on naive cells, this knockdown promotes sensitivity under chronic rapamycin treatment as evidenced by reduced neurosphere formation. TLE4 mediate rapamycin resistance through activation of the quiescent population. Our results indicate that chronic mTOR specific inhibition imparts TLE4 mediated resistance through both ERK activation and inhibition of mTOR. The identification of this role for TLE4 in rapamycin resistance provides a novel target for mTOR pathway-associated chemotherapeutic agents.

Rapamycin Resistance Is Linked to Defective Regulation of Skp2

The mammalian target of rapamycin (mTOR) plays a role in controlling malignant cellular growth. mTOR inhibitors, including rapamycin (sirolimus), are currently being evaluated in cancer trials. However, a significant number of tumors are rapamycin resistant. In this study, we report that the ability of rapamycin to downregulate Skp2, a subunit of the ubiquitin protein ligase complex, identifies tumors that are sensitive to rapamycin. RNA interference (RNAi)-mediated silencing of Skp2 in human tumor cells increased their sensitivity to rapamycin in vitro and inhibited the growth of tumor xenografts in vivo. Our findings suggest that Skp2 levels are a key determinant of antitumor responses to mTOR inhibitors, highlighting a potentially important pharmacogenomic marker to predict sensitivity to rapamycin as well as Skp2 silencing strategies for therapeutic purposes.

PIK3CA/PTEN Mutations and Akt Activation As Markers of Sensitivity to Allosteric mTOR Inhibitors

We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance. Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed. Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC(50) > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146). PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.

Abstract 727: NF-κB Mediates Resistance to Rapamycin in Neuroblastoma

Abstract Neuroblastoma is the most common extracranial pediatric solid tumor and is the most prevalent malignancy of infancy. We were interested in the potential use of rapamycin for the treatment of neuroblastoma. However, 7/8 of neuroblastoma cell lines were found to be rapamycin resistant in vitro, independently of MYCN status. The sensitive cell line (CHLA90) and 2 of the most resistant cell lines (SKNAS [non-MYCN amplified] and NB1691 [MYCN amplified]) were further analyzed to characterize their response to rapamycin. Electromobility shift assay analysis showed that treatment of resistant cells with rapamycin activated NF-κB, while NF-κB activation was reduced in sensitive cells. Immunoblotting with specific NF-κB antibodies showed that rapamycin induced p50 but not p65 nuclear translocation in resistant cells, suggesting that the NF-κB complex activated by rapamycin represents p50 homodimers. Furthermore, immunocytochemical analysis showed nuclear translocation the p50 protein following treatment with rapamycin. To attempt to overcome rapamycin resistance, we tested the combination of two different NF-κB inhibitors (Velcade, a proteasome inhibitor and BMS-345541, a highly selective IκB kinase inhibitor) with rapamycin. Sub-lethal concentrations that resulted in NF-κB inhibition of both agents were identified and these doses were used in combination with rapamycin treatment. A synergy index (SI) was calculated and in both cell lines significant synergy (SI&amp;lt;0 and 95% confidence interval&amp;lt;0) between rapamycin and the two NF-κB inhibitory agents tested, was observed. Orthotopic neuroblastoma mouse models were established in the retroperitoneal space of SCID mice. Tumors were sized matched into cohorts of 5-10 mice each. Treatment groups included vehicle only, single agent (Velcade: 0.5mg/kg, 3x/w; rapamycin: 5mg/kg, 3x/w) and combination groups. Mice were sacrificed after 2 weeks of treatment and final tumor volumes calculated. In SKNAS, while Velcade and rapamycin individually had only modest effects on tumor volume (Control 6.6±3.2cm3; Velcade 4.5±1.8cm3; Rapamycin 2.6±0.95cm3), combination treatment was significantly more efficacious than either monotherapy (0.99±0.24cm3) (p&amp;lt;0.0002). In NB1691, Velcade has no effect on tumor size (Control 2.68±0.53cm3; Velcade 2.67±0.58cm3) while rapamycin had a modest effect on tumor volume (1.28±0.34cm3). The combination treatment was significantly more efficacious than either monotherapy (0.71±0.15cm3) (p&amp;lt;0.008). A synergy index (SI) was calculated and in both models significant synergy (SI&amp;lt;0 and 95% confidence interval&amp;lt;0) between rapamycin and Velcade was observed. In conclusion, the efficacy of rapamycin treatment of neuroblastoma may be limited by cellular resistance to the cytostatic and cytotoxic effects of rapamycin. However resistance can be overcome by NF-κB inhibitors, which results in significantly synergistic inhibition of tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2011-727

Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells

A high-fat diet and obesity are associated with increased risk of liver cancer. Because increased delivery of NEFA to the liver occurs in these conditions, we investigated the involvement of the unsaturated fatty acid oleate in hepatocarcinoma cell proliferation using human-derived hepatocarcinoma cell lines as model systems. Western blotting, FACS analysis and [(3)H]thymidine incorporation were used to study the signalling pathways and the proliferation of cells cultured for up to 72 h with or without a concentration of oleate considered to be relevant to human pathophysiology (50 μmol/l) or a concentration considered elevated (1 mmol/l). In HepG2 cells, proliferation was increased in the presence of 50 μmol/l oleate, but was decreased at 1 mmol/l. This differential effect was correlated with the activation of the mammalian target of rapamycin complex 1 (mTORC1) and with increased translation of cell cycle regulators. Oleate-mediated mTORC1 activation required phospholipase D activation, which produces phosphatidic acid and is known to render mTORC1 rapamycin resistant. Remarkably, rapamycin resistance was found to affect specifically the mTORC1/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) branch of the mTORC1 pathway in the presence of 50 μmol/l oleate. Furthermore, inhibition of phosphatidic acid production abolished oleate-induced increases in mTORC1 activity and cyclin A production. Importantly, the same differential effects of oleate on mTORC1 activation, cell cycle regulators and rapamycin resistance were found in SK-Hep1 cells. Oleate stimulates mTORC1 activation and rapamycin resistance. We propose that rapamycin-derived mTOR inhibitors are likely to be of limited therapeutic use to restrain hepatic tumour growth, particularly in the context of associated obesity.

MTOR regulates skeletal muscle regeneration in vivo through kinase-dependent and kinase-independent mechanisms

Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.

P70 Ribosomal S6 Kinase Is Required for Airway Smooth Muscle Cell Size Enlargement but Not Increased Contractile Protein Expression

We examined the contribution of p70 ribosomal S6 kinase (p70S6K) to airway smooth muscle hypertrophy, a structural change found in asthma. In human airway smooth muscle cells, transforming growth factor (TGF)-beta, endothelin-1, and cardiotrophin-1 each induced phosphorylation of p70S6K and ribosomal protein S6 while increasing cell size, total protein synthesis, and relative protein abundance of alpha-smooth muscle actin and SM22. Transfection of myocytes with siRNA against either p70S6K or S6, or infection with retrovirus encoding a kinase-dead p70S6K, reduced cell size and protein synthesis but had no effect on contractile protein expression per mg total protein. Infection with a retrovirus encoding a constitutively active, rapamycin-resistant (RR) p70S6K increased cell size but not contractile protein expression. siRNA against S6 decreased cell size in myocytes expressing RR p70S6K. Finally, TGF-beta treatment, but not RR p70S6K expression, increased KCl-induced fractional shortening. Together, these data suggest that p70S6K activation is both required and sufficient for airway smooth muscle cell size enlargement but not contractile protein expression. Further, ribosomal protein S6 is required for p70S6K-mediated cell enlargement. Finally, we have shown for the first time in a functional cell system that p70S6K-mediated myocyte enlargement alone, without preferential contractile protein expression, is insufficient for increased cell shortening.

Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III.

Golgi Manganese Transport Is Required for Rapamycin Signaling in Saccharomyces cerevisiae

The Pmr1 Golgi Ca2+/Mn2+ ATPase negatively regulates target of rapamycin complex (TORC1) signaling, the rapamycin-sensitive TOR complex in Saccharomyces cerevisiae. Since pmr1 causes resistance to rapamycin and tor1 causes hypersensitivity, we looked for genetic interactions of pmr1 with tor1. Deletion of TOR1 restored two wild-type phenotypes. Loss of TOR1 restored the ability of the pmr1 strain to grow on media containing 2 mm MnCl2 and conferred wild type as well as the wild-type sensitivity to rapamycin. Mn2+ additions to media partially suppressed rapamycin resistance of wild type and pmr1 tor1, suggesting that Tor1 and Tor2 are regulated by manganese. We parsed the roles of Ca2+ and Mn2+ transport and the compartments in rapamycin response using separation-of-function mutants available for Pmr1. A strain containing the D53A mutant (Mn2+ transporting) of Pmr1 is rapamycin sensitive, but the Q783A mutant (Ca2+ transporting) strain is rapamycin resistant. Mn2+ transport into the Golgi lumen appears to be required for rapamycin sensitivity. Overexpression of Ca2+ pump SERCA1, Ca2+/H+ antiporter Vcx1, or a Mn2+ transporting mutant of Vcx1 (Vcx1-M1) failed to restore rapamycin sensitivity, and loss of Pmr1 but not other transporters of Ca2+ or Mn2+ results in rapamycin resistance. Overexpression of Ccc1, a Fe2+ and Mn2+ transporter that has been localized to Golgi and the vacuole, does restore rapamycin sensitivity to pmr1Delta. We conclude that Mn2+ in the Golgi inhibits TORC1 signaling.

The Yeast Phosphotyrosyl Phosphatase Activator Is Part of the Tap42–Phosphatase Complexes

Phosphotyrosyl phosphatase activator PTPA is a type 2A phosphatase regulatory protein that possesses an ability to stimulate the phosphotyrosyl phosphatase activity of PP2A in vitro. In yeast Saccharomyces cerevisiae, PTPA is encoded by two related genes, RRD1 and RRD2, whose products are 38 and 37% identical, respectively, to the mammalian PTPA. Inactivation of either gene renders yeast cells rapamycin resistant. In this study, we investigate the mechanism underling rapamycin resistance associated with inactivation of PTPA in yeast. We show that the yeast PTPA is an integral part of the Tap42-phosphatase complexes that act downstream of the Tor proteins, the target of rapamycin. We demonstrate a specific interaction of Rrd1 with the Tap42-Sit4 complex and that of Rrd2 with the Tap42-PP2Ac complex. A small portion of PTPA also is found to be associated with the AC dimeric core of PP2A, but the amount is significantly less than that associated with the Tap42-containing complexes. In addition, our results show that the association of PTPA with Tap42-phosphatase complexes is rapamycin sensitive, and importantly, that rapamycin treatment results in release of the PTPA-phosphatase dimer as a functional phosphatase unit.