Rapamycin-induced Apoptosis Research Articles

Effect of resveratrol combined with atorvastatin on re-endothelialization after drug-eluting stents implantation and the underlying mechanism

AimsTo explore whether the combination of atorvastatins and resveratrol is superior to each individual drug alone regarding re-endothelialization after drug-eluting stents (DESs) implantation. Materials and methodsNinety-four rabbits were randomized into control, atorvastatin, resveratrol, and combined medication groups. Abdominal aorta injury was induced via ballooning, followed by DES implantation. Neointimal formation and re-endothelialization after stent implantation were assessed via optical coherence tomography and scanning electron microscopy. The effects of resveratrol and atorvastatin on bone marrow-derived mesenchymal derived stem cells (BMSCs) were assessed. Key findingsCompared with the findings in the resveratrol and atorvastatin groups, the neointimal area and mean neointimal thickness were greater in the combined medication group, which also exhibited improved re-endothelialization. Compared with the effects of monotherapy, combined treatment further protected BMSCs against rapamycin-induced apoptosis and improved cell migration. Combined medication significantly upregulated Akt, p-Akt, eNOS, p-eNOS, and CXCR4 expression in BMSCs compared with the effects of monotherapy, and these effects were abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. SignificanceThe combination of atorvastatin and resveratrol has the potential of accelerating re-endothelialization after stent implantation, reducing the risk of thrombosis and improving the safety of DESs.

Mitochondrial dysfunction contributes to Rapamycin-induced apoptosis of Human Glioblastoma Cells - A synergistic effect with Temozolomide.

Mammalian target of rapamycin (mTOR) is upregulated in a high percentage of glioblastomas. While a well-known mTOR inhibitor, rapamycin, has been shown to reduce glioblastoma survival, the role of mitochondria in achieving this therapeutic effect is less well known. Here, we examined mitochondrial dysfunction mechanisms that occur with the suppression of mTOR signaling. We found that, along with increased apoptosis, and a reduction in transformative potential, rapamycin treatment significantly affected mitochondrial health. Specifically, increased production of reactive oxygen species (ROS), depolarization of the mitochondrial membrane potential (MMP), and altered mitochondrial dynamics were observed. Furthermore, we verified the therapeutic potential of rapamycin-induced mitochondrial dysfunction through co-treatment with temzolomide (TMZ), the current standard of care for glioblastoma. Together these results demonstrate that the mitochondria remain a promising target for therapeutic intervention against human glioblastoma and that TMZ and rapamycin have a synergistic effect in suppressing glioblastoma viability, enhancing ROS production, and depolarizing MMP.

Rapamycin Induces Apoptosis of K562 Cells through EZH2/Hedgehog Signaling Pathway

To investigate the mechanism of rapamycin-induced apoptosis of chronic myelogenous leukemia cells. The chronic granulocytic leukemia K562 cells were divided into 3 groups: A, B and C group were treated with rapamycin of 10, 15 and 20 nmol/L, repectively for 24 h, while the K562 cells in control group were not treated with rapamycin. The effect of rapamycin on the proliferation of K562 cells was detected by MTT, and the effect of rapamycin on the apoptosis of K562 cells was detected by AnnexinV-FITC/PI double staining. The expression level of EZH2/Hedgehog signaling pathway genes in K562 cells was detected by RT-PCR, and Western blot was used to detect the levels of apoptotic protein and the related signaling pathway proteins in K562 cells. The MTT assay showed that the different concentration of rapamycin had obvious inhibitory effects on the cells, and the survival rate of cells in group C was 37.6%±3.4%, which was significantly lower than that of the other groups (P＜0.05). The apoptosis rate of cells in group C was 93.1%±8.1%, which was significantly higher than that of the other groups (P＜0.05). By Western blot, it was found that the relative expression levels of Caspase-3 and BAX protein in group C were 0.36 ± 0.04 and 0.39±0.06, respectively, which were significantly higher than those in other groups (P＜0.05), and the level of BCL-2 protein was 0.17±0.03, which was significantly lower than that of other groups (P＜0.05). By RT-PCR, it was found that the mRNA levels of EZH2 and Hedgehog genes in A, B and C groups were significantly lower than those in the control group (P＜0.05), but mRNA level of Ptch1 gene was significantly higher than that of the control (P＜0.05). By Western blot, it was found that the expression levels of EZH2 and Hedgehog protein in A, B and C groups were significantly lower than that in the control group (P＜0.05), but the level of Ptch1 protein was higher than that of the control (P＜0.05). The relative levels of EZH2 and Hedgehog protein in group C were 0.21 ±0.03 and 0.16±0.05 respectively, which were significantly lower than those in other groups (P＜0.05), and Ptch1 protein level were 0.46 ±0.06, significantly higher than that of other groups (P＜0.05). Rapamycin can inhibit the protein expression of EZH2 in leukemic cells, thus interfere with the activation of Hedgehog signaling pathway, promote the expression of apoptotic protein, reduce the level of anti apoptotic protein, and eventually induce apoptosis of leukemia cells.

Chloroquine Enhances Rapamycin-induced Apoptosis in MG63 Cells

We previously showed that the use of autophagy inhibitors in combination with chemotherapy can enhance anticancer effects in sarcoma cell lines. In this study, we investigated the combined effect of the autophagy inhibitor chloroquine and the mTOR inhibitor rapamycin on MG63 osteosarcoma cells. Effects of chloroquine and/or rapamycin on cell proliferation were assessed by WST-1 assays. Effects of chloroquine and/or rapamycin on the mTOR pathway components, autophagy, and apoptosis were investigated by western blot, flow cytometry, and fluorescence microscopy using immunocytochemical staining of LC3 and Annexin V-FITC/propidium iodide. Rapamycin suppressed cell growth and inhibited the mTOR pathway. Rapamycin promoted autophagy by blocking the mTOR pathway, and chloroquine enhanced apoptosis by blocking autophagy. Chloroquine enhances the effects of rapamycin in inducing apoptosis via autophagy inhibition in MG63 cells. Thus, the combined therapy of chloroquine and rapamycin may be a potent treatment for osteosarcoma.

Breast and renal cancer—Derived endothelial colony forming cells share a common gene signature

BackgroundNeovascularisation supports the metastatic switch in many aggressive solid cancers. Tumour neovessels are mostly lined by endothelial cells sprouting from nearby capillaries, but they could also be contributed by circulating endothelial progenitor cells (EPCs). However, scant information is available about tumour-derived EPCs. MethodsWe carried out the first thorough, unbiased comparison of phenotype, function and genotype of normal versus tumour-derived endothelial colony forming cells (ECFCs), a truly endothelial EPC subtype. We used healthy donors–derived ECFCs (N-ECFCs) as control for breast cancer (BC)– and renal cell carcinoma (RCC)–derived ECFCs. ResultsWe found that both BC- and RCC-ECFCs belong to the endothelial lineage. Normal and tumour-derived ECFCs did not differ in terms of proliferative and tubulogenic rates. However, RCC-ECFCs were more resistant to rapamycin-induced apoptosis, whereas BC-ECFCs were more sensitive as compared with N-ECFCs. Gene expression profiling revealed 382 differentially expressed genes (DEGs; 192 upregulated and 150 downregulated) and 71 DEGs (33 upregulated, 38 downregulated) when comparing, respectively, BC- and RCC-ECFCs with N-ECFCs. Nonetheless, BC- and RCC-derived ECFCs shared 35 DEGs, 10 of which were validated by qRT-PCR; such 35 DEGs are organised in a gene network centred on FOS. ConclusionThese results provide the first clear-cut evidence that BC- and RCC-derived ECFCs exhibit an altered gene expression profile as compared with N-ECFCs; yet, they share a common gene signature that could highlight novel and more specific targets to suppress tumour vascularisation.

Regulation of ULK1 Expression and Autophagy by STAT1

Autophagy involves the lysosomal degradation of cytoplasmic contents for regeneration of anabolic substrates during nutritional or inflammatory stress. Its initiation occurs rapidly after inactivation of the protein kinase mammalian target of rapamycin (mTOR) (or mechanistic target of rapamycin), leading to dephosphorylation of Unc-51-like kinase 1 (ULK1) and autophagosome formation. Recent studies indicate that mTOR can, in parallel, regulate the activity of stress transcription factors, including signal transducer and activator of transcription-1 (STAT1). The current study addresses the role of STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. We show that STAT1-deficient human fibrosarcoma cells exhibited enhanced autophagic flux as well as its induction by pharmacological inhibition of mTOR. Consistent with enhanced autophagy initiation, ULK1 mRNA and protein levels were increased in STAT1-deficient cells. By chromatin immunoprecipitation, STAT1 bound a putative regulatory sequence in the ULK1 5'-flanking region, the mutation of which increased ULK1 promoter activity, and rendered it unresponsive to mTOR inhibition. Consistent with an anti-apoptotic effect of autophagy, rapamycin-induced apoptosis and cytotoxicity were blocked in STAT1-deficient cells but restored in cells simultaneously exposed to the autophagy inhibitor ammonium chloride. In vivo, skeletal muscle ULK1 mRNA and protein levels as well as autophagic flux were significantly enhanced in STAT1-deficient mice. These results demonstrate a novel mechanism by which STAT1 negatively regulates ULK1 expression and autophagy.

Synergistic roles of p53 and HIF1α in human renal cell carcinoma-cell apoptosis responding to the inhibition of mTOR and MDM2 signaling pathways.

IntroductionmTOR and MDM2 signaling pathways are frequently deregulated in cancer development, and inhibition of mTOR or MDM2 independently enhances carcinoma-cell apoptosis. However, responses to mTOR and MDM2 antagonists in renal cell carcinoma (RCC) remain unknown.Materials and methodsA498 cells treated with MDM2 antagonist MI-319 and/or mTOR inhibitor rapamycin were employed in the present study. Cell apoptosis and Western blot analysis were performed.Results and conclusionWe found that the MDM2 inhibitor MI-319 induced RCC cell apoptosis mainly dependent on p53 overexpression, while the mTOR antagonist rapamycin promoted RCC cell apoptosis primarily through upregulation of HIF1α expression. Importantly, strong synergistic effects of MI-319 and rapamycin combinations at relatively low concentrations on RCC cell apoptosis were observed. Depletion of p53 or HIF1α impaired both antagonist-elicited apoptoses to differential extents, corresponding to their expression changes responding to chemical treatments, and double knockdown of p53 and HIF1α remarkably hindered MI-319- or rapamycin-induced apoptosis, suggesting that both p53 and HIF1α are involved in MDM2 or mTOR antagonist-induced apoptosis. Collectively, we propose that concurrent activation of p53 and HIF1α may effectively result in cancer-cell apoptosis, and that combined MDM2 antagonists and mTOR inhibitors may be useful in RCC therapy.

Apoptosis and antitumor effects induced by the combination of an mTOR inhibitor and an autophagy inhibitor in human osteosarcoma MG63 cells.

The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy. Although it remains under debate whether chemotherapy-induced autophagy in tumor cells is a protective response or is invoked to promote cell death, recent studies indicate that autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents and that blocking autophagy can trigger apoptosis. The aim of this study was to examine the effects of rapamycin, an mTOR inhibitor, on MG63 osteosarcoma cells. We further examined whether the combination of rapamycin and the small molecule inhibitor of autophagy Spautin-1 (specific and potent autophagy inhibitor-1) enhanced the rapamycin-induced apoptosis in MG63 cells. We examined the effects of rapamycin treatment on cell proliferation, phosphorylation of mTOR pathway components, and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without Spautin-1 on the induction of apoptosis by western blot analysis and immunohistochemical staining. We found that rapamycin inhibited cell proliferation and decreased the phosphorylation of mTOR pathway components in MG63 cells. Rapamycin induced the apoptosis of MG63 cells, and this apoptosis was enhanced by Spautin-1. It was considered that Spautin-1 suppressed the protective mechanism induced by rapamycin in tumor cells and induced apoptosis. Therefore, the combination of an mTOR inhibitor and an autophagy inhibitor may be effective in the treatment of osteosarcoma because it effectively induces the apoptotic pathway.

Protective Effect of Silymarin against Rapamycin-induced Apoptosis and Proliferation Inhibition in Endothelial Progenitor Cells

For this study, peripheral blood samples were collected from human volunteers. Mononuclear cells (MNC) were separated by density centrifugation and were induced to differentiate into endothelial progenitor cells (EPCs) in vitro. Different concentrations of rapamycin and silymarin were introduced to the EPCs over 24 hours and then EPCs were analyzed for proliferation, migration, apoptosis and angiogenesis. Compared with the control group, rapamycin (1, 10, 100 ng/mL) inhibited the proliferation and migration of EPCs in a concentration dependent manner (P<0.05). Silymarin (50, 100 μg/mL) enhanced the proliferation and migration of EPCs and inhibited apoptosis in a concentration dependent manner (P<0.05). By adding rapamycin (1 ng/mL) and silymarin (25, 50, 100 μg/mL) over 24 hours, silymarin inhibited the pro-apoptotic effect of rapamycin on EPCs, and reversed the inhibition of proliferation, migration and angiogenesis of EPCs by rapamycin (P<0.05).

Rapamycin-Induced apoptosis in HGF-stimulated lens epithelial cells by AKT/mTOR, ERK and JAK2/STAT3 pathways.

Hepatocyte growth factor (HGF) induced the proliferation of lens epithelial cells (LECs) and may be a major cause of posterior capsule opacification (PCO), which is the most frequent postoperative complication of cataract surgery. To date, several agents that can block LECs proliferation have been studied, but none have been used in clinic. Recently, accumulating evidence has suggested rapamycin, the inhibitor of mTOR (mammalian target of Rapamycin), was associated with the induction of apoptosis in LECs. The purpose of our study was to investigate the potential effects of rapamycin on HGF-induced LECs and the underlying mechanisms by which rapamycin exerted its actions. Using cell proliferation, cell viability and flow cytometric apoptosis assays, we found that rapamycin potently not only suppressed proliferation but also induced the apoptosis of LECs in a dose-dependent manner under HGF administration. Further investigation of the underlying mechanism using siRNA transfection revealed that rapamycin could promote apoptosis of LECs via inhibiting HGF-induced phosphorylation of AKT/mTOR, ERK and JAK2/STAT3 signaling molecules. Moreover, the forced expression of AKT, ERK and STAT3 could induce a significant suppression of apoptosis in these cells after treatment of rapamycin. Together, these findings suggested that rapamycin-induced apoptosis in HGF-stimulated LECs is accompanied by inhibition of AKT/mTOR, ERK and JAK2/STAT3 pathways, which supports its use to inhibit PCO in preclinical studies and provides theoretical foundation for future possible practice.

The combination of rapamycin and MAPK inhibitors enhances the growth inhibitory effect on Nara-H cells

The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy, and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) is well known to induce autophagy. Autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents, and blocking autophagy can trigger apoptosis. In the present study, we demonstrate that an mTOR inhibitor, rapamycin, induces autophagy in the Nara-H malignant fibrous histiocytoma (MFH) cell line through the activation of ERK1/2. Rapamycin-induced apoptosis was enhanced following the inhibition of the MEK/ERK pathway. In the Nara-H cells, we examined the effects of rapamycin treatment on cell proliferation and on the phosphorylation of the mTOR pathway components and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without the MEK inhibitor, U0126, on the induction of apoptosis by using fluorescence microscopy. Rapamycin inhibited Nara-H cell proliferation and decreased the phosphorylation of the mTOR pathway in the Nara-H cells. Rapamycin induced the apoptosis of Nara-H cells, and this apoptosis was enhanced by U0126. Simultaneously, phospho-ERK1/2 was activated by rapamycin. The present study demonstrates that rapamycin induces autophagy in Nara-H cells by activating the MEK/ERK signaling pathway, and the rapamycin-induced apoptosis can be enhanced by the MEK inhibitor, U0126. These results suggest that self-protective mechanisms involving mTOR inhibitors in Nara-H cells are prevented by the inhibition of the MEK/ERK pathway. The combination of an mTOR inhibitor (e.g., rapamycin) and an MEK inhibitor (e.g., U0126) may offer effective treatment for MFH, as this combination effectively activates apoptotic pathways.

A Unique Herpesviral Transcriptional Program in KSHV-Infected Lymphatic Endothelial Cells Leads to mTORC1 Activation and Rapamycin Sensitivity

Immunosuppression therapy following organ transplantation is a significant factor in the development and progression of Kaposi's sarcoma-associated herpesvirus (KSHV)-induced posttransplant Kaposi's sarcoma (KS). Switching from cyclosporine to the mTOR inhibitor rapamycin is reported to promote KS regression without allograft rejection. Examining the underlying molecular basis for this clinical observation, we find that KSHV infection selectively upregulates mTOR signaling in primary human lymphatic endothelial cells (LECs), but not blood endothelial cells (BECs), and sensitizes LECs to rapamycin-induced apoptosis. Viral transcriptome analysis revealed that while infected BECs display conventional latency, KSHV-infected LECs support a radically different program involving widespread deregulation of both latent and lytic genes. ORF45, a lytic gene selectively expressed in infected LECs, is required for mTOR activation and critical for rapamycin sensitivity. These studies reveal the existence of a unique herpesviral gene expression program corresponding to neither canonical latency nor lytic replication, with important pathogenetic and therapeutic consequences.

Low-Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

Regulatory T cells (Treg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote Treg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human Treg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic Treg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2−/−Il2rg−/− mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4+ but not CD8+ T lymphocytes were sensitive to Treg and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of Treg-based immunosuppressive protocols in clinical practice. By inhibiting TA, Treg and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival

NF1 Inactivation in Adult Acute Myelogenous Leukemia

This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML). We analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for recurrent genomic microdeletions using ultra-high density Affymetrix single nucleotide polymorphism 6.0 array-based genomic profiling. Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of approximately 0.9-Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analysis of all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired truncating frameshift mutations in two patients. These NF1 mutations were already present in the hematopoetic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort using fluorescence-activated cell sorting sorted blasts as a source of RNA identified six patients (one with a NF1 mutation) with absent NF1 expression. The NF1 null states were associated with increased Ras-bound GTP, and short hairpin RNA-mediated NF1 suppression in primary AML blasts with wild-type NF1 facilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike blasts with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mammalian target of rapamycin (mTOR) signaling for survival. Finally, colony formation in methylcellulose ex vivo of NF1 null CD34+/CD38- cells sorted from AML bone marrow samples was inhibited by low-dose rapamycin. NF1 null states are present in 7 of 95 (7%) of adult AML and delineate a disease subset that could be preferentially targeted by Ras or mammalian target of rapamycin-directed therapeutics.

Invasive Glioblastoma Cells Acquire Stemness and Increased Akt Activation

Glioblastoma multiforme (GBM) is the most frequent and most aggressive brain tumor in adults. The dismal prognosis is due to postsurgery recurrences arising from escaped invasive tumor cells. The signaling pathways activated in invasive cells are under investigation, and models are currently designed in search for therapeutic targets. We developed here an in vivo model of human invasive GBM in mouse brain from a GBM cell line with moderate tumorigenicity that allowed simultaneous primary tumor growth and dispersal of tumor cells in the brain parenchyma. This strategy allowed for the first time the isolation and characterization of matched sets of tumor mass (Core) and invasive (Inv) cells. Both cell populations, but more markedly Inv cells, acquired stem cell markers, neurosphere renewal ability, and resistance to rapamycin-induced apoptosis relative to parental cells. The comparative phenotypic analysis between Inv and Core cells showed significantly increased tumorigenicity in vivo and increased invasion with decreased proliferation in vitro for Inv cells. Examination of a large array of signaling pathways revealed extracellular signal-regulated kinase (Erk) down-modulation and Akt activation in Inv cells and an opposite profile in Core cells. Akt activation correlated with the increased tumorigenicity, stemness, and invasiveness, whereas Erk activation correlated with the proliferation of the cells. These results underscore complementary roles of the Erk and Akt pathways for GBM proliferation and dispersal and raise important implications for a concurrent inhibitory therapy.

Abstract 3059: The Tmeff2 protein functions as a tumor suppressor and interacts with Sarcosine dehydrogenase

Abstract Tmeff2 is a gene with an expression profile limited to the brain and the prostate but shown to be frequently upregulated in prostate cancer. It encodes a type I transmembrane protein with a short cytoplasmic tail containing a potential G-protein activation domain and an extracellular domain containing two follistatin (FS) modules and an epidermal growth factor-like (EGF) domain. Although these features are suggestive of a function in signal transduction, the role of the Tmeff2 in prostate cancer, or the mechanism(s) that lead to its overexpression during the disease are unclear. In the present study, we evaluated the functional role of Tmeff2 in prostate tumorigenesis. To investigate the function of the gene, Tmeff2 was overexpressed in TRAMP C1 murine prostate cancer cells and HEK293T cells, and the effects of overexpression on cellular growth and tumorigenicity were assessed. Tmeff2 overexpression in HEK293T cells almost completely abolished the ability of these cells to form colonies in soft agar and resulted in a 20-30% reduction in proliferation rates in these and TRAMP C1 cells, indicating that the gene may function as a tumor suppressor in prostate cancer cells. Moreover, although TMEFF2 overexpression alone did not induce apoptosis, it sensitized these cell lines to staurosporine or rapamycin-induced apoptosis, causing nearly a three-fold increase in the percentage of apoptotic cells. In order to assess the molecular mechanism(s) by which Tmeff2 may suppress cellular growth and tumorigenicity, we screened Tmeff2 affinity complexes by mass spectrometry to search for candidate functional partner(s) of Tmeff2. Our results showed an interaction with Sarcosine Dehydrogenase (SARDH) an enzyme involved in the metabolism of sarcosine. This interaction was further confirmed by co-immunoprecipitation analysis. Importantly, sarcosine has recently been described as a biomarker for prostate cancer as it was shown to be highly increased in prostate and metastatic prostate cancer. In addition, it may also play a role in the biology of the cancer since it promotes cell invasion when added to benign prostate epithelial cells. In summary, the results presented here suggest that Tmeff2 functions as a tumor suppressor for prostate cancer. Its interaction with SARDH may represent a mechanism by which Tmeff2 modulates tumorigenicity by altering the levels of sarcosine. Further analyses are necessary to determine the value of Tmeff2 as a prognostic marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3059.

Protection from Rapamycin-Induced Apoptosis by Insulin-Like Growth Factor-I Is Partially Dependent on Protein Kinase C Signaling

Rapamycin-induced apoptosis in sarcoma cells is inhibited by insulin-like growth factor-I (IGF-I) through a signaling pathway independent of Ras-extracellular signal-regulated kinase 1/2 and Akt. IGF-I induces Bad phosphorylation (Ser112, Ser136, and Ser155) in a pathway involving phosphoinositide 3' kinase (PI3K) and protein kinase C (PKC; mu, epsilon, or theta) resulting in sequestering Bad from mitochondria and subsequently interacting with 14-3-3gamma in the cytosol. Gene knockdown of Bad, Bid, Akt1, Akt2, PKC-mu, PKC-epsilon, or PKC-theta was achieved by transient transfection using small interfering RNAs. Results indicate that IGF-I signaling to Bad requires activation of PI3K and PKC (mu, theta, epsilon) but not mTOR, Ras-extracellular signal-regulated kinase 1/2, protein kinase A, or p90(RSK). Wortmannin blocked the phosphorylation of PKC-mu (Ser744/Ser748), suggesting that PI3K is required for the activation of PKCs. PKCs phosphorylate Bad under in vitro conditions, and the association of phosphorylated Bad with PKC-mu or PKC-epsilon, as shown by immunoprecipitation, indicated direct involvement of PKCs in Bad phosphorylation. To confirm these results, cells overexpressing pEGFP-N1, wt-Bad, or Bad with a single site mutated (Ser112Ala; Ser136Ala; Ser155Ala), two sites mutated (Ser(112/136)Ala; Ser(112/155)Ala; Ser(136/155)Ala), or the triple mutant were tested. IGF-I protected completely against rapamycin-induced apoptosis in cells overexpressing wt-Bad and mutants having either one or two sites of phosphorylation mutated. Knockdown of Bid using small interfering RNA showed that Bid is not required for rapamycin-induced cell death. Collectively, these data suggest that IGF-I-induced phosphorylation of Bad at multiple sites via a pathway involving PI3K and PKCs is important for protecting sarcoma cells from rapamycin-induced apoptosis.

Mtor and PI3K Inhibitors Block Myeloma Cell Growth in a Synergistic Manner.

Abstract 1843Poster Board I-869Introduction: The mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signaling pathways. In multiple myeloma, mToR is involved in the phosphoinositide-3-kinase (PI3K)-AKT pathway which can be activated by the loss of the tumor suppressor phosphatase and tensin homolog (PTEN) or by stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). Inhibitors of the mToR pathway (sirolimus/rapamycin, everolimus and temsirolimus) are approved for immunosuppression and/or cancer treatment. However, the clinical activity of mToR inhibitors may be limited by the fact that, after inhibition of the rapamycin-sensitive mToR-Raptor complex, AKT is activated by the rapamycin-insensitive mToR-Rictor complex. In this regard, the inhibitory effect of mToR inhibitors was evaluated in combination with PI3K inhibitors in vitro and in vivo. Results: Rapamycin and everolimus induced a dose-dependent growth inhibition in six human malignant plasma cell lines. Growth inhibition was mediated by G1 cell cycle arrest and in a subset of cell lines by induction of apoptosis. Overexpression of Bcl-XL or Mcl-1 proteins did not prevent from apoptosis induction by mToR inhibitors, nor did sensitivity to rapamycin-induced apoptosis correlate with the p53 mutation status. In the INA-6 SCID mouse xenograft model, treatment with rapamycin resulted in a significant survival benefit compared to untreated mice. Six out of 14 rapamycin treated mice did not develop plasmacytomas during the observation period of 149 days. Remarkably, short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the plasma cell tumor. Explanted tissue showed apoptotic plasma cells, a finding confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase (PARP). The combination of rapamycin and the PI3K inhibitor Ly294002 led to an increase of growth inhibition in all tested plasma cell lines. The additional growth inhibition by Ly294002 appeared to be due to AKT activation upon mToR inhibition by the rapamycin-insensitive Rictor complex, indicated by increased AKT phosphorylation at Ser473 as determined by Western blot analysis. Conclusion: Clinical trials currently evaluate mToR inhibitors for their potential to expand treatment options for myeloma patients. The data presented here suggest that a combination of mToR inhibitors with PI3K inhibitors may lead to additive therapeutic chances. Disclosures:Guenther:Novartis: Consultancy, Research Funding. Gramatzki:Novartis: Consultancy, Research Funding, Speakers Bureau.

Genome-Wide Analysis of Microdeletions and Identification of NF1 Inactivation in Acute Myelogenous Leukemia.

Abstract 165Genomic aberrations are of dominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML). To further our understanding of such aberrations in AML, we analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for subchromosomal copy number changes and allele identities using ultra-high-density Affymetrix SNP 6.0 array-based genomic profiling. A total of 358 somatically acquired copy number changes were detected in 95 AML genomes. We detected 16 losses and 22 gains of entire chromosomes, 285 subchromosomal losses and 35 subchromosomal gains. No recurrent high-level amplifications or recurrent homozygous deletions were identified. Eight of the 34 AML cases (24%) with normal karyotype each had one lesion detected through 6.0 array profiling, all but one of which was less than 4Mb in length.Focusing on microdeletions as potential indicators of the locations of novel tumor suppressor genes or genes with importance to AML biology, we identified 60 deletions that were less than 1 Mb in length and 158 deletions of less than 5 Mb, the vast majority of which were undetectable by conventional cytogenetics.Through fine mapping of microdeletions on 17q, we identified Neurofibromin 1 (NF1) null states due to mutations or absent expression in ∼7% of AML. NF1 mutations were present in the hematopoetic stem cell compartment (CD34+/CD38- cell population) and siRNA-mediated NF1 suppression using recombinant lentiviruses significantly increased colony formation of primary AML blasts in methylcellulose. Further, AML blasts without functional NF1 displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival.As an additional validation of using microdeletions to guide pathogenetic gene discovery, we identified deletions involving RUNX1, IRF8, Core Binding Factor Beta (CBFB) and Casitas B-cell lymphoma B (CBLB), genes known to be altered in AML. IRF8 expression was found to be absent in ∼30% of all AML but sequencing of all coding exons of IRF8 of 48 AML cases did not disclose somatically acquired mutations.In summary, this comprehensive description of subchromosomal copy number changes and microdeletions in adult AML substantially adds to our knowledge of the pathological anatomy of the AML genome and should inform future searches for novel genes with importance to AML biology. Disclosures:Malek:Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival

The mTOR (mammalian target of rapamycin) inhibitor rapamycin caused growth arrest in both androgen-dependent and androgen-independent prostate cancer cells; however, long-term treatment induced resistance to the drug. The aim of this study was to investigate methods that can overcome this resistance. Here, we show that rapamycin treatment stimulated androgen receptor (AR) transcriptional activity, whereas suppression of AR activity with the antiandrogen bicalutamide sensitized androgen-dependent, as well as AR-sensitive androgen-independent prostate cancer cells, to growth inhibition by rapamycin. Further, the combination of rapamycin and bicalutamide, but not the individual drugs, induced significant levels of apoptosis in prostate cancer cells. The net effect of rapamycin is determined by its individual effects on the mTOR complexes mTORC1 (mTOR/raptor/GbetaL) and mTORC2 (mTOR/rictor/sin1/GbetaL). Inhibition of both mTORC1 and mTORC2 by rapamycin-induced apoptosis, whereas rapamycin-stimulation of AR transcriptional activity resulted from the inhibition of mTORC1, but not mTORC2. The effect of rapamycin on AR transcriptional activity was mediated by the phosphorylation of the serine/threonine kinase Akt, which also partially mediated apoptosis induced by rapamycin and bicalutamide. These results indicate the presence of two parallel cell-survival pathways in prostate cancer cells: a strong Akt-independent, but rapamycin-sensitive pathway downstream of mTORC1, and an AR-dependent pathway downstream of mTORC2 and Akt, that is stimulated by mTORC1 inhibition. Thus, the combination of rapamycin and bicalutamide induce apoptosis in prostate cancer cells by simultaneously inhibiting both pathways and hence would be of therapeutic value in prostate cancer treatment.