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The receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) pathway is frequently altered in cancer, but the underlying mechanism leading to tumorigenesis by activated mTOR remains less clear. Here we show that mTOR is a positive regulator of Notch signaling in mouse and human cells, acting through induction of the STAT3/p63/Jagged signaling cascade. Furthermore, in response to differential cues from mTOR, we found that Notch served as a molecular switch to shift the balance between cell proliferation and differentiation. We determined that hyperactive mTOR signaling impaired cell differentiation of murine embryonic fibroblasts via potentiation of Notch signaling. Elevated mTOR signaling strongly correlated with enhanced Notch signaling in poorly differentiated but not in well-differentiated human breast cancers. Both human lung lymphangioleiomyomatosis (LAM) and mouse kidney tumors with hyperactive mTOR due to tumor suppressor TSC1 or TSC2 deficiency exhibited enhanced STAT3/p63/Notch signaling. Furthermore, tumorigenic potential of cells with uncontrolled mTOR signaling was suppressed by Notch inhibition. Our data therefore suggest that perturbation of cell differentiation by augmented Notch signaling might be responsible for the underdifferentiated phenotype displayed by certain tumors with an aberrantly activated RTK/PI3K/AKT/mTOR pathway. Additionally, the STAT3/p63/Notch axis may be a useful target for the treatment of cancers exhibiting hyperactive mTOR signaling.

Experiments on the effects of external and internal exhaust gas recirculation (EGR) on combustion and emission performance during a cold start process were investigated in a 135 single-cylinder DI diesel engine. Combustion was improved during the initial ignition cycles by introducing internal or external EGR. The addition of an appropriate amount of internal or external EGR can promote the combustion stability significantly. However, excessive amounts of external EGR could lead to extremely unstable combustion or even misfiring. An appropriate amount of internal or external EGR decreased smoke opacity effectively during a cold start. External EGR reduced NOx emissions effectively while internal EGR led to an increase in NOx emissions due to thermal effects.

Through experiments conducted on a single cylinder direct injection (DI) diesel engine, effects of exhaust gas recirculatoin (EGR) on combustion and emission during cold start were investigated. Combustion of first firing cycle can be promoted significantly by introducing EGR. In experiments, when partially closed choking valve and partially or fully opened EGR valve, peak cylinder pressure of first firing cycle was about 45% higher than that under normal condition without EGR, and the start of combustion (SOC) was also much earlier. EGR also had effects on combustion stability. In the case, which kept 50% or 100% opening of EGR valve (OEV) and kept 100% opening of choking valve (OCV), more stable combustion process was achieved when common rail pressure decreased during cold start. However, excessive amount of EGR led to extreme unstable combustion and even misfiring. Opacity and NO emissions were also analyzed in detail. In the case with maximum EGR, the lowest average opacity, which was less than 4%, was achieved during initial several firing cycles of cold start. But in the later phase, excessive amount of EGR led to a great deal of white smoke emission. NO emission during initial phase of cold start is mainly affected by increase in fuel amount of injection. When combustion became stable gradually, EGR showed significant effect on NO reduction.

This paper focuses on the effects of internal and cooled external exhaust gas recirculation (EGR) on the combustion and emission performance of diesel fuel homogeneous charge compression ignition (HCCI). The use of fuel injection before the top center (TC) of an exhaust stroke and the negative valve overlap (NVO) to form the homogeneous mixture achieves low NOx and smoke emissions HCCI. Internal and external EGR are combined to control the combustion. Internal exhaust gas recirculation (IEGR) benefits to form a homogeneous mixture and reduces smoke emission further, but lower the high load limits of HCCI. Cooled external EGR can delay the start of combustion (SOC) effectively, which is very useful for high cetane fuel (diesel) HCCI because these fuels can easily self-ignited, making the SOC earlier. External EGR can avoid the knock combustion of HCCI at high load, which means it can expand the high load limit. HCCI maintains low smoke emission at various EGR rates and various loads compared with a conventional diesel engine because there are no fuel-rich volumes in the cylinder.

Mechanistic target of rapamycin (mTOR) integrates multiple extracellular and intracellular signals to regulate cell growth and survival. Hyperactivation of mTOR has been observed in various cancers. Regulation of mTOR activity is thus of importance in physiological processes and tumor development. Here, we present pyruvate dehydrogenase kinase 4 (PDK4) as a novel regulator of mTORC1 signaling. mTORC1 activity was augmented with PDK4 overexpression and reduced by PDK4 suppression in various cell lines. Furthermore, PDK4 bound to cAMP-response element-binding protein (CREB) and prevented its degradation. The enhanced CREB consequently transactivated the expression of Ras homolog enriched in brain (RHEB), a direct key activator of mTORC1, independent of AMP-activated protein kinase or tuberous sclerosis complex protein 2. PDK4 potentiated the mTORC1 effectors hypoxia-inducible factor 1α and pyruvate kinase isozymes M2 and promoted aerobic glycolysis (Warburg effect). Knockdown of PDK4 suppressed the tumor development of cancer cells with activated mTORC1. The abundance of PDK4 dictated the responsiveness of cells to the mTOR inhibitor, rapamycin. Combinatory suppression of mTOR and PDK4 exerted synergistic inhibition on cancer cell proliferation. Therefore, PDK4 promotes tumorigenesis through activation of the CREB-RHEB-mTORC1 signaling cascade.

This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1(-/-)) was found. Majority of Tie2-Cre/Tsc1(-/-) embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1(-/-) embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

Metastatic medullary thyroid cancer (MTC) is a rare, but often aggressive, thyroid malignancy with a 5-year survival rate of 28% and few effective therapeutic options. Adoptive T-cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CARTs) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4), which associates with the RET receptor tyrosine kinase, as a putative antigenic target for CAR-based therapy of MTC. Using RNA in situ hybridization (RNAscope) and quantitative RT-PCR, we show that GFRα4 is highly expressed in MTC. Normal tissue expression of GFRα4 mRNA is restricted to parafollicular cells (C-cells) within the thyroid, the normal cell type from which MTC originates, and normal thymus.Based upon the highly restricted expression, we generated two high affinity single chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by selecting a naïve rabbit antibody library by phage display. Second generation CARs bearing the CD137 costimulatory domain were constructed using these GFRα4-specific scFvs. GFRα4-specific CARTs trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to control pre-established TT cell tumors in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CARTs, and support this molecule as a promising target for adoptive T cell immunotherapy and other antibody-based therapy of MTC. Citation Format: Vijay G. Bhoj, Selene Nunez-Cruz, Kenneth Zhou, Dimitrios Arhontoulis, Michael Feldman, Keith Mansfield, Haiyong Peng, Christoph Rader, Don L. Siegel, Michael C. Milone. GDNF family receptor alpha 4 (GFRa4)-targeted adoptive T-cell immunotherapy for medullary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2295.

The protein complex of tuberous sclerosis complex (TSC)1 and TSC2 tumor suppressors is a key negative regulator of mammalian target of rapamycin (mTOR). Hyperactive mTOR signaling due to the loss-of-function of mutations in either TSC1 or TSC2 gene causes TSC, an autosomal dominant disorder featured with benign tumors in multiple organs. As the ubiquitous second messenger calcium (Ca(2+)) regulates various cellular processes involved in tumorigenesis, we explored the potential role of mTOR in modulation of cellular Ca(2+) homeostasis, and in turn the effect of Ca(2+) signaling in TSC-related tumor development. We found that loss of Tsc2 potentiated store-operated Ca(2+) entry (SOCE) in an mTOR complex 1 (mTORC1)-dependent way. The endoplasmic reticulum Ca(2+) sensor, stromal interaction molecule 1 (STIM1), was upregulated in Tsc2-deficient cells, and was suppressed by mTORC1 inhibitor rapamycin. In addition, SOCE repressed AKT1 phosphorylation. Blocking SOCE either by depleting STIM1 or ectopically expressing dominant-negative Orai1 accelerated TSC-related tumor development, likely because of restored AKT1 activity and enhanced tumor angiogenesis. Our data, therefore, suggest that mTORC1 enhancement of store-operated Ca(2+) signaling hinders TSC-related tumor growth through suppression of AKT1 signaling. The augmented SOCE by hyperactive mTORC1-STIM1 cascade may contribute to the benign nature of TSC-related tumors. Application of SOCE agonists could thus be a contraindication for TSC patients. In contrast, SOCE agonists should attenuate mTOR inhibitors-mediated AKT reactivation and consequently potentiate their efficacy in the treatment of the patients with TSC.

Malignant melanoma, characterized by early distant metastasis to the lungs and brain, is a leading cause of mortality related to skin cancer. Cell fusion and the subsequent aneuploidy, commonly observed in melanoma, are associated with poor prognosis. However, the pathological consequences of cell fusion in melanoma remain unknown. Therefore, the present study aims to investigate the pathological consequences of cell fusion in melanoma and the mechanism of melanoma metastasis.Phytohemagglutinin-polyethylene glycol (PHA-PEG) fusion method was developed for the fusion of tumor cells. Melanoma cells were fused through the improved PHA-PEG fusion method and obtained by fluorescence-activated cell sorting. DNA content was analyzed through flow cytometry. Cell proliferation rate was detected by cell culture in vitro, and the cell number was counted daily. To detect the tumor growth rate in vivo, cells were injected subcutaneously and the tumor volumes were measured using a vernier caliper. To analyze the tumor metastatic potential, cells were injected intravenously, and the collected lung-metastasis samples were weighed by an electronic balance and the surface nodules were counted.We established an improved phytohemagglutinin-polyethylene glycol fusion method and successfully obtained stable melanoma tumor-tumor cell fusion hybrids. Cell size, DNA content, and chromosome numbers of the fusion hybrids were approximately twice those of the parents. The metastatic potential of the fusion hybrids was dramatically enhanced, in contrast to their proliferation rate. Their metastasis was specific to the lungs.We developed a highly efficient cell fusion method that can be applied in many fields, particularly cancer research. Our study has proven that tumor-tumor cell fusion hybrids in melanoma can acquire enhanced and specific metastatic potential. Thus, blockage of cell fusion may be a new strategy for melanoma metastasis therapy.

Background & Aims Levels of the Golgi protein 73 (GP73) increase during development of hepatocellular carcinoma (HCC); GP73 is a serum marker for HCC. However, little is known about the mechanisms or effects of GP73 during hepatic carcinogenesis. Methods GP73 was overexpressed from a retroviral vector in HepG2 cells, which were analyzed in proliferation and migration assays. Xenograft tumors were grown from these cells in nude mice. The effects of monoclonal antibodies against GP73 were studied in mice and cell lines. GP73 −/– , GP73 +/− , and GP73 +/+ mice were given injections of diethylnitrosamine to induce liver injury. Levels of GP73 were reduced in MHCC97H, HCCLM3, and HepG2.215 cell lines using small hairpin RNAs; xenograft tumors were grown in mice from MHCC97H–small hairpin GP73 or MHCC97H-vector cells. We used microarray analysis to compare expression patterns between GP73-knockdown and control MHCC97H cells. We studied the effects of the mechanistic target of rapamycin (mTOR) inhibitor rapamycin on GP73 expression in different cancer cell lines and on growth of tumors in mice. Levels of GP73 and activated mTOR were quantified in human HCC tissues. Results Xenograft tumors grown from HepG2 cells that expressed GP73 formed more rapidly and more metastases than control HepG2 cells in mice. A monoclonal antibody against GP73 reduced proliferation of HepG2 cells and growth of xenograft tumors in mice. GP73 −/– mice had less liver damage after administration of diethylnitrosamine than GP73 +/− or GP73 +/+ mice. In phosphatase and tensin homolog–null mouse embryonic fibroblasts with constitutively activated mTOR, GP73 was up-regulated compared with control mouse embryonic fibroblasts; this increase was reversed after incubation with rapamycin. Expression of GP73 also was reduced in HCC and other cancer cell lines incubated with rapamycin. mTORC1 appeared to regulate expression of GP73 in cell lines. Activated mTOR correlated with the level of GP73 in human HCC tissues. Injection of rapamycin slowed the growth of xenograft tumors from MHCC97H-vector cells, compared with MHCC97H–short hairpin GP73 cells. Conclusions Increased expression of GP73 promotes proliferation and migration of HCC cell lines and growth of xenograft tumors in mice. mTORC1 regulates the expression of GP73, so GP73 up-regulation can be blocked with rapamycin. mTOR inhibitors or other reagents that reduce the level or activity of GP73 might be developed for the treatment of HCC.

Background & Aims Aberrant activation of the signal transducer and activator of transcription (Stat)3 and overexpression of polo-like kinase (PLK)1 each have been associated with cancer pathogenesis. The mechanisms and significance of dysregulation of Stat3 and PLK1 in carcinogenesis and cancer progression are unclear. We investigated the relationship between Stat3 and PLK1 and the effects of their dysregulation in esophageal squamous cell carcinoma (ESCC) cells. Methods We used immunoblot, quantitative reverse-transcription polymerase chain reaction, immunochemistry, chromatin immunoprecipitation, mobility shift, and reporter assays to investigate the relationship between Stat3 and PLK1. We used colony formation, fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling, and xenograft tumor assays to determine the effects of increased activation of Stat3 and PLK1 in proliferation and survival of ESCC cells. Results Stat3 directly activated transcription of PLK1 in esophageal cancer cells and mouse embryonic fibroblast cell NIH3T3. PLK1 then potentiated the expression of Stat3; β-catenin was involved in PLK1-dependent transcriptional activation of Stat3 . This mutual regulation between Stat3 and PLK1 was required for proliferation of esophageal cancer cells and resistance to apoptosis in culture and as tumor xenografts in mice. Furthermore, phosphorylation of Stat3 and overexpression of PLK1 were correlated in a subset of ESCC. Conclusions Stat3 and PLK1 control each other's transcription in a positive feedback loop that contributes to the development of ESCC. Increased activity of Stat3 and overexpression of PLK1 promote survival and proliferation of ESCC cells in culture and in mice.

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe discovery of non-peptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used for discovery of other classes of antigen surrogates.