Sustained Cell Proliferation Research Articles

Unveiling the effect of three-dimensional bioactive fibre mesh scaffolds functionalized with silanol groups on bacteria growth.

The need to replace or repair deteriorating bones and simultaneously prevent the formation of bacteria biofilm without impairing local tissue integration has pushed scientists to look for new designs and processing methods to develop innovative biomaterials. Silicon-based biomaterials, widely studied for application in bone regeneration, have demonstrated antibacterial properties. Herein, the aim of this work is to investigate the potential of the functionalization of biomaterials surfaces with silanol groups to prevent the bacterial biofilm formation. For that, we evaluated the adherence and biofilm formation of Escherichia coli (E. coli, Gram negative) and Staphylococcus aureus (S. aureus, Gram positive) on starch-based scaffolds. Three-dimensional fibre meshes scaffolds were developed by wet-spinning and functionalized with silanol (Si-OH) groups using a calcium silicate solution as a nonsolvent. The functionalization of the scaffolds was confirmed by X-ray photoelectron spectroscopy. The developed scaffolds showed no biocide activity against the bacterial tested, although the colony-forming units (CFU) mL(-1) counts were significant lower between 4 and 12 h of incubation for both bacteria. The adherence of E. coli and S. aureus to the scaffolds was also investigated. After a growth period of 12 h, the SPCL scaffolds functionalized with Si-OH groups showed a reduced bacterial adherence of E. coli and S. aureus. The functionalized scaffolds showed a positive effect in preventing the formation of biofilm in the case of S. aureus, however, in the case of E. coli this was not observed, suggesting that silanol groups may only have a positive effect in preventing the proliferation of gram-positive bacteria. The in vitro biological assessment of the functionalized materials showed that these materials sustained cell proliferation and induced their osteogenic differentiation. The outcome of this work suggests that the presence of Si-OH groups in SPCL scaffolds maintained bactericidal activity against S. aureus. Further research is still needed in order to understand the full antibacterial potential of Si-OH groups. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2189-2199, 2016.

Postnatal Muscle Growth Is Dependent on Satellite Cell Proliferation Which Demonstrates A Specific Requirement for Dietary Protein

Extrauterine growth faltering is a serious problem for premature neonates due in large part to insufficient nutrient intake secondary to physiological immaturity and feeding intolerance. Growth of lean mass is especially compromised in these patients due to poor muscle growth. In neonates, short‐term supplementation with leucine of a reduced protein, but energy adequate, feed promoted muscle protein synthesis by stimulating mTORC1 activity similar to feeding a high protein feed. Whether sustained long‐term leucine supplementation of a protein limiting diet can enhance muscle growth is unknown. Five‐d‐old pigs (n = 8–11/diet) were fed by gastric catheter a milk replacement diet with either a high protein (HP) or restricted protein (RP) content or RP supplemented with leucine to the same level as in the HP diet (RPL); all feeds were isocaloric. Pigs were fed every 4 h for 21 d. BrdU (25 mg/(kg.d)) was given as an i.v. bolus every 12 hrs over 3 d from day 6. After 21 d, longissimus dorsi muscle was collected and muscle mass, BrdU+ myonuclei and sublaminal Pax7+ nuclear frequency in muscle cross‐sections were quantified (>1000 fibers/pig). Average fiber cross‐sectional area (CSA) was correlated (r=0.83, P=0.001) with muscle weight and was lower in RP compared to HP pigs (P=0.01), with no improvement in muscle mass with leucine supplementation. Total myonuclear number/fiber also was lower in RP and RPL compared to HP (P=0.01) and was correlated to CSA (r=0.77; P=0.001), with no difference in nuclear domain size (fiber CSA/myonucleus) among groups. There were 40% fewer Pax7+ nuclei in RP and RPL than HP muscles (P=0.001) and their frequency was predictive of myonuclear number/fiber (r=0.79, P=0.001). RP and RPL muscle on average had 30% fewer BrdU+ myonuclei than HP (P=0.003) muscle; the average number of myonuclei per fiber was correlated to the number of BrdU+ nuclei (r=0.76; P=0.001) and the average ratio was similar among groups. The results demonstrate that in the immature muscle fiber hypertrophy is dependent on sustained satellite cell proliferation and myonuclear accretion. This process is highly sensitive to protein intake even when concurrent energy and leucine intakes are optimized for growth.Support or Funding InformationStudies were funded by NIH grants: HD072891, AR 46308, AR44474; USDA NIFA 2013‐67015‐20438; USDA/ARS/CSREES 6250‐51000‐55

Mir-765 promotes cell proliferation by downregulating INPP4B expression in human hepatocellular carcinoma.

microRNAs (miRNAs) dysregulation is widely involved in cancer progression and contributed to sustained cell proliferation by directly targeting multiple targets. Therefore, better understanding the underlying mechanism of miRNA in carcinogenesis may improve diagnostic and therapeutic strategies for malignancy. In our study, we found that mir-765 is upregulated in both hepatocellular carcinoma (HCC) cell lines and tissues, compared to human normal liver cell line and adjacent non-cancerous tissues, respectively. Overexpression of mir-765 increased HCC cells proliferation and tumorigenicity, whereas inhibition of mir-765 reverses this effect. Furthermore, we demonstrated that INPP4B as a direct target of mir-765 and ectopic expression of mir-765 repressed INPP4B expression, resulting in upregulation of p-AKT, Cyclin D1, and downregulation of p-FOXO3a, p21 expression in HCC. Strikingly, we found that silencing the expression of INPP4B is the essential biological function of miR-765 during HCC cell proliferation. Collectively, our findings reveal that miR-765 is a potential onco-miR that participates in carcinogenesis of human HCC by suppressing INPP4B expression, and might represent a potential therapeutic target for HCC patients.

Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein.

Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells.

Regulation of limited N-terminal proteolysis of APE1 in tumor via acetylation and its role in cell proliferation.

Mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a ubiquitous and multifunctional protein, plays an essential role in the repair of both endogenous and drug-induced DNA damages in the genome. Unlike its E.coli counterpart Xth, mammalian APE1 has a unique N-terminal domain and possesses both DNA damage repair and transcriptional regulatory functions. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the cellular and molecular mechanisms that alter APE1 functions during tumorigenesis are largely unknown. Here, we show the presence of full-length APE1 and N-terminal truncated isoforms of APE1 in tumor tissue samples of various cancer types. However, primary tumor tissue has higher levels of acetylated APE1 (AcAPE1) as well as full-length APE1 compared to adjacent non-tumor tissue. We found that APE1 is proteolytically cleaved by an unknown serine protease at its N-terminus following residue lysine (Lys) Lys6 and/or Lys7 and after Lys27 and Lys31 or Lys32. Acetylation of these Lys residues in APE1 prevents this proteolysis. The N-terminal domain of APE1 and its acetylation are required for modulation of the expression of hundreds of genes. Importantly, we found that AcAPE1 is essential for sustained cell proliferation. Together, our study demonstrates that increased acetylation levels of APE1 in tumor cells inhibit the limited N-terminal proteolysis of APE1 and thereby maintain the functions of APE1 to promote tumor cells' sustained proliferation and survival.

High Expression of Orai1 Enhances Cell Proliferation and is Associated with Poor Prognosis in Human Colorectal Cancer.

Store-operated Ca2+ entry (SOCE) is the predominant Ca2+ influx mechanism in non-excitable cells and regulates a variety of cellular functions. As the essential channel pore-forming protein of SOCE, Orai1 has recently been implicated in carcinogenesis and tumor progression in multiple malignancies. However, the role of Orai1 in colorectal cancer (CRC) has not been investigated. The expression of Orai1 in 21 CRC specimens was examined by immunohistochemistry (IHC) staining, Western blot, and qRT-PCR. The results were compared with paired non-tumor tissues. The expression of Orai1 in additional specimens of 129 CRC patients was examined by IHC staining and the IHC scores were compared with clinicopathological features. After knock-down of the expression of Orai1 in LoVo cells, cell proliferation was examined using a MTT assay and colony formation assay. Flow cytometry was also used to detect apoptosis. The expression of Orai1 was upregulated in human CRC tissues, high expression of Orai1 was closely associated with depth of tumor invasion, lymph node metastasis, and perineural invasion, and patients with high expression of Orai1 had shorter overall survival. Moreover, the expression of Orai1 was also upregulated in CRC cell lines, knockdown of Orai1 inhibited cell proliferation, the effect of growth inhibition was not due to enhanced apoptosis, and stromal interaction molecule1 (STIM1) did not take part in the regulation of CRC cell proliferation. Orai1 is crucial to sustained CRC cell proliferation, high expression of Orai1 is associated with tumor progression and poor prognosis, and Orai1 may be a promising target for prognosis and treatment of CRC.

Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel

An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone.

Mathematical models of cancer metabolism.

Metabolism is essential for life, and its alteration is implicated in multiple human diseases. The transformation from a normal to a cancerous cell requires metabolic changes to fuel the high metabolic demands of cancer cells, including but not limited to cell proliferation and cell migration. In recent years, there have been a number of new discoveries connecting known aberrations in oncogenic and tumour suppressor pathways with metabolic alterations required to sustain cell proliferation and migration. However, an understanding of the selective advantage of these metabolic alterations is still lacking. Here, we review the literature on mathematical models of metabolism, with an emphasis on their contribution to the identification of the selective advantage of metabolic phenotypes that seem otherwise wasteful or accidental. We will show how the molecular hallmarks of cancer can be related to cell proliferation and tissue remodelling, the two major physiological requirements for the development of a multicellular structure. We will cover different areas such as genome-wide gene expression analysis, flux balance models, kinetic models, reaction diffusion models and models of the tumour microenvironment. We will also highlight current challenges and how their resolution will help to achieve a better understanding of cancer metabolism and the metabolic vulnerabilities of cancers.

Organic Pollutants and Telomere Length: A New Facet of Carcinogenesis

Organic Pollutants and Telomere Length: A New Facet of Carcinogenesis

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response.

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

TMOD-14CHARACTERIZING SIGNALING PATHWAYS IN A MOUSE MODEL OF GLIOBLASTOMA

TMOD-14. CHARACTERIZING SIGNALING PATHWAYS IN A MOUSE MODEL OF GLIOBLASTOMA Matthaeus Ware, Kaitlin Thomas, Jessica DePetro, Carmen Binding, Michael Blough, and Gregory Cairncross; Southern Alberta Cancer Research Institute, University of Calgary, Calgary, AB, Canada INTRODUCTION: Glioblastoma (GBM) is a fatal brain cancer, characteristically ‘driven’ by mutations in multiple tyrosine kinase and tumour suppressor pathways. We have developed a new murine model of GBM in which the sequence of molecular events and signaling pathways that underlie human GBM can be explored. In our model, cells from the subventricular zone (SVZ) of p53-/mice become growth factor independent when cultured in serum-free media supplemented with platelet-derived growth factor-AA (PDGF-AA). When these cells are implanted into the brains of immunecompetent p53 wild-type mice they form tumours that closely resemble human GBM. Here, we explored the mechanism of sustained cell proliferation in the transformed state. METHODS: Phospho-receptor tyrosine kinase (RTK) arrays were used to evaluate RTK phosphorylation, non-RTK arrays to examine pathway activation, and PDGFR-a inhibitors to assess the role of PDGFR-a in sustaining cell viability in pre-transformed and transformed cells. RESULTS: PDGFR-a was phosphorylated in pre-transformed SVZ cells and remained phosphorylated in transformed cells proliferating in the absence of exogenous PDGF-AA. An inhibitor of PDGFR-a that blocked PDGF-AA binding decreased the viability of pre-transformed SVZ cells but had no effect on transformed cells. In contrast, Imatinib, an inhibitor of PDGFR-a phosphorylation, blocked the proliferation of both. Downstream proteins, ERK1/2 and WNK1, and the transcription factor, CREB, were activated in transformed cells. CONCLUSION: In a PDGF-AA initiated mouse model of GBM, exogenous growth factor independent proliferation of p53-null SVZ cells and tumorigenicity are associated with persistent phosphorylation (i.e., activation) of PDGFR-a. The finding that transformed cells are insensitive to inhibition by a PDGFR-a blocking antibody, but respond to Imatinib, is consistent with receptor autophosphorylation. These observations may be applicable to human GBM where over-expression of PDGF-AA appears to be an early event in the pathogenesis of proneural and other subtypes of GBM. Neuro-Oncology 17:v226–v229, 2015. doi:10.1093/neuonc/nov237.14 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

SDF-1 liposomes promote sustained cell proliferation in mouse diabetic wounds.

Chronic skin wounds are a common complication of diabetes. When standard wound care fails to heal such wounds, a promising approach consists of using decellularized matrices and other porous scaffold materials to promote the restoration of skin. Proper revascularization is critical for the efficacy of such materials in regenerative medicine. Stromal cell-derived factor-1 (SDF-1) is a chemokine known to play a key role for angiogenesis in ischemic tissues. Herein we developed nanosized SDF-1 liposomes, which were then incorporated into decellularized dermis scaffolds used for skin wound healing applications. SDF-1 peptide associated with liposomes with an efficiency of 80%, and liposomes were easily dispersed throughout the acellular dermis. Acellular dermis spiked with SDF-1 liposomes exhibited more persistent cell proliferation in the dermis, especially in CD31(+) areas, compared to acellular dermis spiked with free SDF-1, which resulted in increased improved wound closure at day 21, and increased granulation tissue thickness at day 28. SDF-1 liposomes may increase the performance of a variety of decellularized matrices used in tissue engineering.

Catherine Pasquier Memorial Award Lecture (shared): Critical role of oxidative and nitrosative stress in pathophysiology: Inflammation, cell injury and cancer

The presence of chronic inflammation associated with sustained cell death and proliferation turnover is a potential tissue environment for cancer development. In this situation, oxidative and nitrosative stress are the major driving force playing a key role either as a preventing or inducer/promoter of tumorigenesis. During inflammatory response, activated macrophages release a great variety of protease that degraded extracellular matrix, inflammatory mediators that plays a relevant role in chemotaxis and cell signaling, as well as oxygen and nitrogen reactive species (ROS and RNS, respectively) actively involved in cell defense through oxidation of essential biological molecules such as DNA, carbohydrates, proteins and lipids. Iron-loosely bound plays a relevant role in promoting oxidative-dependent reactions leading to cell damage. In this sense, we have shown that locally iron overload induced an accumulation of neutrophils with increased NADPH-derived superoxide anion generation in exudates from rats submitted to carrageenan-induced granuloma model. The increase of lipid peroxidation products was associated with the presence of iron-loosely bound, reduction of antioxidant status in inflammatory exudates and blood, as well as induction of hepatic acute phase response. A similar pro-inflammatory and oxidant reaction was observed by intraperitoneal iron-dextran administration in animals with Mycobacterium butyricum-induced adjuvant arthritis. The administration of a diet devoid of polyunsaturated fatty acids reduced the substrate for lipid peroxidation, and consequently a decrease of conjugated diennes and thiobarbituric acid-reactive substances in plasma and hepatic microsomal fraction were observed in animals with adjuvant arthritis. The acute phase response induced by different inflammatory mediators in hepatocytes has been studied in detail. The induction of carrageenan-induced granuloma drastically reduced the expression of drug metabolizing enzymes (DME) (CYP3A1, CYP4A, CYP2B1, CYP2D and CYP2E1) and their related-activities in rat livers. Cytokines (IL-1α, IL-6 and TNF-α) exerted a specific timely-dependent regulation of DME expression (CYP1A1, CYP1A2 and CYP3A4) and secretion of acute phase reactants (albumin, ferritin, fibrinogen and ceruloplasmin) in cultured primary human hepatocytes. Although TNF-α induces acute phase response and liver injury in different experimental settings such as sepsis, we have also shown that TNF-α-dependent induction of nitric oxide synthase type II (NOS-2) hepatic expression during PGE1 pre-administration drastically reduces D-galactosamine (D-GalN)-induced liver injury in rats. The pre-administration of PGE1 also reduced cell death induced by D-GalN in primary culture of human and rat hepatocytes. However, the cytoprotective properties of PGE1 do not involve a reduction of D-GalN-induced ROS production. Nitric oxide (NO) has demonstrated to exert pro- and anti-apoptotic properties according to local concentration, target cell, as well as the presence of other ROS such as superoxide anion generating high reactive peroxynitrite. We have observed that moderate increase of PGE1-dependent NO prevented further potent NF-kB-dependent NOS-2 expression and apoptosis induced by D-GalN in cultured rat hepatocytes. However, the administration of PGE1 at advanced stages of cell injury or high doses of NO donor increased cell death in D-GalN-treated human and rat hepatocytes. The induction of apoptosis by D-GalN was associated with ROS production, mitochondrial dysfunction and post-translation (S-nitrosylation and Tyrosine nitration) modifications of proteins involved in unfolded protein response, enzymatic antioxidants, cell metabolism and death in cultured human hepatocytes. In consequences, the administration of antioxidants such as N-acetylcysteine, Q10 and MnTBAB reduced ROS generation, mitochondrial dysfunction and apoptosis induced by D-GalN and glycochenodeoxycholate (GCDCA) in cultured human hepatocytes. The cytoprotective properties of α-tocopherol appears to mostly related to gene regulation (NOS-2, PPAR-α and carnitine palmitoyl transferase) rather than to its antioxidant activity in D-GalN-induced cell death in hepatocytes. The reduction of cell death by α-tocopherol was also associated with reduction of CYP7A1, Na+-taurocholate co-transporting polypeptide (NTCP), and heme oxygenase-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and Tyrosine nitration, and reduced Taurocholic acid uptake in hepatocytes. The pro-apoptotic properties of high NO donor concentration have been successfully applied to exert antitumoral activity in liver cancer cells. In this sense, the administration of NO donor or NOS-3 overexpression induces p53-dependent cell death receptor expression and apoptosis in hepatoma cell lines. The first generation adenovirus specifically designed to induce NOS-3 overexpression in liver cancer cells induced oxidative and nitrosative stress, DNA damage, p53, cell death receptors expression and their S-nitrosylation, and apoptosis that was correlated to a reduction of cell proliferation and growth of tumors orthotopically implanted in fibrotic livers. The S-nitrosylation of cell death receptors (TNF-α, CD95 and TRAIL-R1) plays a critical role on the shift from extrinsic to intrinsic cell death pathways during antitumoral molecular therapy (Sorafenib Tosylate) in liver cancer cells. In conclusion, new data are emerging that elucidate the extraordinary role that plays ROS- and RNS-dependent posttranslational target modifications in regulating intracellular cell signaling.

Abstract 4369: Rearrangements of the erythropoietin receptor are recurrent in Ph-like acute lymphoblastic leukemia and are sensitive to Jak2 inhibition

Abstract Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a diverse range of genetic alterations that activate cytokine receptor and kinase signaling. We recently identified (Roberts KG, NEJM 2014) rearrangements of the erythropoietin receptor (EPOR) with immunoglobulin heavy (IGH) or kappa (IGK) chain loci in 154 Ph-like ALL. Here, we aimed to investigate their spectrum and recurrence in an extended cohort, as well as their pathogenic role and therapeutic potential in childhood Ph-like ALL. Methods: Whole transcriptome sequencing, real-time quantitative PCR (qPCR) and Sanger sequencing were performed to detect and map the EPOR rearrangements in 307 Ph-like ALL. Wild-type or EPOR rearranged alleles were expressed in IL-3-dependent mouse hematopoietic Ba/F3 cells and IL-7-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6. Proliferation and downstream EPOR signaling were examined in absence or presence of erythropoietin (EPO). Leukemic growth was monitored in C57BL/6 mice transplanted with Arf-/- pre-B cells expressing rearranged EPOR. Luciferase-marked xenografts were established in NOD-SCID-IL2R gamma (NSG) null mice. Signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitors were assessed ex vivo and in vivo. Results: EPOR rearrangements were identified in 8.3% of Ph-like ALL as the result of: i) inversion and cryptic insertion under the promoter region of IGH/IGK; ii) reciprocal translocation; or iii) fusion to 5′ LAIR1. All rearrangements resulted in ectopic overexpression of a C-terminal truncated receptor, confirmed by qPCR, that retained the phosphorylation site required for STAT5 activation, but lacked multiple tyrosine residues required for normal negative regulation. The rearranged alleles were expressed at higher and more prolonged levels than wild-type EPOR in Ba/F3 and Arf-/- mouse pre-B cells, and sustained cell proliferation with increased STAT5 phosphorylation following exogenous EPO stimulation. In vivo, the truncated receptor promoted leukemia development in C57BL/6 mice. Xenografted EPOR-rearranged leukemic cells exhibited high levels of truncated EPOR, STAT5 phosphorylation and ex vivo sensitivity to JAK2 inhibition using ruxolitinib, fedratinib, momelotinib, or pacritinib alone or in combination with daunrobucin, vincristine or dexamethasone (synergistic effect). The efficacy of Jak2 inhibition and the synergistic effect of combining ruxolitinib and dexamethasone were subsequently demonstrated in vivo. Conclusions: EPOR rearrangements are recurrent in Ph-like ALL and result in ectopic expression of truncated EPOR and activation of JAK2-STAT5 signaling pathway, sustaining leukemia development in vivo. Xenografted EPOR-rearranged leukemia cells exhibit ex vivo and in vivo sensitivity to JAK2 inhibitors alone or in combination with chemotherapy. Citation Format: Ilaria Iacobucci, Kathryn G. Roberts, Yongjin Li, Jinghui Zhang, Richard C. Harvey, Debbie Payne-Turner, Marcus Valentine, Kelly McCastlain, John Easton, I-Ming Chen, Michael Rusch, Steven M. Kornblau, Marina Konopleva, Elisabeth Paietta, Jacob M. Rowe, Ching-Hon Pui, Julie M. Gastier-Foster, Shalini Reshmi, Mignon L. Loh, Cheryl Willman, James R. Downing, Stephen P. Hunger, Charles G. Mullighan. Rearrangements of the erythropoietin receptor are recurrent in Ph-like acute lymphoblastic leukemia and are sensitive to Jak2 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2015-4369

Abstract 2095: DEPTOR is a direct NOTCH1 target that regulates proliferation, metabolism and glucocorticoid resistance in T cell leukemia

Abstract DEP-Domain Containing mTOR-Interacting Protein (DEPTOR) is frequently overexpressed in multiple myelomas and required for cell survival. However, its implications in other hematological malignancies and the precise mechanisms of regulation remain largely undefined. Here, we systematically analyzed DEPTOR expression in human acute lymphoblastic leukemia (ALL), a hematological malignancy accounting for 80% of pediatric leukemia. Whereas substantially inhibited in B-ALLs, unexpectedly, expression of DEPTOR is significantly elevated in a subset of T-ALLs where NOTCH1 is aberrantly activated. Mechanistically, we show DEPTOR expression is dependent on NOTCH signaling in T-ALL cell lines and primary specimens, and demonstrate NOTCH1 directly binds to and activates human DEPTOR promoter. DEPTOR depletion in T-ALL cells abolishes proliferation, attenuates glucose metabolism, enhances apoptosis and sensitizes resistant cells to γ-secretase inhibitor or dexamethasone treatments. Conversely, ectopically expressed DEPTOR promotes proliferation and aerobic glycolysis, and confers dexamethasone resistance. DEPTOR functions through AKT-dependent mechanisms to sustain cell proliferation and drug resistance, while the AKT-independent roles of DEPTOR appear to promote glucose metabolism. We therefore define a novel mechanism that, in addition to the previously reported HES1/PTEN-mediated regulation of AKT, NOTCH1 alternatively activates AKT through inducing the downstream target gene DEPTOR. Our findings thereby highlight DEPTOR as a critical mediator in leukemogenesis and suggest DEPTOR expression may serve as a potential biomarker predicting glucocorticoid resistance and targeting DEPTOR may achieve better therapeutic benefits. Citation Format: Yufeng Hu, Hexiu Su, Qi Ye, Zhaojing Wang, Liang Huang, Qian Wang, Shuangyou Liu, Suning Chen, Jianfeng Zhou, Peng Li, Guoliang Qing, Hudan Liu. DEPTOR is a direct NOTCH1 target that regulates proliferation, metabolism and glucocorticoid resistance in T cell leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2095. doi:10.1158/1538-7445.AM2015-2095

The comparison between dual inhibition of mTOR with MAPK and PI3K signaling pathways in KRAS mutant NSCLC cell lines.

KRAS mutations are found in 15-25 % of patients with lung adenocarcinoma, and they lead to constitutive activation of KRAS signaling pathway that results in sustained cell proliferation. Currently, there are no direct anti-KRAS therapies available. Therefore, it is rational to target the downstream molecules of KRAS signaling pathway, which are mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK) and PI3K pathway (PI3K-AKT-mTOR). Here, we examined the inhibition of both these pathways alone and in combination and analyzed the anti-proliferative and apoptotic events in KRAS mutant NSCLC cell lines, A549 and Calu-1. Cytotoxicity was determined by MTT assay after the cells were treated with LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), and RAD001 (mTOR inhibitor) for 24 and 48 h. The expression levels of p-ERK, ERK, AKT, p-AKT, p53, cyclinD1, c-myc, p27(kip1), BAX, BIM, and GAPDH were detected by western blot after 6 and 24 h treatment. Although PI3K/mTOR inhibition is more effective in cytotoxicity in A549 and Calu-1 cells, MEK/mTOR inhibition markedly decreases cell proliferation protein marker expressions. Our data show that combined targeting of MEK and PI3K-AKT with mTOR is a better option than single agents alone for KRAS mutant NSCLC, thus opening the possibility of a beneficial treatment strategy in the future.

Retrospective outcomes in patients with BCR-ABL positive Acute Lymphoid Leukemia without Allogeneic stem cell transplantation at the Houston Methodist

S176 Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a variety of rearrangements and other genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We recently identified recurring rearrangements of the erythropoietin receptor (EPOR) into the immunoglobulin heavy (IGH) or kappa (IGK) chain loci. The goal of this study was to examine the role of these alterations in kinase signaling and lymphoid transformation.Methods:Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of EPO. EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Luciferase-marked xenografts of human EPORrearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases out 154 Ph-like ALL (5.2%) harbored rearrangements of EPOR into either the IGH or IGK loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR, resulting in overexpression of a C-terminal truncated receptor that retains the phosphorylation site required for STAT5 activation, but lacking multiple residues required for negative EPOR regulation. Notably, the location of the truncation sites is similar to those arising from inherited mutations in primary familial congenital polycythemia. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Xenograft leukemic cells from EPOR-rearranged cases exhibited high levels of mutant EPOR on the cell surface, constitutive pSTAT5 and noteworthy ex vivo and in vivo sensitivity to the JAK2 inhibitor ruxolitinib. Conclusions: Rearrangements of truncated EPOR into the IGH/IGK chain loci define a new subgroup of Phlike ALL and unexpectedly implicate EPOR signaling as an important factor influencing B-lymphoid malignancies. Finally, these findings suggest that aberrant EPOR signaling is amenable to JAK-STAT5 inhibition in cases harboring these rearrangements.

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

Metabolomic profile of glycolysis and the pentose phosphate pathway identifies the central role of glucose-6-phosphate dehydrogenase in clear cell-renal cell carcinoma.

The analysis of cancer metabolome has shown that proliferating tumor cells require a large quantities of different nutrients in order to support their high rate of proliferation. In this study we analyzed the metabolic profile of glycolysis and the pentose phosphate pathway (PPP) in human clear cell-renal cell carcinoma (ccRCC) and evaluate the role of these pathways in sustaining cell proliferation, maintenance of NADPH levels, and production of reactive oxygen species (ROS). Metabolomic analysis showed a clear signature of increased glucose uptake and utilization in ccRCC tumor samples. Elevated levels of glucose-6-phosphate dehydrogenase (G6PDH) in association with higher levels of PPP-derived metabolites, suggested a prominent role of this pathway in RCC-associated metabolic alterations. G6PDH inhibition, caused a significant decrease in cancer cell survival, a decrease in NADPH levels, and an increased production of ROS, suggesting that the PPP plays an important role in the regulation of ccRCC redox homeostasis. Patients with high levels of glycolytic enzymes had reduced progression-free and cancer-specific survivals as compared to subjects with low levels. Our data suggest that oncogenic signaling pathways may promote ccRCC through rerouting the sugar metabolism. Blocking the flux through this pathway may serve as a novel therapeutic target.

Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes

Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.