C-myc mRNA Research Articles

Synergistic interactions between PLK1 and HDAC inhibitors in non-Hodgkin's lymphoma cells occur in vitro and in vivo and proceed through multiple mechanisms.

Interactions between the polo-like kinase 1 (PLK1) inhibitor volasertib and the histone deacetylase inhibitor (HDACI) belinostat were examined in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells in vitro and in vivo. Exposure of DLBCL cells to very low concentrations of volasertib in combination with belinostat synergistically increased cell death (apoptosis). Similar interactions occurred in GC-, ABC-, double-hit DLBCL cells, MCL cells, bortezomib-resistant cells and primary lymphoma cells. Co-exposure to volasertib/belinostat induced a marked increase in M-phase arrest, phospho-histone H3, mitotic errors, cell death in M-phase, and DNA damage. Belinostat diminished c-Myc mRNA and protein expression, an effect significantly enhanced by volasertib co-exposure. c-Myc knock-down increased DNA damage and cell death in response to volasertib, arguing that c-Myc down-regulation plays a functional role in the lethality of this regimen. Notably, PLK1 knock-down in DLBCL cells significantly increased belinostat-induced M-phase accumulation, phospho-histone H3, γH2AX, and cell death. Co-administration of volasertib and belinostat dramatically reduced tumor growth in an ABC-DLBCL flank model (U2932) and a systemic double-hit lymphoma model (OCI-Ly18), accompanied by a pronounced increase in survival without significant weight loss or other toxicities. Together, these findings indicate that PLK1/HDAC inhibition warrants attention as a therapeutic strategy in NHL.

Inducing goat pluripotent stem cells with four transcription factor mRNAs that activate endogenous promoters

BackgroundTraditional approaches for generating goat pluripotent stem cells (iPSCs) suffer from complexity and low preparation efficiency. Therefore, we tried to derive goat iPSCs with a new method by transfecting exogenous Oct4, Sox2, Klf4 and c-Myc mRNAs into goat embryonic fibroblasts (GEFs), and explore the mechanisms regarding the transcription regulation of the reprogramming factors in goat iPSCs induction.ResultsmRNAs of the four reprogramming factors were transfected into GEFs, and were localized in nucleus with approximately 90% transfection efficiency. After five consecutive transfections, GEFs tended to aggregate by day 10. Clones appeared on day 15–18, and typical embryonic stem cell -like clones formed on day 20. One thousand AKP staining positive clones were achieved in 104 GEFs, with approximately 1.0% induction efficiency. Immunofluorescence staining and qRT-PCR detection of the ESCs markers confirmed the properties of the goat iPSCs. The achieved goat iPSCs could be cultured to 22nd passage, which showed normal karyotype. The goat iPSCs were able to differentiate into embryoid bodies with three germ layers. qRT-PCR and western blot showed activated endogenous pluripotent factors expression in the later phase of mRNA-induced goat iPSCs induction. Epigenetic analysis of the endogenous pluripotent gene Nanog revealed its demethylation status in derived goat iPSCs. Core promoter regions of the four reprogramming factors were determined. Transcription factor binding sites, including Elf-1, AP-2, SP1, C/EBP and MZF1, were identified to be functional in the core promoter regions of these reprogramming genes. Demethylation and deacetylation of the promoters enhanced their transcription activities.ConclusionsWe successfully generated goat iPSCs by transfection of Oct4, Sox2, Klf4 and c-Myc mRNAs into GEFs, which initiated the endogenous reprogramming network and altered the methylation status of pluripotent genes. Core promoter regions and functional transcription binding sites of the four reprogramming genes were identified. Epigenetic regulation was revealed to participate in mRNA induced iPSCs formation. Our study provides a safe and efficient approach for goat. iPSCs generation.

In vitro evaluation of ruthenium complexes for photodynamic therapy

BackgroundPhotodynamic therapy (PDT) is a promising anti-tumor treatment strategy. Photosensitizer is one of the most important components of PDT. In this work, the anticancer activities of PDT mediated by six new ruthenium porphyrin complexes were screened. The mechanisms of the most efficacious candidate were investigated. MethodsPhotocytotoxicity of the six porphyrins was tested. The most promising complex, Rup-03, was further investigated using Geimsa staining, which indirectly detects reactive oxygen species (ROS) and subcellular localization. Mitochondrial membrane potential (MMP), cell apoptosis, DNA fragmentation, c-Myc gene expression, and telomerase activities were also assayed. ResultsRup-03 and Rup-04 had the lowest IC50 values. Rup-03 had an IC50 value of 29.5±2.3μM in HepG2 cells and 59.0±6.1μM in RAW264.7 cells, while Rup-04 had an IC50 value of 40.0±3.8μM in SGC-7901 cells. The complexes also induced cellular morphological changes and impaired cellular ability to scavenge ROS, and accumulated preferentially in mitochondria and endoplasmic reticulum. Rup-03 reduced MMP levels, induced apoptosis, and repressed both c-Myc mRNA expression and telomerase activity in HepG2 cells. ConclusionsAmong six candidates, Rup-03-mediated PDT is most effective against HepG2 and RAW264.7, with a similar efficacy as that of Rup-04-mediated PDT against SGC-7901 cells. Repression of ROS scavenging activities and c-Myc expression, which mediated DNA damage-induced cell apoptosis and repression of telomerase activity, respectively, were found to be involved in the anticancer mechanisms of Rup-03.

Molecular Mechanisms Underlying the Inhibitory Effects of Qingzaojiufei Decoction on Tumor Growth in Lewis Lung Carcinoma.

Objective: Qingzaojiufei decoction (QD) is an empirical herbal formula from traditional Chinese medicine that is used for the treatment of lung-related diseases. However, the effect of QD on the growth of lung tumor cells has not been investigated. The aim of this study was to examine the antitumor activity of QD in Lewis lung carcinomas (LLC) in vivo and in vitro, and to elucidate the underlying mechanisms. Methods: The LLC cells were used to assess the antitumor activity of QD by Cell Counting Kit-8 assay in vitro. In vivo, mice were randomly assigned to 5 groups (n = 10/group): the model control (MC) group was intragastrically administered physiological saline (0.9% NaCl) twice daily from day 2 after tumor implantation for 2 weeks. The QD groups were intragastrically administered QD twice daily from 2 weeks before to 2 weeks after tumor implantation for 4 weeks. The mRNA levels were detected by quantitative polymerase chain reaction, the proteins expression was determined by immunohistochemistry or western blotting. Results: Compared with the model group, QD showed inhibition of proliferation of LLC cells and reductions in tumor weight and proliferating cell nuclear antigen protein expression. Furthermore, QD up-regulated p53 mRNA expression, and downregulated c-myc and Bcl-2 mRNA expression, while MMP-9, VEGF, and VEGFR protein expression was suppressed. Phosphorylated ERK1/2 levels were also reduced by QD in a dose-dependent manner. Conclusion: Our findings suggest that QD inhibited lung tumor growth and proliferation, by activation of tumor suppressor genes, inactivation of oncogenes, suppressing the potential for invasion and metastasis, and attenuating angiogenesis. The ERK/VEGF/MMPs signaling pathways may play an important role in QD-induced inhibition of malignant tumor cell proliferation.

LncRNA, TUG1 regulates the oral squamous cell carcinoma progression possibly via interacting with Wnt/β-catenin signaling

Oral squamous cell carcinoma (OSCC) is one the most common cancer affecting the head and neck region, and the molecular mechanisms underlying OSCC development is largely unknown. Long non-coding RNAs (lncRNAs) are emerging as key regulators in tumor development. The present study aimed to investigate the role of lncRNA, taurine upregulated gene 1 (TUG1) in OSCC development. The mRNA and protein expression levels were determined by qRT-PCR and western blotting; flow cytometry and ELISA experiments were employed to examine the cell apoptosis; CCK-8 assay, MTT assay, colony formation assay, and cell invasion assay was used to determine cell growth, cell proliferation and cell invasion, respectively. qRT-PCR results showed that TUG1 was up-regulated in both OSCC tissues and cell lines. The high expression level of TUG1 was significantly correlated with TNM stage, lymph node metastasis and tumor grade in OSCC patients. CCK-8 assay, MTT assay, colony formation assay, and cell invasion assay results showed that knock-down of TUG1 by siRNA transfection suppressed cell growth, cell proliferation, and cell invasion in OSCC cell lines (Tca8113 and TSCCA). The cell apoptosis was induced in Tca8113 and TSCCA cells transfected with TUG1 siRNA. In addition, knock-down of TUG1 in Tca8113 and TSCCA cells significantly suppressed the mRNA and protein expression levels of β-catenin, cyclin D1, and c-myc. Wnt/β-catenin pathway activator (LiCl) reversed the TUG1 knock-down effect on cell proliferation, cell invasion and cell apoptosis in Tca8113 and TSCCA cells. In summary, knock-down of TUG1 suppressed cell growth, proliferation and invasion, and also induced apoptosis of OSCC possibly via targeting Wnt/β-catenin signaling. Our data suggest that knock-down of TUG1 may represent a novel therapeutic target for the management of OSCC.

A Novel Combination RNAi toward Warburg Effect by Replacement with miR-145 and Silencing of PTBP1 Induces Apoptotic Cell Death in Bladder Cancer Cells.

Bladder cancer is one of the most difficult malignancies to control. We explored the use of a novel RNA-interference method for a driver oncogene regulating cancer specific energy metabolism by the combination treatment with a small interfering RNA (siRNA) and a microRNA. After transfection of T24 and 253JB-V cells with miR-145 and/or siR-PTBP1, we examined the effects of cell growth and gene expression by performing the trypan blue dye exclusion test, Western blot, Hoechst 33342 staining, reverse transcription polymerase chain reaction (RT-PCR), and electron microscopy. The anti-cancer effects of xenograft model mice with miR-145 and/or siR-PTBP1 were then assessed. The combination treatment induced the deeper and longer growth inhibition and reduced the levels of both mRNA and protein expression of c-Myc and polypyrimidine tract-binding protein 1 (PTBP1) more than each single treatment. Notably, the combination treatment not only impaired the cancer specific energy metabolism by inhibiting c-Myc/PTBP1/PKMs axis but also inactivated MAPK/ERK and PI3K/AKT pathways examined in vitro and in vivo. Furthermore, the combination treatment induced apoptosis or autophagy; but, in some cells, apoptotic cell death was accompanied by autophagy, because the condensation of chromatin and many autophagosomes were coexistent. This combination treatment could be a novel RNA-interference strategy through the systemic silencing of the Warburg effect-promoting driver oncogene PTBP1 in bladder cancer cells.

Upregulation of Far Upstream Element-Binding Protein 1 (FUBP1) Promotes Tumor Proliferation and Tumorigenesis of Clear Cell Renal Cell Carcinoma

ObjectiveThe far upstream element (FUSE)-binding protein 1 (FUBP1) is a transactivator of human c-myc proto-oncogene transcription, with important roles in carcinogenesis. However, the expression pattern and potential biological function of FUBP1 in clear cell renal cell carcinoma (ccRCC) is yet to be established.MethodsFUBP1 expression was detected in ccRCC tissues and cell lines by real-time RT-PCR, Western blot analysis, and immunohistochemistry. The correlations of FUBP1 mRNA expression levels with clinicopathological factors were evaluated. The biological function of FUBP1 during tumor cell proliferation was studied by MTS, colony formation, and soft-agar colony formation. The effects of FUBP1 on cell cycle distribution and apoptosis were analyzed by flow cytometry. Western blot analysis was used to identify the potential mechanism of FUBP1 regulating cell cycle and apoptosis.ResultsThe levels of FUBP1 mRNA and protein expression were upregulated in human ccRCC tissues compared with adjacent noncancerous tissues. High levels of FUBP1 mRNA expression were associated with higher tumor stage and tumor size. FUBP1 knockdown inhibited cell proliferation and induced cell cycle arrest and apoptosis. Meanwhile, the expression levels of c-myc and p21 mRNA were correlated with that of FUBP1 mRNA.ConclusionsFUBP1 acts as a potential oncogene in ccRCC and may be considered as a novel biomarker or an attractive treatment target of ccRCC.

SUMO-1 Gene Silencing Inhibits Proliferation and Promotes Apoptosis of Human Gastric Cancer SGC-7901 Cells

Background: It has been reported that blocking small ubiquitin-like modifier (SUMO) conjugation by silencing SUMO gene remarkably decreased tumor growth in vivo. However, few studies have examined the relationship between SUMO gene silencing and gastric cancer (GC). The study aims to explore the effects of SUMO-1 gene silencing on GC cell proliferation and apoptosis. Methods: GC cells were cultured and divided into 5 groups: the blank group (without any transfection or treatment), the empty vector group (transfected with empty vector), the shRNA-SUMO-1-1 group (transfected with shRNA-SUMO-1-1 plasmid), the shRNA-SUMO-1-2 group (transfected with shRNA-SUMO-1-2 plasmid), and the shRNA-SUMO-1-3 group (transfected with shRNA-SUMO-1-3 plasmid). Cell Counting Kit-8 (CCK-8) assay was performed to examine cell proliferation. Annexin V/PI staining combined with flow cytometry were used to detect cell apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to measure the mRNA and protein expressions of SUMO-1, P53, Bcl-2 and c-myc, respectively. Results: SUMO-1 mRNA and protein expressions were decreased after transfecting with shRNA-SUMO-1. Compared with the blank group, the shRNA-SUMO-1-1 group presented a remarkable decreased proliferation of SGC-7901 cells. Significant increase in cell apoptosis rate was observed. Bcl-2, c-myc and P53 expressions were declined after transfecting with shRNA-SUMO plasmid. Conclusion: Our study provided evidence that SUMO-1 gene silencing could decrease proliferation and promote apoptosis in GC cells.

The circadian clock gene PER2 plays an important role in tumor suppression through regulating tumor-associated genes in human oral squamous cell carcinoma.

Low expression of the clock gene PER2 is closely related to carcinogenesis and the development of cancer; however, the mechanism of the low expression of PER2 that led to cell malignant transformation remains unclear. This study used RNA interference (RNAi) technology to silence PER2 in SCC15 human oral squamous cell carcinoma (OSCC) cells. Then it was found that the ability of cancer cell proliferation, migration, and invasion were markedly increased (P<0.05), and the ability of cancer cell apoptosis and the number of cells in G1/G0 phase were markedly reduced (P<0.05) after PER2 knockdown. PER2 knockdown increased the expression of Ki-67, MDM2, c-Myc, Bcl-2, MMP2, and VEGF mRNA (P<0.05), and decreased the expression of p53, Bax, and TIMP-2 mRNA (P<0.05). The invivo experiments also proved that the tumorigenicity of SCC15 cells was significantly enhanced after PER2 silence (P<0.05). Overall, these results show that PER2 through the regulation of the numerous important downstream tumor-related genes, plays a major role in tumor suppression, and it may be a novel molecular target for cancer treatment.

Protective Effect of Curcumin Against Oxidative Stress-Induced Injury in Rats with Parkinson’s Disease Through the Wnt/ β-Catenin Signaling Pathway

Background/Aims: The study aimed to investigate the protective effect of curcumin against oxidative stress-induced injury of Parkinson’s disease (PD) through the Wnt/β-catenin signaling pathway in rats. Methods: The successfully established PD rat models and normal healthy rats were randomly assigned into the 6-hydroxydopamine (6-OHDA), the curcumin (Cur) and the control groups. Immunohistochemistry was used to detect the positive expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and glial fibrillary acidic protein (GFAP). Deutocerebrum primary cells were extracted and classified into the control, 6-OHDA, Cur (5, 10, 15 µmol/L), Dickkopf-1 (DKK-1) and Cur + DKK-1 groups. MTT assays, adhesion tests and TUNEL staining were used to assess cell viability, adhesion and apoptosis, respectively. Western blotting and qRT-PCR were used to examine the protein and mRNA expressions of Wnt3a and β-catenin and the c-myc and cyclinD1 mRNA expressions. Results: TH and DAT expressions in the Cur group were elevated and GFAP was reduced compared with the 6-OHDA group. Curcumin enhanced viability, survival and adhesion and attenuated apoptosis of deutocerebrum primary cells by activating the Wnt/β-catenin signaling pathway. Higher Wnt3a and β-catenin mRNA and protein expressions and c-myc and cyclinD1 mRNA expressions, enhanced superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) contents, decreased malondialdehyde (MDA) content and elevated mitochondrial membrane potential (∆ψm) were found in the 10 and 15 µmol/L Cur groups compared with the 6-OHDA group. However, opposite tendencies were found in the Cur + DKK-1 group compared to the 10 µmol/L Cur group. Conclusion: This study suggests that curcumin could protect against oxidative stress-induced injury in PD rats via the Wnt/β-catenin signaling pathway.

Down-regulation of PINCH-1 Reduces Expression of EGFR, ERK1/2 and c-Myc, and Leads to Loss of Cell Viability in Colon Cancer Cells

Particularly interesting new cysteine-histidine rich protein (PINCH) is related to poor outcome in colorectal cancers. Here, the relationship between PINCH-1 and cell survival in colon cancer cells was analyzed and the signaling pathways regulated by PINCH-1 by using PINCH-1 siRNA. KM12C cells were treated with PINCH-1 siRNA or control siRNA. Cell number was analyzed by crystal violet staining and caspase-3 activity was assessed using a fluorescent substrate. PINCH-1 extra- and intracellular pathways in KM12C cells were investigated, using phospho-kinase/phospho-receptor tyrosine kinase (RTK) antibody arrays. The expression of c-Myc was evaluated by Quantitative real-time PCR (qPCR) and Western blot analysis. Cell number was significantly decreased (P=0.003) and the caspase-3 activity increased (P=0.019) in PINCH-1 depleted KM12C cultures compared to siRNA cultures. In PINCH-1 silenced KM12C cells, the levels of EGFR and ERK1/2 were significantly decreased (P=0.008, P=0.003, respectively) compared to their controls, as were the c-Myc mRNA and protein expressions (P=0.0073, P=0.0002, respectively). Down-regulation of PINCH-1 reduced the cell survival and lowers the levels of EGFR, ERK1/2 and c-Myc in colon cancer cells. PINCH-1 is essential for cell survival, and may be a future target for anticancer therapy.Â

Global and MYC-Specific Translation Is Enhanced in Activated Chronic Lymphocytic Leukemia Cells Carrying NOTCH1 C.7541_7542delct Mutations

Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D’Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay (’Click-iT’ assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF<1% in all cases) or additional genetic lesion other than 13q deletion. CLL cells were incubated with 7.5 μg/ml CpG-ODN 2006 for 24 hours and assays were performed at baseline, 3 and 24 hours. NOTCH1 pathway γ-secretase inhibition was performed with DAPT GSi. Results: The CLLΔCT cohort had higher sIgM levels (range 31-372 MFI, median 81 MFI) and signaling capacity (Fab’2 anti-IgM induced intracellular calcium mobilization sIgM [iCa2+] range 47-54%, median 51) than the CLLWT cohort (sIgM levels range 19-288 MFI, median 47 MFI; IgM iCa2+ range 2-78%, median 25%). Following TLR9-mediated cell activation, the CLLΔCT cohort had sustained NICD (NOTCH1-intracellular cleaved domain) protein accumulation for up to 24 hours and expressed higher NOTCH1 target gene HES1 (hairy enhancer of split) transcript levels than in the CLLWT cohort. These data indicated NOTCH1 canonical pathway sustainment in the CLLΔCT upon activation. Global mRNA translation after 24 hours in the presence of CpG was 11.5 fold higher than that without CpG in the CLLΔCT cohort and only 4 fold higher in the CLLWT cohort, revealing significantly higher levels of translation in CLLΔCT than in CLLWT (p=0.03). CpG-induced global mRNA translation in the CLLWT cohort was similar to that in the CLLΔCT cohort treated with CHX. By using CpG-induced global mRNA translation in the presence of CHX inhibitor as background levels for each group, DAPT GSiat 2.5 to 10 μM showed from 47% to 63% inhibition of the residual CpG-induced global translation in CLLΔCT (p<0.05), but no effect in CLLWT. Remarkably, c-MYC mRNA translation after 3 hour culture with CpG was higher in CLLΔCT than in CLLWT (p= 0.02), and a similar trend was maintained in the cases investigated at 24 hour. Treatment of CLLΔCT cells with DAPT GSi decreased expression of c-MYC in a dose-dependent manner. Conclusion: NOTCH1ΔCT mutations associate with a very aggressive clinical behavior in CLL. These results now indicate that pro-activating mutations of NOTCH1 pathway associate with increased global mRNA translation and c-MYC expression. They highlight a mechanism by which NOTCH1 pathway may induce c-MYC overexpression in CLL, likely leading to increased proliferation and survival. The association of increased NOTCH1 variant allele frequency with sIgM levels and signaling capacity indicate that these mechanisms are predominant in the less anergic subgroup of U-CLL and make NOTCH1 mediated c-MYC translation an attractive target for therapeutic inhibition. DisclosuresSteele:Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.

Inhibition of c-Myc By Apto-253 As an Innovative Therapeutic Approach to Induce Cell Cycle Arrest and Apoptosis in Acute Myeloid Leukemia

Abstract The c-Myc multifunctional transcription factor protein, a product on the c-myc proto-oncogene, contributes to the pathogenesis of many types of human cancers through mechanisms of proliferation, apoptosis, cell cycle progression and cellular senescence. c-Myc is frequently overexpressed in acute myeloid leukemia, yet strategies to effectively modulate c-Myc function do not exist. We evaluated inhibition of c-myc gene expression by APTO-253, a small molecule anticancer agent that is being developed clinically for the treatment of acute myelogenous (myeloid) leukemia (AML) and high risk myelodysplastic syndromes (MDS). We first confirmed that c-Myc mRNA level were significantly higher in AML cell lines as compared to peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. However, the c-Myc expression in AML cells was inhibited by APTO-253 in dose-dependent and time-dependent manners at both the mRNA and protein levels. Likewise, APTO-253 was found to induce AML cell apoptosis in dose-dependent and time-dependent manners as demonstrated by positive Annexin-V staining and increases in cleaved poly (ADP-ribose) polymerase (c-PARP). APTO-253 induced AML cells arrest at G1/G0 phase of cell cycle by increasing p21 expression and decreasing expression of cyclin D3 and cyclin-dependent kinases 4/6 (CDK4/6). For the p53 positive cell lines MV4-11 and EOL-1, p53 was also increased by APTO-253 at early time points (less than 6-hour treatment), suggesting that p53-dependent cell cycle arrest and apoptosis is mechanistically operative as a consequence of treatment with APTO-253. Importantly, we demonstrated that APTO-253 selectively targeted tumor cells but not normal healthy cells, with MV4-11 AML cells and normal PBMCs having IC50s of 0.25±0.03µM and more than 100µM, respectively. Our previous studies (56th ASH abstract #4813) showed that APTO-253 induces the Krüppel-like Factor 4 (KLF4) transcription factor and was effective and well tolerated as a single agent in multiple AML xenograft models without causing bone marrow suppression. Taken together, our results suggested that APTO-253 may serve as an effective and safe agent for AML chemotherapy, and that APTO-253 mechanistically inhibits c-Myc expression in AML cells and subsequently induces cell cycle arrest and apoptosis. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Cells Export c-Myc in Extracellular Vesicles Driving a Proliferation of Immune-Suppressive Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) play a critical role in promoting immune tolerance and disease growth. We have previously shown that MDSCs are expanded in patients with AML and can be induced from healthy donor PBMCs by co-culture with leukemic cells; a mechanism dependent on expression of the MUC1-C oncoprotein. We sought to elucidate the precise mechanism by which MUC1-C signaling mediates the expansion of this immune suppressive population of immature myeloid cells.We have previously demonstrated that AML cells release membrane bound extracellular vesicles, which traffic to co-cultured cells. We hypothesized that AML EVs may mediate the expansion of MDSCs. MOLM-14 and THP-1 AML EVs were isolated using the ExoQuick precipitation technique, and analyzed by flow cytometry, and compared to size standardized beads, demonstrating particles between 200-300nM in diameter. Furthermoreisolated AML EVs were visualised using Transmission Electron Microscopy demonstrating multiple rounded structures measuring 100-200nM in diameter and bound by darkly staining membrane. Subsequently, healthy donor PBMCs were cultured for three days with GFP tagged AML EVs and then quantified for CD33+/HLADR-/CD11b+ MDSCs and HLADR+/CD11c+ antigen presenting myeloid cells by flow cytometry. In the PBMCs co-cultured with EVs, the proportion of MDSCs increased 8-fold, whilst the proportion of HLADR+/CD11c+ antigen presenting myeloid cells decreased by 10 fold (n=3, p<0.05).We subsequently investigated how MUC1-C signaling, necessary for the expansion of MDSCs, might alter AML extracellular vesicles composition. We evaluated AML EVs for the presence of the pro-proliferative oncoprotein c-Myc by immune-blotting, demonstrating that AML cells secrete EVs containing c-Myc, which is abrogated by downregulation of MUC1-C. Furthermore, EVs containing MUC1 and c-myc led to an up-regulation of the c-Myc downstream targets cyclin D2 and cyclin E1 in co-cultured MDSCs, indicating that c-Myc containing EVs may drive MDSC proliferation. Critically, EVs from MUC1-C silenced AML cells failed to elicit this increase in c-Myc and cyclin D2 and E1 expression in EV exposed MDSCs. Interestingly, exposure of MDSCs to AML EVs lead to an increased expression of PD-L1, which was abrogated in EVs from MUC1-C silenced AML cells.We then sought to determine how MUC1 signaling promotes c-Myc signaling in AML. MUC1-C silencing did not alter c-Myc mRNA levels suggesting a post-transcriptional level of regulation. Micro RNAs are small non-encoding RNA molecules involved in post-translational regulation of gene expression. MiR34a, a known p53 inhibitor, has been implicated in regulating the expansion of MDSCs and it is known that tumor cells suppress MiR34a expression as part of their self-protective armoury. Furthermore, MiR34a is a predicted negative regulator of c-Myc, due to a complementary sequence for MiR34a in the c-Myc promoter region. Using qPCR, we have demonstrated that MUC1-C silencing results in increased expression of MiRNA34a. Furthermore, over-expression of MiR34a in AML cells led to a dramatic down-regulation of c-Myc protein expression, and conversely silencing of MiR34a led to a significant upregulation of c-Myc expression, confirming that MiR34a regulates c-Myc expression in AML. To confirm MiR34a as a critical negative regulator of MDSC expansion, MiR34a altered cells were interrogated for their ability to elicit an expansion of MDSCs in co-cultured PBMCs. Overexpression of MiR34a in AML cells partially abrogated their ability to induce MDSCs from co-cultured donor PBMCs. In concert, silencing of MiR34a in MUC1-C silenced AML cells, recapitulated their ability to induce MDSCs in this model.Taken together, this study illustrates a novel role of the MUC1-C and c-Myc oncoproteins in driving MDSC proliferation and MDSC PD-L1 expression. We have demonstrated that AML EVs alter the tumor microenvironment away from antigen presentation capable myeloid cells and towards immature immune suppressive MDSCs. DisclosuresArnason:Gilead: Consultancy. Küfe:Genus Oncology: Equity Ownership. Rosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding.

A Novel BRD4 Inhibitor CA2 Suppresses MM Cell Proliferation in an Orthotopic Myeloma Mouse Model

We previously demonstrated that a novel BRD4 inhibitor, CA2, suppressed MM cell proliferation in vitro in the 57th ASH Annual Meeting. In the present study, we investigated the inhibitory mechanisms of CA2 on myeloma cell proliferation in vitro and in vivo. CA2 has a unique quinolinone core and inhibited the proliferation of MM cell lines, MM.1s, AMO-1, NCI-H929, OPM-2, and IM-9. As expected, CA2 suppressed the expression of c-MYC mRNA and protein in a dose- and time-dependent manner. In addition, CA2 decreased BRD4 binding to c-MYC promoters and c-MYC enhancers in a ChIP assay using IM-9 cells and an anti-BRD4 antibody (Figure 1). Flow cytometric Annexin-V/propodium iodide staining analyses demonstrated that CA2 induced apoptosis in MM cells. Taken together, CA2 may induce apoptosis in MM cells by inhibiting the binding of BRD4 to promoters and enhancers of c-MYC gene thereby suppressing c-MYC expression.We next performed a pharmacokinetic study of CA2 in mice. Half-lives (t1/2) of CA2 following oral administration (50 mg/kg) and subcutaneous administration (10 mg/kg) were approximately 61 min and 139.8 min, respectively. The values of area under the curve for oral and subcutaneous administrations were 179,000 hr·ng/mL and 31,000 hr·ng/mL, respectively. Lastly, we assessed the in vivo effects of CA2 using orthotopic MM mouse model, where IM-9Luc cells were inoculated intravenously through the tail veins of SCID mice that had received 2 Gy of irradiation. The mice in the vehicle-control arm died of MM by approximately 30 days after transplantation; IM-9Luc cells engrafted in many organs including bone marrow of spine and femurs, kidneys, ovaries, and stomach. CA2 treatment consisting of 20 cycles of 100 mg/kg/day b.i.d. oral dosing resulted in reduced luminescence intensity of IM-9Luc cells and prolonged the survival of mice (mean 33.7 days) compared to the vehicle-control arm (mean 27.1 days; p=0.029, Figure 2). In conclusion, CA2 is a BRD4 inhibitor with a novel structure and could be a promising molecular targeting-agent against MM. [Display omitted] [Display omitted] DisclosuresYoshioka:ConverGene LLC: Employment. Kado:Japan Community Health Care Organization Kyoto Kuramaguchi Medical Center: Employment. Strovel:ConverGene LLC: Employment.

357P Targeting dual metabolic pathway specifically killed malignant lymphoma cells

Background The metabolic reprogramming is a remarkably different metabolic character among tumor cells and is linked to higher proliferation, stronger resistance to chemotherapy, more adaptive survival advantages and aggressive tumor progression. Malignant tumor cells prefer glycolysis rather than aerobic oxidation even under aerobic surroundings. Furthermore, this altered metabolism is an active adjustment regulated by PI3K/Akt/mTOR pathway, HIF-1α and c-MYC in a reversible manner. Methods Raji and SU-DHL4 cells were treated with different concentrations of rapamycin or oligomycin alone or the two drugs in combination. Cell proliferation was detected by CCK-8. The mRNA and protein expression levels of oncogene C-myc, hypoxia inducible factor-1a (HIF-1a), as well as key enzymes and proteins related to glycolysis pathway including hexokinase II (HKII), lactic dehydrogenase (LDHA) and succinate dehydrogenase (SDHA) were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Glucose consumption and lactic acid generation were examined by Glucose (hexokinase, HK) Assay Kit and Lactate Assay Kit, respectively. Apoptosis and cell cycle distribution were analyzed by FCM. Results The mTORC1 inhibitor rapamycin (RAPA) and the proteasome inhibitor bortezomib (BTZ) hampered glycolysis in malignant B-cell lymphoma cells successfully. Either glycolysis inhibitors RAPA and BTZ or the aerobic oxidation inhibitor oligomycin (OM) selectively inhibited the proliferation of Raji and SU-DHL4 cells in a dose-dependent manner. In contrast, no difference was observed in normal lymphocytes. Furthermore, rapamycin and oligomycin combined with doxorubicin or bortezomib in combination with oligomycin, indicating attenuating both the glycolysis and the aerobic oxidation pathway at the same time, synergistically inhibited cell proliferation and induced cell apoptosis, and thus increased the sensitivity of malignant B-cell lymphoma cells toward doxorubicin and bortezomib, via blocking the dual metabolism. Synergistically inhibition on mRNAs and proteins (HIF-1a, HKII, LDHA, SDHA) was involved. Conclusions Inhibiting the dual metabolic pathway may provide a novel strategy to cure malignant B-cell lymphoma. Legal entity responsible for the study Renji Hospital, Shanghai Jiaotong University School of Medicine Funding National Natural Science Foundation of China (NSFC), Grant 81172253, Grant 81570177 Disclosure All authors have declared no conflicts of interest.

Effect of BRD4 Inhibitor GSK525762A on Proliferation and Apoptosis of SUP-B15 Cells In Vitro and Its Possible Mechanism

To investigate the effect of BRD4 inhibitor GSK525762A on the proliferation and apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia SUP-B15 cells and its mechanism. SUP-B15 cells were treated with different concentration of GSK525762A, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining. The transcripts of anti-apoptotic genes C-MYC, CDK6 and BCL-2 were detected by real-time PCR. GSK525762A could inhibit significantly SUP-B15 cell proliteration in dose-and time-dependent manner; GSK525762A treatment could induce apoptosis of SUP-B15 cells. The levels of C-MYC,CDK6 and BCL-2 mRNA transcripts in SUP-B15 cells were reduced in GSK525762A-treated group. The GSK525762A can remarkably inhibit proliferation and induce apoptosis of SUP-B15 cells. The down-regulation of apoptosis-related genes C-MYC,CDK6 and BCL-2 may be involved in the process of apoptosis.

Bile acid: a potential inducer of colon cancer stem cells.

BackgroundAlthough the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer.MethodsNormal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of β-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA.ResultsWe found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial–mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF.ConclusionsOur results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/β-catenin signaling and thus could be considered promoters of colon cancer.

MicroRNA let-7g inhibited hypoxia-induced proliferation of PASMCs via G0/G1 cell cycle arrest by targeting c-myc

AimsPulmonary hypertension (PH) is a proliferative disorder characterized by enhanced proliferation and suppressed apoptosis of intrapulmonary vascular smooth muscle cells. Recently, network-based bioinformatics have identified let-7 family, a tumor suppressive microRNA, regulate multiple interacting targets relevant to PH. However, the role of let-7 in vascular homeostasis in PH remains unknown. Thus, we wanted to investigate the role of let-7 in hypoxia-induced PASMCs proliferation and the underlying mechanism in hypoxic pulmonary hypertension (HPH). Main methodsThe male Sprague–Dawley (SD) rats were exposed to hypoxia (10% O2) for 21days to induce HPH. The expression of let-7 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Primary rat PASMCs were exposed to hypoxia (3% O2). MTS and EDU were performed to evaluate PASMCs proliferation. The mRNA and protein expression of c-myc, Bmi-1 and p16 were determined by qRT-PCR and Western blotting, respectively. The functions of let-7g on PASMCs proliferation, c-myc, Bmi-1 and p16 expression were assessed by let-7g mimic and inhibitor transfection. Key findingsAmong let-7 family members, only let-7b and let-7g were significantly down-regulated in remodeled pulmonary artery in HPH rats. Furthermore, only let-7g level was decreased in hypoxic PASMCs. Either hypoxia or let-7g inhibitor stimulated proliferation of PASMCs, let-7g mimic inhibited hypoxia-induced PASMCs proliferation. C-myc was the target of let-7g in PASMCs. Transfect of let-7g mimic inhibited hypoxia-induced c-myc, Bmi-1 up-regulation and p16 down-regulation, which ultimately controls cell cycle progression. SignificanceLoss of inhibition on c-myc-Bmi-1-p16 signaling pathway by let-7g may lead to PASMCs proliferation and vascular remodeling in HPH.

Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo.

BackgroundTissue-specific stem/progenitor cells are found in various adult tissues and may have the capacity for lineage-specific differentiation, facilitating applications in autologous transplantation. Stage-specific embryonic antigen 4 (SSEA-4), an early embryonic glycolipid antigen, is expressed in cells derived from adult human pancreas exocrine tissue. Here, we examined the characteristics and lineage-specific differentiation capacity of SSEA-4+ cells.MethodsHuman adult partial pancreas tissues were obtained from different donors and cultured in vitro. SSEA-4+ and CA19-9+ cells were isolated from adult human pancreas exocrine cells using magnetic-activated cell sorting, and gene expression was validated by quantitative polymerase chain reaction. To confirm in-vivo differentiation, SSEA-4+ and CA19-9+ cells were transplanted into the dorsal subcutaneous region of mice. Finally, morphological features of differentiated areas were confirmed by immunostaining and morphometric analysis.ResultsSSEA-4-expressing cells were detected in isolated pancreas exocrine cells from adult humans. These SSEA-4+ cells exhibited coexpression of CA19-9, a marker of pancreatic duct cells, but not amylase expression, as shown by immunostaining and flow cytometry. SSEA-4+ cells exhibited higher relative expression of Oct4, Nanog, Klf4, Sox2, and c-Myc mRNAs than CA19-9+ cells. Pancreatic intralobular ducts (PIDs) were generated from SSEA-4+ or CA19-9+ cells in vivo at 5 weeks after transplantation. However, newly formed PIDs from CA19-9+ cells were less abundant and showed an incomplete PID morphology. In contrast, newly formed PIDs from SSEA-4+ cells were abundant in the transplanted area and showed a crowded morphology, typical of PIDs. Sox9 and Ngn3, key transcription factors associated with pancreatic development and regeneration, were expressed in PIDs from SSEA-4+ cells.ConclusionsSSEA-4-expressing cells in the adult human pancreas may have the potential for regeneration of the pancreas and may be used as a source of stem/progenitor cells for pancreatic cell lineage-specific differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0422-0) contains supplementary material, which is available to authorized users.