Effects Of c-Myc Research Articles

KITENIN promotes aerobic glycolysis through PKM2 induction by upregulating the c-Myc/hnRNPs axis in colorectal cancer

PurposeThe oncoprotein KAI1 C-terminal interacting tetraspanin (KITENIN; vang-like 1) promotes cell metastasis, invasion, and angiogenesis, resulting in shorter survival times in cancer patients. Here, we aimed to determine the effects of KITENIN on the energy metabolism of human colorectal cancer cells.Experimental designThe effects of KITENIN on energy metabolism were evaluated using in vitro assays. The GEPIA web tool was used to extrapolate the clinical relevance of KITENIN in cancer cell metabolism. The bioavailability and effect of the disintegrator of KITENIN complex compounds were evaluated by LC–MS, in vivo animal assay.ResultsKITENIN markedly upregulated the glycolytic proton efflux rate and aerobic glycolysis by increasing the expression of GLUT1, HK2, PKM2, and LDHA. β-catenin, CD44, CyclinD1 and HIF-1A, including c-Myc, were upregulated by KITENIN expression. In addition, KITENIN promoted nuclear PKM2 and PKM2-induced transactivation, which in turn, increased the expression of downstream mediators. This was found to be mediated through an effect of c-Myc on the transcription of hnRNP isoforms and a switch to the M2 isoform of pyruvate kinase, which increased aerobic glycolysis. The disintegration of KITENIN complex by silencing the KITENIN or MYO1D downregulated aerobic glycolysis. The disintegrator of KITENIN complex compound DKC1125 and its optimized form, DKC-C14S, exhibited the inhibition activity of KITENIN-mediated aerobic glycolysis in vitro and in vivo.ConclusionsThe oncoprotein KITENIN induces PKM2-mediated aerobic glycolysis by upregulating the c-Myc/hnRNPs axis.

Splicing factor derived circular RNA circCAMSAP1 accelerates nasopharyngeal carcinoma tumorigenesis via a SERPINH1/c-Myc positive feedback loop

BackgroundCircular RNAs play an important role in tumor genesis and progression, but they have not been sufficiently studied in patients with nasopharyngeal carcinoma (NPC).MethodsThe circular RNA, circCAMSAP1, was screened in NPC cells by RNA sequencing analysis. The expression of circCAMSAP1 in NPC tissues was examined by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization. Wound-healing, transwell, MTT and flow cytometry assays, and nude mouse tumor models were used to explore the effect of circCAMSAP1 on proliferation and metastasis of NPC in vitro or in vivo. The downstream proteins regulated by circCAMSAP1 were screened using mass spectrometry. The interaction between circCAMSAP1 and the SERPINH1 mRNA was identified using the circular RNA immunoprecipitation method and the luciferase reporter assay. The interaction between SERPINH1 and transcription factor c-Myc was verified through Co-immunoprecipitation (Co-IP) and immunofluorescence. The effect of c-Myc on the generation of circCAMSAP1 was examined through RT-qPCR and chromatin immunoprecipitation. Finally, the splicing factors that promote the production of circCAMSAP1 were explored by RT-qPCR and RNA immunoprecipitation (RIP).ResultsWe found that circCAMSAP1 was highly expressed in NPC tissues and promoted NPC proliferation and metastasis. Additionally, circCAMSAP1 promoted SERPINH1 expression through improved SERPINH1 mRNA stability by binding to the 3′-untranslated region (3’UTR) of SERPINH1. Highly expressed SERPINH1 reduced the ubiquitination-degradation rate of c-Myc, causing increased tumorigenesis. Meanwhile, c-Myc, cooperating with splicing factor 10 (SRSF10), could also promote CAMSAP1 pre-mRNA transcription and back-splicing, forming a positive feedback of circCAMSAP1 production, resulting in the proliferation and metastasis of NPC.ConclusionsOur findings revealed that circCAMSAP1 promotes NPC proliferation and metastasis by binding to the 3’UTR of SERPINH1, suggesting that the positive feedback of circCAMSAP1-SERPINH1-c-Myc may serve as a prognostic biomarker or therapeutic target in patients with NPC.

C-Myc promotes lymphatic metastasis of pancreatic neuroendocrine tumor through VEGFC upregulation.

Pancreatic neuroendocrine tumor (pNET) is a pancreatic neoplasm with neuroendocrine differentiation. pNET in early stage can be treated with surgical resection with long‐term survival, whereas the prognosis of pNET with locoregional or distant metastasis is relatively poor. Lymphangiogenesis is essential for tumor metastasis via the lymphatic system and may overhead distant metastasis. c‐Myc overexpression is involved in tumorigenesis. The role of c‐Myc in lymphangiogenesis is unclear. In this study, we evaluated the mechanism and effect of c‐Myc on lymphangiogenesis of pNET via interaction of lymphatic endothelial cells (LECs) and pNET cells. Lymph node metastasis was evaluated in pNET xenograft mice. Potential target agents to inhibit lymph node metastasis were evaluated in an animal model. We found that vascular endothelial growth factor C (VEGFC) expression and secretion was increased in pNET cell lines with c‐Myc overexpression. c‐Myc transcriptionally upregulates VEGFC expression and the secretion of pNET cells by directly binding to the E‐box of the VEGFC promoter and enhances VEGF receptor 3 phosphorylation and the tube formation of LECs. c‐Myc overexpression is associated with lymph node metastasis in pNET xenograft mice. Combinational treatment with an mTOR inhibitor and c‐Myc inhibitor or VEGFC‐neutralizing chimera protein reduced lymph node metastasis in the mice with c‐Myc overexpression. The mTOR inhibitor acts on lymphangiogenesis by reducing VEGFC expression in pNET cells and inhibiting the tube formation of LECs. In conclusion, mTOR and c‐Myc are important for lymphangiogenesis of pNET and are potential therapeutic targets for prevention and treatment of lymph node metastasis in pNET.

Silencing c-Myc Enhances the Antitumor Activity of Bufalin by Suppressing the HIF-1α/SDF-1/CXCR4 Pathway in Pancreatic Cancer Cells.

BackgroundPancreatic cancer is one of the most aggressive malignancies. Bufalin, a traditional Chinese medicine, has been used to treat pancreatic cancer as an antitumor agent although the mechanism by which it exerts its effects is still unclear. c-Myc has been found to be overexpressed in more than half of human cancers including pancreatic cancer. However, the role of c-Myc in pancreatic cancer cells and its influence in bufalin-treated pancreatic cancer are yet to be clarified. The present study aimed to investigate the role of c-Myc in the antitumor activity of bufalin in pancreatic cancer.Methodsc-Myc siRNA and overexpression plasmid were transfected into pancreatic cancer cells to construct the cell models. c-Myc expression was detected via quantitative real-time polymerase chain reaction and western blot. The effect of c-Myc on bufalin-induced inhibition of cell proliferation was detected via CCK-8 assay. Cell apoptosis and the cell cycle were analyzed via flow cytometry. Cell invasion and migration was detected via Transwell and wound healing assays, respectively. In addition, the effect of bufalin on the suppression of tumor growth in vivo was studied in nude mice model subcutaneously injected with PANC-1 and SW1990 cells. Hematoxylin-eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay were used to evaluate pathological changes in vivo. The expression of HIF-1α/SDF-1/CXCR4 were detected via western blot.ResultsCCK-8 assay showed that bufalin could inhibit the proliferation of pancreatic cancer cell, and c-Myc downregulation enhanced this effect. Similarly, c-Myc downregulation enhanced the effect of bufalin on cell cycle arrest, apoptosis, and the invasion and migration of pancreatic cancer cell in vitro. Further mechanism assay showed that c-Myc enhances the effect by regulating the HIF-1α/SDF-1/CXCR4 signaling pathway. The in vivo studies verified the results that c-Myc enhances the effect of bufalin through regulation of the HIF-1α/SDF-1/CXCR4 pathway.ConclusionsDownregulation of c-Myc enhanced the antitumor activity of bufalin in pancreatic cancer cells by suppressing the HIF-1α/SDF-1/CXCR4 pathway. These findings indicate that c-Myc inhibitors could enhance the clinical therapeutic effect of bufalin and may expand the clinical application of bufalin accordingly.

Oncogenic regulation of tumor metabolic reprogramming.

Development of malignancy is accompanied by a complete metabolic reprogramming closely related to the acquisition of most of cancer hallmarks. In fact, key oncogenic pathways converge to adapt the metabolism of carbohydrates, proteins, lipids and nucleic acids to the dynamic tumor microenvironment, conferring a selective advantage to cancer cells. Therefore, metabolic properties of tumor cells are significantly different from those of non-transformed cells. In addition, tumor metabolic reprogramming is linked to drug resistance in cancer treatment. Accordingly, metabolic adaptations are specific vulnerabilities that can be used in different therapeutic approaches for cancer therapy. In this review, we discuss the dysregulation of the main metabolic pathways that enable cell transformation and its association with oncogenic signaling pathways, focusing on the effects of c-MYC, hypoxia inducible factor 1 (HIF1), phosphoinositide-3-kinase (PI3K), and the mechanistic target of rapamycin (mTOR) on cancer cell metabolism. Elucidating these connections is of crucial importance to identify new targets and develop selective cancer treatments that improve response to therapy and overcome the emerging resistance to chemotherapeutics.

Abstract 3519: The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs

Abstract Several clinical studies have shown a correlation between elevated c-Myc levels and a better response to chemotherapy. Although elevated c-Myc might indirectly cause such chemosensitivity, it is possible that a direct effect is occurring due to underlying molecular mechanisms. The purpose of the current study was to determine whether c-Myc overexpression could acutely cause chemosensitivity. The c-Myc protein is known to activate DNA replication origins, but the mechanism for this is unknown. Origins are regulated by the loading of MCM2-7 hexamers, which form the core of the helicase that starts the initiation of replication. Mammalian cells load more MCM complexes than are necessary to complete one round of replication. The excess MCM complexes function as backups to allow recovery of replication when forks stall during drug treatment. Consistent with this, we found that loss of Mcm4 or Mcm7 sensitizes PDAC cell lines to 5-FU and Gemcitabine, two S phase stress drugs that are used to treat PDAC patients but are not highly effective on their own. Similar to depletion of backup MCM complexes via loss of Mcm4 or Mcm7, elevated c-Myc expression over-activates backup MCMs, leaving fewer available to recover from drug treatment. Cells containing overexpressed c-Myc display increased chemosensitivity to Gemcitabine that is not seen in cells containing normal c-Myc levels or a non-functional c-Myc mutant. These results demonstrate that reduction in backup MCM complexes, either by direct downregulation or by oncogene driven over-activation (via c-Myc), render cells sensitive to chemotherapy drugs. These results suggest that development of drugs that target MCM functionality may be useful in combination with S-phase stress drugs currently used in the clinic. These results also demonstrate that c-Myc has the ability to acutely cause chemosensitivity and c-Myc elevated tumors may respond better to chemotherapy due to the c-Myc effects on MCM complexes. Citation Format: Victoria Bryant, Mark G. Alexandrow. The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2014-3519

Chromatin dynamics at the hTERT promoter during transcriptional activation and repression by c-Myc and Mnt in Xenopus leavis oocytes

The transcription factors c-Myc and Mnt regulate gene expression through dimerization with Max and binding to E-boxes in target genes. While c-Myc activates gene expression via recruitment of histone modifying complexes, Mnt acts as a transcriptional repressor. Here, we used the Xenopus leavis oocyte system to address the effect of c-Myc and Mnt on transcription and chromatin remodeling over the E-box region in the human telomerase reverse transcriptase (hTERT) promoter. As expected we found elevated and decreased levels of hTERT transcription upon exogenously expressed c-Myc/Max and Mnt/Max, respectively. In addition, we confirmed binding of these heterodimers to both E-boxes already enriched with H3K9ac and H4K16ac. These chromatin marks were further enhanced upon c-Myc/Max binding followed by increased DNA accessibility in the E-box region. In contrast, Mnt/Max inhibited Myc-induced transcription and mediated repression through complete chromatin condensation and deacetylation of H3K9 and H4K16 across the E-box region. Importantly, Mnt was able to counteract c-Myc mediated activation even when expressed at low levels, suggesting Mnt to act as a strong repressor by closing the chromatin structure. Collectively our data demonstrate that the balance between c-Myc and Mnt activity determines the transcriptional outcome of the hTERT promoter by modulation of the chromatin architecture.

Effect of c-myc on p21Cipl and apoptosis of breast cancer cells via a p53,independent manner

Objective To investigate the effect of mutant （T58A） and wild type （ WT） c-myc onp21Clpl and apoptosis of breast cancer cells in a p53-independent way. Methods Lentiviral vectors contai-ning c-myc T58A and WT were transfected into breast cancer cells HCC1937 （ cell transfection rate exceeds85% ） . Untreated cells （ group A） and cells transfected by lentivirus without c-myc gene （group B） servedas blank control group and transfection control group respectively. Cells stably expressing c-myc T58A（group C） and WT （group D） served as experiment groups. All the cells were then transfected by p21Cipi/siRNA. RT-PCR and Western blotting were used to detect the mRNA and protein levels of c-myc andp21Cipl,and the apoptosis was examined by using TdT-mediated dUTP nick end labeling （TUNEL） . Re-sults As compared with groups A and B,the c-myc was overexpressed in groups C and D （ all p 〈0. 01 ） . Before transfection, the p21c,pl in group C was highest （P 〈0. 01 ）,while group D had the lowestexpression of p21Cipl （p 〈0. 01 ） ; the apoptosis rate in group C was lower than in other groups, and that ingroup D was the highest： （5. 5 ±0. 5）% , （5. 8 ±0. 3）% , （2. 8 ±0. 3）% and （9. 8 ±0. 8）% in groupsA,B,C and D, respectively （p 〈0.01）. After transfection, the apoptosis rate in all groups were im-proved obviously （P 〈0.05）. Conclusion Normally,in a non-p53 background,c-myc WT can suppressthe expression of p21c,pI,resulting in the increased apoptosis rate, but after mutation （T58A）,the abilityof c-myc to induce apoptosis is decreased. Key words: Breast cancer; c-myc ; p21 cip1 ; Apoptosis

WNT/β-Catenin-Signaling Pathway Stimulates the Proliferation of Cultured Adult Human Sertoli Cells via Upregulation of C-myc Expression

The role of WNT/β-catenin-signaling pathway is critical in mouse Sertoli cell maturation and tumorigenesis. This study aims to examine the effects of WNT/β-catenin signaling on the cultured adult human Sertoli cells and the underlying molecular mechanisms. Glycogen synthase kinase 3β (GSK-3β) inhibitors, SB216763 and lithium chloride (LiCl), were used to activate WNT/β-catenin-signaling pathway. 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry were used to analyze the proliferation and cell cycle of cultured human Sertoli cells, respectively. C-myc expression was accessed by immunofluorescence, real-time polymerase chain reaction and Western blot. The effects of c-myc on Sertoli cell proliferation were investigated by RNA interference technology and BrdU incorporation assay. The results showed activation of WNT/β-catenin signaling stimulated human Sertoli cell proliferation. Obvious increases in c-myc messenger RNA and protein expression were observed after SB216763 and LiCl treatments. Knockdown of c-myc expression attenuated the ability of WNT/β-catenin signaling to stimulate the proliferation of human Sertoli cells. WNT/β-catenin signaling enhances human Sertoli cell proliferation via upregulation of c-myc expression.

Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis

IntroductionRetinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/β in favor of RARγ.MethodsThe effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis.ResultsModulation of the RARα/β to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death.ConclusionsThe functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/β balance suggests that prevalence of RARγ over-RARα/β expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials.See related editorial by Garattini et al http://breast-cancer-research.com/content/14/5/111

Abstract 2226: Mechanisms for cross-talk between Notch1 and AKT signaling in T-cell acute lymphoblastic leukemia

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL) has a variable prognosis, with many patients exhibiting high-risk features at diagnosis, including older age and high presenting white blood cell counts. Activating mutations involving the Notch1 heterodimerizeration and PEST domains occur in more than 50% of T-ALL cases. Increased Notch1 activity promotes survival and proliferation of leukemic cells, in part by transcriptionally repressing PTEN (via HES1). This provides compelling rationale for the use of gamma secretase inhibitors (GSIs) to block Notch1 activation and to restore PTEN levels, which antagonize AKT signaling. Further, PTEN mutations are implicated in GSI resistance. PTEN activity is also modulated by an assortment of posttranslational modifications and Notch1 may regulate PTEN at the posttranslational level. To investigate the impact of Notch1 on AKT signaling, independent of its transcriptional regulation of PTEN, we used PTEN-null Jurkat cells with constitutively active Notch1 and conditional ectopic PTEN expression under control of a tetracycline (tet)-inducible promoter. Induction of PTEN was accompanied by increased phosphorylation of AKTT308, increased insulin-like growth factor 1 receptor (IGF1R) protein and transcripts, and increased Notch1. Increased AKTT308 phosphorylation was blocked with an IGF1R inhibitor (GSK1904259A) and was abolished in Jurkat cells expressing a tet-inducible G129R PTEN phosphatase-inactive mutant. This suggests that PTEN can activate AKT signaling by modulating IGF1R levels. In Jurkat cells, inhibition of Notch1, either through GSI treatment or shRNA knockdown (KD), resulted in increased phosphorylation of AKTT308. Although this result could involve Notch1-dependent decreased cMyc, reflecting indirect effects of cMyc on mTOR1 and negative feedback on AKT, this was not impacted by treatment with a cMyc inhibitor (10058-F4) or by ectopic overexpression of cMyc, suggesting that other effectors and mechanisms are involved. Microarray analysis was performed on wild-type and KD Jurkat cells. Using a 1.5-fold cutoff, 1208 differentially regulated genes were identified. These include PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1) and prokineticin-2 (PROK2), both of which could significantly impact AKT phosphorylation in response to Notch1. By real-time RT-PCR, PHLPP1 was significantly decreased (∼4-fold), and PROK2 was significantly increased (∼6-fold) in Notch1 KD cells compared to wild-type cells. Studies are underway to characterize the potential roles of PHLPP1 and PROK2 in mediating the inhibitory effects of Notch1 on AKT activation in T-ALL. Better understanding of the cross-talk between Notch1 and AKT in T-ALL may identify rational therapeutic strategies for combining GSIs with inhibitors of AKT, mTOR, or IGF1R, and/or for designing therapies based on capacities for constitutive Notch1 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2226. doi:1538-7445.AM2012-2226

C-MYC Drives PBK Expression in High-Grade Malignant Lymphomas Through E2F1

Abstract 1569 Background:PBK (PDZ-binding kinase) is a serine-threonine kinase that was originally cloned as an interaction partner with the human tumor suppressor hdlg, as an IL-2 inducible gene in cytotoxic T lymphocytes and as an expressed sequence tag in Burkitt's lymphoma (BL) cells. PBK/TOPK mRNA is expressed in normal testicular and embryonic tissues, proliferating neural progenitor cells, various tumor cell lines and in aggressive hematopoietic neoplasms. Previously E2F1 and CREB/ATF were identified as transcriptional regulators of PBK (Leuk Res, 2006). Recently, we reported that PBK interacts with tumor suppressor p53 via its DNA binding domain and modulates its function as a transcription factor (Oncogene, 2010). These lines of evidence point to PBK as a novel oncoprotein with possible relevance as a therapeutic target. c-MYC is the hall mark for Burkitt's lymphoma, and it is highly expressed in various other hematological malignancies. E2F1 is one of the transcriptional targets of c-MYC. The association of PBK expression with aggressive hematologic neoplasms and BL in particular, suggested a possible role of a c-MYC-E2F1-PBK axis in the pathogenesis of hematological malignancies. Study Aim:To investigate the frequency of PBK expression in a larger number of hematopoietic neoplasms and determine whether c-MYC has a role in PBK expression. Methods:Immunohistochemistry for PBK, E2F1 and c-MYC expression on malignant lymphoma tissue microarrays (US Biomax) was conducted. The slides were scored by a blinded hematopathologist (XFZ) according to the following criteria: For staining (0–10), 0= no tumor cells were stained; 1 = 10% tumor cells were stained; 10= 100% tumor cells were stained. For intensity (0–4), 0= no staining; 1= weak; 2=intermediate; 3=strong; 4=very strong. Spearman rank correlation coefficient analysis was used to measure relationships in expression among c-MYC, E2F1 and PBK. To confirm the results from immunohistochemistry assay, effects of c-MYC on expression of E2F1 and PBK were investigated by knocking down c-MYC in Burkitt’s lymphoma cell lines and overexpressing c-MYC in HCT116 cells, followed by Western blot analysis for E2F1 and PBK levels. Results:PBK was expressed in 100% of BL samples (N=8), 80% of Diffuse large B-cell Lymphoma (DLBCL) (N=10), 40% of myeloma samples (N=10), 50% of Hodgkins (HL) samples (N=10) but 0/3 Follicular lymphoma (FL) samples, 1/3 T-cell lymphoma samples and 0/4 normal lymph nodes. Among all 44 cases of malignant lymphoma, staining of PBK, E2F1 and c-MYC were significantly positively correlated: Spearman rank correlation coefficient (ρ) between c-MYC and E2F1 is 0.455 (p=0.0028), ρ=0.874 for E2F1 and PBK(p<0.0001), and ρ=0.537 for c-MYC and PBK(p=0.0004); Staining intensity of these three proteins were also significantly positively correlated: ρ=0.407 for c-MYC and E2F1 (p=0.0077), ρ=0.849 for E2F1 and PBK(p<0.0001),and ρ=0.479 for c-MYC and PBK (p=0.0017). In 10 cases of (DLBCL), staining of PBK, E2F1 and c-MYC were significantly positively correlated: Spearman rank correlation coefficients are 0.721, 0.929, and 0.797 for c-MYC and E2F1(p=0.0319), E2F1 and PBK(p<0.0057), c-MYC and PBK(p=0.0177), respectively; In terms of staining intensity of these proteins, Spearman rank correlation coefficients are 0.849, 0.963, and 0.908 for c-MYC and E2F1(p=0.0114), E2F1 and PBK(p=0.0041), c-MYC and PBK(p=0.0068), respectively, in 8 cases of Burkitt’s lymphoma, staining intensity of PBK was found to be correlated to that of c-MYC (ρ=0.755, p=0.0492). Gene knockdown experiments in BL2, BJAB, Raji, Daudi cells showed that inhibition of c-MYC expression could downregulate both E2F1 and PBK, whereas overexpression of c-MYC in HCT116 cells increased expression of E2F1 and PBK. [Display omitted] Conclusions and Future Directions:PBK protein was highly expressed in a high proportion of Burkitt lymphoma and diffuse large B-cell lymphoma and in a lesser % of Hodgkin lymphoma and myeloma cases, suggesting that PBK is expressed in a relatively high proportion of aggressive hematologic neoplasms. Expression of PBK was correspondent to and subjected to regulation by c-MYC and E2F1 in these lymphomas. These new results in combination with our earlier data as well as the adverse prognostic implications of c-MYC expression in malignant lymphomas suggest that PBK function may be a rational target for novel anti-neoplastic therapeutics. Disclosures:No relevant conflicts of interest to declare.

C-Myc Attenuates The Ability of Doxorubicin to Reduce The Colony Formation Partially Through Regulating Nbs1 in U2OS Cells*

c-Myc is a ubiquitous transcription factor that regulates a wide variety of genes involved in the control of cell proliferation,differentiation and apoptosis.It was demonstrated that over-expressions of c-Myc and Nbs1 attenuated the ability of doxorubicin in reduction of colony formation in U2OS cells,and it was found that this effect of c-Myc was associated with Nbs1.It can be confirmed that Nbs1 is a target gene of c-Myc.Chromatin immunoprecipitation assays reveal that c-Myc recruited the histone acetyltransferase p300 complex to the promoter region of Nbs1 gene,resulting in an increased level of acetylated histone H4.Furthermore,it was found that the two proximal E-box elements located in Nbs1 promoter region were essential for the c-Myc binding.Thus,it was concluded that c-Myc attenuates the effect of doxorubicin partially through regulating Nbs1,implicating its roles in repair of DNA breakage induced by doxorubicin.

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.

Effect of siRNA targeting c-Myc and VEGF on human colorectal cancer cells

To investigate the biological behavioral effects of specific siRNA expression plasmids targeted against c-Myc and vascular endothelial growth factor (VEGF) on human colorectal cancer cell line Volo. The expression plasmids with small interfering RNA (siRNA) aiming at c-Myc and VEGF were designed and constructed respectively, then transfected into Volo cells by eukaryocyte transfection technique. The protein expressions of c-Myc and VEGF were detected by Western blotting. Cellular proliferation, apoptosis, cycle distribution and invasion character were analyzed by tetrazolium bromide colorimetry (MTT), flow cytometry (FCM), TUNEL assay and matrigel invasion assay respectively. Enzymatic digestion and DNA sequencing confirmed that the c-Myc and VEGF specific siRNA expression plasmids were constructed successfully. After plasmids were transfected into cells, the protein expressions of c-Myc and VEGF were significantly down-regulated respectively as compared with control group (P<0.01). The cellular proliferation inhibitory rates in c-Myc siRNA group, VEGF siRNA group and c-Myc+VEGF group were (59.20+/-5.05)%, (32.31+/-3.48)% and (75.81+/-7.89)% respectively, which were higher than that in control group [(6.80+/-1.45)%] (all P<0.05). The cell apoptosis rate in above 3 groups were (40.50+/-4.37)%, (21.30+/-2.98)% and (62.59+/-9.66)% respectively, which were higher than that in control group [(2.90+/-0.36)%] (all P<0.05). The cell invasion rates in VEGF siRNA group and c-Myc+VEGF siRNA group were (7.34+/-3.65)% and (2.80+/-1.02)%, which were lower than that in control group [(18.57+/-7.46)%] (P<0.05). The effect of c-Myc+VEGF siRNA group was greater. The specific siRNA efficiently silences the expression of c-Myc and VEGF, subsequently, suppresses the cell proliferation, triggers the cell apoptosis and inhibits the cell invasiveness in these transfected colorectal cancer Volo cells. In addition, the synergism of siRNA-c-Myc and siRNA-VEGF in transfected cells can be found.

Effect of c-MYC and E2F1 Gene Silencing and of 5-Azacytidine Treatment on Telomerase Activity in Pancreatic Cancer-Derived Cell Lines

Background:The gene promoter region of human telomerase reverse transcriptase (hTERT) contains binding sites for c-myc and E2F1 as well as CpG islands, suggesting regulation by genetic factors and epigenetically by methylation. Hence, the effect of the demethylating agent 5-azacytidine and silencing of c-MYC and E2F1 genes on its expression and consequently on telomerase activity were studied in pancreatic cancer-derived cell lines. Methods: MlaPaCa-2 and PANC-1 cell lines were transfected with SiRNA against E2F1 and c-MYC genes separately as well as along with 5-azacytidine treatment. The hTERT gene methylation status was determined by methylation-specific PCR and telomerase activity quantitated by TRAP-PCR-ELISA. Results: Demethylation by 5-azacytidine resulted in hTERTinhibition with a reduction in telomerase activity to 37–49% of controls. Silencing of E2F-1 or c-MYC also decreased the hTERT transcript and telomerase activity with a more pronounced effect with respect to c-MYC silencing. There was a synergistic effect of demethylation and gene silencing on the inhibition of hTERT mRNA expression which resulted in undetectable levels of telomerase activity. Conclusion: Telomerase activity, which is necessary for cellular immortalization, can be shut down by a combined approach using SiRNA-mediated gene silencing and demethylating agents, which has therapeutic implications.

Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.

Oncogene Expression and Cellular Radiation Resistance: A Modulatory Role for c-myc.

Background: Being able to predict the response of tumors to radiation therapy would improve the decision-making process involved in choosing treatment options for cancer. Expression of certain oncogenes and/or inactivation of tumor suppressor genes has been shown to alter cellular radiation responses; however, it is still not clear what marker or combination of markers would best indicate a radioresistant tumor, or whether such screening would be clinically useful. Current choices of markers are derived mainly from in vitro studies on cell survival after irradiation. In general, expression of transforming oncogenes increases cellular radioresistance. This was also demonstrated in this study for v-abl, bcr abl, v-Ha-ras, v-mos, and v-fes expressed in rat-1 cells. There are, however, conflicting data. Some of the discrepancies may in part be due to interactions between the oncogene-activated signals and other intrinsic or activated pathways. One downstream pathway that is required for oncogene-induced transformation involves c-myc. There is evidence that in some systems myc expression can potentiate ras-induced radiation resistance. Myc may therefore play an important role in determining tumor radioresistancy in the context of other oncogenes. Methods and Results: In this study, the role of c-myc in modulating intrinsic and oncogene-induced cellular radiation responses was investigated in more detail. Retroviral vectors were used to express c-myc and dominant negative mutant c-myc genes in rat 1 cells, with and without ca-transfection of v-abl as measured by clonogenic assay, rat 1. Cells infected with c-myc or v-abl were more resistant to irradiation than neo-transfected cells or control cells; however, cells doubly infected were not resistant, even though they had an increased transformation index. This indicates that transformation-related events per se do not necessarily lead to radiation resistance. It also suggests that the effects of c-myc on radioresistance may depend on what other pathways are activated. This conclusion was strengthened by the finding that expression of a dominant negative c-myc (dn-myc) mutant gene blocked v-abl-induced radiation resistance, but on its own made rat-1 cells more resistant to radiation. Conclusions: The apparently contradictory effects of c-myc in either enhancing or reducing radioresponsiveness may be explained by the dualistic roles of c-myc in promoting signal transduction pathways resulting in either cell proliferation or death, depending on what other pathways are activated. The studies indicate that it will be difficult to predict tumor response to radiation purely by examining expression of transforming oncogenes and it is likely that a number of markers will need to be examined to derive a reliable indication of tumor radiation response.

C-MYC Induces KIR Expression Via a Novel Control Region Upstream of the Conventional Adult KIR Promoter.

KIR determine whether NK cells will be alloreactive against targets. Although epigenetic control is thought to play a role in KIR expression, the mechanism of KIR repertoire formation is unknown. Recently, a probabilistic transcriptional mechanism has been shown to control the acquisition of mouse Ly49 receptors using a switch region upstream of each Ly49 promoter (S. Anderson). Based on this information, the DNA upstream of known KIR genes was scanned for similar motifs to test the hypothesis that similar control mechanisms are operant in human NK cells. A putative c-myc/AML binding site was found approximately 1Kb upstream of the traditional KIR promoters. Initiation of transcription in this region was observed by RNAse protection, and spliced KIR mRNAs originating in the upstream region were cloned. To look at the effects of c-myc on KIR expression, we transduced NK92 cells using retroviral murine stem cell virus vectors containing either c-myc/eGFP or eGFP alone. Cells expressing high levels of c-myc were 18% KIR positive as compared to 0.9% of control cells. To further look at the effect of c-myc on KIR from primary cells, we transduced CD34+ progenitors isolated from umbilical cord blood with c-myc/eGFP and eGFP vectors. eGFP+ cells were plated on the murine fetal liver line AFT024 and cultured with IL-15, IL-7, IL-3, Flt3 ligand, and c-kit ligand, all known to induce NK cell differentiation. Although c-myc is an oncogene, it did not promote autonomous growth of NK cells in the absence of exogenous cytokines. Proliferation was significantly increased in single cell progenitors over-expressing c-myc with 739.3x103 NK cell progeny compared to 0.56x103 from each control cell after 21 days (p<0.0001). At this early time point, 21.68 ± 3.25% of c-myc cells were KIR positive while 0.0% of control cells were KIR positive (p<0.0001) suggesting that c-myc targets KIR expression. Furthermore, bulk-transduced cells over expressing c-myc had 3.9 times the level of early non-coding transcripts originating from the upstream promoter as compared to eGFP transduced cells. A direct effect of c-myc on the upstream promoter was supported by ChIP assays demonstrating increased binding to the c-myc/AML site. The presence of these upstream transcripts directly correlates with increased conventional KIR transcripts as shown by Q-RT-PCR and an increase of KIR expression as shown by flow cytometry. These data show that c-myc play an important role in the differentiation of NK cells and in KIR acquisition. Experiments are currently in progress to look at the effects of the competing AML site on KIR expression and to investigate how non-coding RNA affects adult KIR expression. Understanding the mechanisms of KIR expression may lead to development of novel strategies to manipulate NK cells for therapeutic benefit.

Myc Antagonizes Ras-mediated Growth Arrest in Leukemia Cells through the Inhibition of the Ras-ERK-p21Cip1 Pathway

Even though RAS usually acts as a dominant transforming oncogene, in primary fibroblasts and some established cell lines Ras inhibits proliferation. This can explain the virtual absence of RAS mutations in some types of tumors, such as chronic myeloid leukemia (CML). We report that in the CML cell line K562 Ras induces p21Cip1 expression through the Raf-MEK-ERK pathway. Because K562 cells are deficient for p15INK4b, p16INK4a, p14ARF, and p53, this would be the main mechanism whereby Ras up-regulates p21 expression in these cells. Accordingly, we also found that Ras suppresses K562 growth by signaling through the Raf-ERK pathway. Because c-Myc and Ras cooperate in cell transformation and c-Myc is up-regulated in CML, we investigated the effect of c-Myc on Ras activity in K562 cells. c-Myc antagonized the induction of p21Cip1 mediated by oncogenic H-, K-, and N-Ras and by constitutively activated Raf and ERK2. Activation of the p21Cip1 promoter by Ras was dependent on Sp1/3 binding sites in K562. However, mutational analysis of the p21 promoter and the use of a Gal4-Sp1 chimeric protein strongly suggest that c-Myc affects Sp1 transcriptional activity but not the binding of Sp1 to the p21 promoter. c-Myc-mediated impairment of Ras activity on p21 expression required a transactivation domain, a DNA binding region, and a Max binding region. Moreover, the effect was independent of Miz1 binding to c-Myc. Consistent with its effect on p21Cip1 expression, c-Myc rescued cell growth inhibition induced by Ras. The data suggest that in particular tumor types, such as those associated with CML, c-Myc contributes to tumorigenesis by inhibiting Ras antiproliferative activity.