c-Myc Target Genes Research Articles

Abstract 281: Loss of Krueppel-like Factor 15 (KLF15) Leads to Altered Wnt-dependent Gene Regulation in Hearts With Systolic Dysfunction

Background & Aim: Developmentally conserved pathways, such as the Wnt/β-catenin pathway, are upregulated in adult heart diseases. Here, we aimed to elucidate the role of Wnt regulation via KLF15 for adult heart homeostasis. Results: We identified the transcription factor KLF15, a factor previously identified to contribute to cardiac fibrosis and hypertrophy, as a cardiac specific Wnt/β-catenin inhibitor. Accordingly, a constitutive Klf15 KO mouse model revealed specific cardiac upregulation of the Wnt target genes Tcf7l2 and cMyc (n=6, p<0.001). Serial echocardiography showed reduced systolic function starting at 16 weeks in the Klf15 KO hearts (n=10, p<0.05). Genome-wide sequencing studies of Klf15 KO vs. WT hearts at the postnatal age day P10, week 4 and 20 showed that gene expression differs increasingly at 4 and 20 weeks of age, respectively. This is in line with the increasing KLF15 expression after birth, reaching significant level at P13 (n=3/embryonal, fetal, and neonatal stage; each 5-8 pooled hearts). Accordingly, gene set enrichment analysis revealed activation of the Wnt signaling in Klf15 KO hearts at 4 and 20 weeks but not at P10 (p<0.001). This activation was milder in 20 weeks vs. 4 weeks, which may be explained by a concomitant upregulation of Wnt repressors, as Shisa3 and Dact3 with so far unknown function in the mammalian heart (n=9, p<0,01). Wnt activation was accompanied by upregulation of developmental genes and transcriptional regulators at 4 weeks and followed by upregulation of stress factors such as ANP, BNP, and Ankrd1 at 20 weeks. Moreover, we found the Wnt targets TCF7L2 and AXIN2 significantly upregulated in human ventricular biopsies from dilated (n≥5, p<0.05) and ischemic cardiomyopathy (n≥5, p<0.001) vs. non-failing, further underscoring the relevance of the Wnt pathway for human cardiac cellular homeostasis as well. Conclusion: A Wnt-dependent gene signature may precede the expression of stress factors and functional deterioration due to deletion of its cardiac repressor KLF15. Our data indicate that KLF15 is an important regulator of the Wnt pathway and is relevant for cardiac homeostasis and function in the postnatal heart.

Baicalein suppresses the viability of MG-63 osteosarcoma cells through inhibiting c-MYC expression via Wnt signaling pathway

The major reason responsible for the poor prognosis of osteosarcoma is the malignant proliferation of osteosarcoma cells. The activated Wnt/β-catenin signaling induces c-MYC gene transcription and results in osteocytes' carcinomatous change, which contributes to osteosarcoma development, so c-MYC gene is one of the therapeutic targets. The role of multiple botanical extracts in the expression of β-catenin's target gene c-MYC in osteosarcoma MG-63 cells was tested by cellomics high content screening. Baicalein was identified as the most effective one which can inhibit the proliferation and promote the apoptosis of MG-63 cells in a dose-dependent manner by cell counting kit-8 test and fluorescence-activated cell sorting, respectively. This process was associated with the decreased levels of β-catenin and its target gene c-MYC, identified by q-PCR and Western blotting, respectively. When MG-63 cells were treated with both baicalein and JNK inhibitor SP600125, the apoptosis and expression of c-MYC were not significantly decreased. After the construct pcDNA3.1-BANCR (BRAF-regulated lncRNA 1) was transfected into MG-63 cells, RT-PCR, Western blotting and CCK-8 assay showed that BANCR was positively correlated with baicalein. These results indicated that baicalein inhibited osteosarcoma cell proliferation and promoted apoptosis by targeting c-MYC gene through Wnt signaling, in which JNK and BANCR were also involved as well as β-catenin, suggesting a new potential mechanism for us to better understand the inhibiting effect of baicalein on osteosarcoma.

Fibulin-5 inhibits Wnt/β-catenin signaling in lung cancer.

Metastatic lung cancer is incurable and a leading cause of cancer death in the United States. However, the molecular mechanism by which lung cancer cells invade other tissues has remained unclear. We previously identified fibulin-5, an extracellular matrix protein, as a frequently silenced gene in lung cancer and a suppressor of cell invasion. In this study, we found fibulin-5 functions by inhibiting the Wnt/β-catenin pathway. The Cancer Genome Atlas (TCGA) datasets show a strong association between loss of fibulin-5 expression and poor outcomes of lung cancer patients, and also activation of the Wnt target genes MMP-7 and c-Myc. Fibulin-5 impedes Wnt/β-catenin signaling by inhibiting extracellular signal-regulated kinase (ERK) to activate glycogen synthase kinase-3 β (GSK3β), which downregulates β-catenin and prevents its nuclear accumulation, leading to suppression of MMP-7 and c-Myc expression. These effects of fibulin-5 are mediated by its amino-terminal integrin-binding RGD motif. Fibulin-5 also blocks Wnt/β-catenin signaling in vivo in H460 metastasis and H1299 tumor models. Furthermore, knockdown of β-catenin suppresses metastasis of H460 tumors, while knockdown of GSK3β promotes metastasis of fibulin-5-expressing H1752 tumors. Together, our results suggest that fibulin-5 functions as a metastasis suppressor in lung cancer by modulating tumor microenvironment to suppress Wnt/β-catenin signaling.

The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCF(Fbw7) (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc's nucleolar degradation pathway.

Metadherin contributes to the pathogenesis of chronic lymphocytic leukemia partially through Wnt/β-catenin pathway.

Metadherin (MTDH) is involved in aberrant proliferation, migration, and chemoresistance of tumor cells. It has been demonstrated that it can promote tumor growth by modulation multiple oncogenic signaling pathways. However, MTDH expression, significance, and related mechanism in chronic lymphocytic leukemia (CLL) are still unclear. The objective of this study was to investigate the expression of MTDH in CLL and the involvement of Wnt/β-catenin signaling pathway in MTDH effects. Overexpression of MTDH mRNAs was seen in CLL samples. MTDH expression was associated with Rai stage classification of CLL, and altered levels of β2-MG and lactate dehydrogenase in serum samples from patients. Overexpression of MTDH protein was seen in 87% of CLL samples. Specific siRNAs inhibited MEC-1 cell growth and enhanced cell apoptosis (P<0.05). Inhibition of MTDH expression resulted in decreased expression levels of lymphoid enhancer-binding factor 1 (LEF-1), and its downstream target genes c-myc and cyclin D1. And there was a strong correlation between MTDH and LEF-1 protein expression in 14 patients with CLL. The results demonstrate that MTDH is specifically expressed in B cell of CLL and exert a preservative role through activation of Wnt signaling pathway. Our findings indicated that MTDH may be a potential therapeutic target of CLL.

SOD2 is a C-myc target gene that promotes the migration and invasion of tongue squamous cell carcinoma involving cancer stem-like cells

Our previous studies revealed that manganese superoxide dismutase (SOD2) contributes to the migration and invasion of tongue squamous cell carcinoma (TSCC). The purpose of the current study was to further clarify the mechanisms of SOD2 in the migration and invasion of TSCC. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers (Bmi1, Nanog and ABCG2). We found that UM1 cells (TSCC cells with increased SOD2 expression, migration and invasion abilities) possessed a higher proportion of SP cells, sphere and colony formation, and expressed a higher level of stem cell markers compared to UM2 cells (reduced SOD2 expression, migration and invasion abilities). SOD2 expression as well as migration and invasion abilities were enhanced in SP cells compared to non-SP cells. Knockdown of SOD2 in UM1 cells or SP cells inhibited the migration and invasion abilities, reduced sphere and colony formation, and the expression of stem cell markers. Direct binding of the C-myc protein to the SOD2 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Knockdown of C-myc in UM1 cells inhibited SOD2 expression as well as migration and invasion abilities. Our results indicate that cancer stem-like cells play an important role in the migration and invasion of TSCC. SOD2 is a direct target gene of C-myc and C-myc-SOD2-mediated migration and invasion of TSCC involve cancer stem-like cells.

β-Catenin-Dependent Signaling Pathway Contributes to Renal Fibrosis in Hypertensive Rats.

The mechanism of hypertension-induced renal fibrosis is not well understood, although it is established that high levels of angiotensin II contribute to the effect. Since β-catenin signal transduction participates in fibrotic processes, we evaluated the contribution of β-catenin-dependent signaling pathway in hypertension-induced renal fibrosis. Two-kidney one-clip (2K1C) hypertensive rats were treated with lisinopril (10 mg/kg/day for four weeks) or with pyrvinium pamoate (Wnt signaling inhibitor, single dose of 60 ug/kg, every 3 days for 2 weeks). The treatment with lisinopril reduced the systolic blood pressure from 220 ± 4 in 2K1C rats to 112 ± 5 mmHg (P < 0.05), whereas the reduction in blood pressure with pyrvinium pamoate was not significant (212 ± 6 in 2K1C rats to 170 ± 3 mmHg, P > 0.05). The levels of collagen types I and III, osteopontin, and fibronectin decreased in the unclipped kidney in both treatments compared with 2K1C rats. The expressions of β-catenin, p-Ser9-GSK-3beta, and the β-catenin target genes cyclin D1, c-myc, and bcl-2 significantly decreased in unclipped kidney in both treatments (P < 0.05). In this study we provided evidence that β-catenin-dependent signaling pathway participates in the renal fibrosis induced in 2K1C rats.

A critical role for the regulated wnt-myc pathway in naive T cell survival.

Wnt signaling is involved in T cell development, activation, and differentiation. However, the role for Wnt signaling in mature naive T cells has not been investigated. In this article, we report that activation of Wnt signaling in T cell lineages by deletion of the Apc (adenomatous polyposis coli) gene causes spontaneous T cell activation and severe T cell lymphopenia. The lymphopenia is the result of rapid apoptosis of newly exported, mature T cells in the periphery and is not due to defects in thymocyte development or emigration. Using chimera mice consisting of both wild-type and Apc-deficient T cells, we found that loss of naive T cells is due to T cell intrinsic dysregulation of Wnt signaling. Because Apc deletion causes overexpression of the Wnt target gene cMyc, we generated mice with combined deletion of the cMyc gene. Because combined deletion of cMyc and Apc attenuated T cell loss, cMyc overexpression is partially responsible for spontaneous T cell apoptosis and lymphopenia. Cumulatively, our data reveal a missing link between Wnt signaling and survival of naive T cells.

Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes

Nanog plays an important role in maintaining the pluripotency of murine and human embryonic stem cells. However, the molecular features and transcriptional regulation of the NANOG gene have not been well investigated in pig. Here, we report, for the first time, that porcine NANOG is encoded by a single exon gene (SEG) mapped on chromosome 1 and has two daughter genes, one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1. The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG, but does not encode a functional protein due to deletions and in-frame stop codons. The NANOGP1 contains four exons and three introns, but is short of the homeodomain sequence. Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG, but not from NANOGP1, because the NANOGP1 promoter is highly methylated, as confirmed by global DNA methylation analysis. The NANOG protein encoded by NANOG retains N, H, and C1/W/C2 domains. The H domain is required for nuclear translocation, while the C1/W/C2 domain ensures the NANOG regulatory function. Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2, KLF4, and c-MYC. In conclusion, the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2, and can promote the activation of target pluripotent factors in vivo.

C-MYC Transcriptionally Amplifies SOX2 Target Genes to Regulate Self-Renewal in Multipotent Otic Progenitor Cells

SummarySensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation.

Protein Arginine Methyltransferase 5 Supports MYC, Survivin and Cyclin D1 Activity in Aggressive Lymphomas By Regulating the WNT/β-Catenin Pathway

Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis.Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis.Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells.Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. DisclosuresNo relevant conflicts of interest to declare.

365 Pim-1 kinase: Validated as a therapeutic cancer target for MYC-driven tumours

Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) kinase belongs to a family of constitutively active serine/threonine kinases. The overexpression of Pim-1 can contribute to tumorigenesis, thereby establishing Pim-1 as a proto-oncogene. In B-cell lymphoma, colorectal cancer, pancreatic cancer and other tumours, the over expression of Pim-1 is linked to poor prognosis. Proviral integration experiments suggested the cooperation between Pim-1 and the protooncogene c-myc that was subsequently confirmed by double transgenic mice. Pim-1 acts synergistically with the transcription factor c-myc by phosphorylation and by interaction with c-myc on the chromatin level, leading to elevated trancription of c-myc target genes and cell proliferation. Pim-1 also promotes cell cycle progression. Pim-1-mediated phosphorylation of p27 leads to its binding to 14−3−3 protein, its nuclear export and proteasome-dependent degradation. The aim of our study was to analyze the effect of kinase-inhibition of Pim-1 on tumour growth in a mouse model of haematological malignancy. To this end, we constructed a conditional knock-in mouse model in which a single amino acid exchange can be induced by expression of the Cre-recombinase, leading to expression of an inactive form of Pim-1 kinase. These Pim-1 kinase-dead (KD) mice do not display phenotypic abnormalities or reduced live span even after homozygous exchange by CMV-Cre expression of the wild type form of Pim-1 against the mutated form. As expected, elevated levels of the cell cycle inhibitor p27 can be observed in the organs of Pim-1 kinase-dead mice by immunohistochemistry (IHC). In order to study the effect of the Pim-1 kinase inhibition on tumour development driven by c-myc, we crossed the Pim-1 KD mice with the Em-cmyc transgenic mouse line, which expresses high levels of the oncoprotein c-myc leading to the development of a rapidly growing B-cell lymphoma that resembles the human Burkitt-lymphoma. Even the heterozygous switch to the kinase-dead form of Pim-1 doubles the survival of the Em-c-myc mice; the homozygous switch brings survival back close to normal. On the molecular level, we observed elevated p27-levels in tumours of heteroand homozygous Pim-1 kinase-dead mice carrying the Em-c-myc transgene. The tumours of these mice also displayed areas with reduced levels of c-myc (Western blot and IHC). Based upon these results, we set-up an allogenic Em-c-myc mouse model that allowed us to study the effect of kinase inhibitors in an efficient and standardized way. We can show that novel, selective and potent Pim-1 kinase inhibitors that have been developed in our department are able to efficiently inhibit c-myc-driven tumour growth, as proposed by the genetic ablation of Pim-1 kinase activity. In summary, the presented study proofs inhibition of Pim-1 kinase activity to be a promising strategy to inhibit cancer growth driven by c-myc, one of the most frequently deregulated oncogenes in human cancer.

CS-28 * THE TRANSCRIPTION FACTOR POU3f2 UNIFORMLY EXPRESS IN HUMAN GLIOMAS AND PROMOTES TUMORIGENESIS AND OVERALL GROWTH RATE IN VIVO

The transcription factor POU3f2 is expressed during neurogenesis, and is essential for CNS development. Moreover, POU3f2 expression has been reported as a promoter of proliferation and invasion in malignant melanoma of the skin. Since both melanomas and CNS-malignancies arise in organs of neuroectodermal origin, we investigated whether human gliomas expressed POU3f2 and its potential role in glioma tumorigenesis. Immunohistochemistry results showed that POU3f2 was almost uniformly expressed in human gliomas, although at a varying degree. Microscopy revealed a predominantly nuclear staining pattern, but cytoplasmic immunopositivity for POU3f2 was also detected in some tumors. Moreover, western blot, qPCR and flow cytometry confirmed POU3f2 expression. Two independent observers estimated the nuclear staining index for all tumors, with 61% positive nuclei in GBM specimens, significantly higher than for grade II (37%, p = 0,0004) and grade III (36%, p = 0,0069) tumors. In acutely dissociated tumors, POU3f2 positive cells displayed an increased S- and G2/M-phase fraction. Moreover, overexpression of POU3f2 in the glioma cell lines increased its growth rate and colony formation, suggesting a role in regulating tumor cell proliferation, and hence possibly overall tumor aggressiveness. Cancer pathway reporter array showed that POU3f2 affects the NF-κB, MAPK/JNK and Myc/Max pathways. Overexpression of POU3f2 increase c-Myc expression and probably the POU3f2 drives cell proliferation elicited via activation of transcription of c-Myc target genes that are positive regulators of the cell cycle. On the other hand, POU3f2 regulates phosphorylation of histone H3 and might have an effect on epigenetic regulation through NF-kB signaling pathway. Ongoing studies aim at elucidating the mechanisms underlying the tumor-promoting effect of POU3f2, and will be presented at the meeting.

Abstract 2702: Suppression of c-Myc oncogenic signaling by MK591, an inhibitor of arachidonate 5-lipoxygenase

Abstract Background: Prostate cancer cells continuously generate 5(S)-HETE series of metabolites from arachidonic acid via 5-lipoxygenase (5-Lox) activity, and inhibition of 5-Lox blocks production of metabolites and triggers apoptosis both in androgen-sensitive as well as androgen-independent prostate cancer cells. This apoptosis is prevented by 5(S)-HETE and 5-oxoETE, but not by leukotrienes or metabolites of 12-Lox or 15-Lox, which is consistent with the idea that the 5-HETE series of metabolites are essential survival factors for prostate cancer cells. Thus, 5-Lox emerges as a novel molecular target for prostate cancer therapy. However, details of the downstream signaling mechanisms regulated by 5-Lox metabolites in prostate cancer cells are yet to be fully characterized, and the lack of suitable compounds with in vivo efficacy is hampering proper translational development to target 5-Lox. We addressed this problem by treating LNCaP human prostate cancer cells with MK591, a specific, third-generation inhibitor of 5-Lox activity, and analyzing the effects by gene-expression array, RT-PCR, and Western blot. Methods: Cells were treated with MK591 for eight hours and gene expression was analyzed by Illumina HT-12v4 whole genome gene-expression array. Expression of c-Myc was confirmed by RT-PCR and Western blot. Apoptosis was measured by annexin-V binding, PARP-cleavage, and detecting degradation of chromatin-DNA to nucleosomes. Transcriptional activation of c-Myc was analyzed by nuclear localization, DNA-binding, luciferase-reporter assays and expression of c-Myc-target genes. Results: We observed that MK591 strongly inhibits the expression of c-Myc oncogene in prostate cancer cells, as revealed by the gene expression array. Down-regulation of c-Myc by MK591 was confirmed by RT-PCR and Western blot as well as by the reduction in nuclear-localization and DNA-binding activities of c-Myc proteins. Treatment with MK591 dramatically decreased the Myc-driven E-box-luciferase reporter activity in prostate cancer cells which is effectively prevented by 5-oxoETE, a metabolic product of 5-Lox. Moreover, MK591 substantially reduced the expression of c-Myc target genes (e.g., cyclin D1, CDK4, survivin, Aurora kinase, Gemin4 and MCM10), and blocked the matrigel-invasion and soft-agar colony-formation by prostate cancer cells. Interestingly, while MK591 dramatically affected the Myc function in prostate cancer cells (where Myc is over-activated), it did not inhibit the basal c-Myc functions in non-cancer cells, such as human foreskin fibroblasts (HFF) which do not expresses 5-Lox. Conclusion: Our findings indicate that expression and oncogenic-function of c-Myc in prostate cancer cells are severely down-regulated by the 5-Lox inhibitor, MK591, and suggest that MK591 may be useful to treat advanced, aggressive prostate cancers which are frequently associated with over-activation of the c-Myc oncogene. Citation Format: Sivalokanathan Sarveswaran, Debrup Chakraborty, Jagadananda Ghosh. Suppression of c-Myc oncogenic signaling by MK591, an inhibitor of arachidonate 5-lipoxygenase. [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 2702. doi:10.1158/1538-7445.AM2014-2702

The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus.

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

MiR-506 suppresses proliferation of hepatoma cells through targeting YAP mRNA 3'UTR.

MiR-506 is a miRNA involved in carcinogenesis of several kinds of cancer. In this study, we explored whether miR-506 played a critical role in hepatocellular carcinoma (HCC). Twenty HCC and adjacent normal liver tissue samples were collected. Human hepatoma cell lines HepG2 and H7402 were used for in vitro studies. The expression of miR-506 and transcriptional co-activator YAP was examined using qRT-PCR. Western blot analysis was used to measure the expression of YAP and its target genes. Luciferase reporter gene assay was used to identify YAP as a target gene of miR-506. MTT and EdU assays were carried out for functional analysis. The expression of miR-506 was significantly lower in HCC than in adjacent normal liver tissues. Bioinformatics analysis revealed that YAP mRNA might be one of the targets of miR-506, and miR-506 in HCC tissues was found to be negatively correlated with YAP (r=-0.605). In both HepG2 and H7402 cells, miR-506 dose-dependently down-regulated YAP and its target genes c-Myc and the connective tissue growth factor (CTGF). Luciferase reporter gene assays demonstrated that miR-506 targeted the wild type 3'UTR of YAP mRNA, but not a 3'UTR with a mutant seed site. Furthermore, miR-506 significantly inhibited the proliferation of HepG2 and H7402 cells, while anti-miR-506 enhanced the cell proliferation, which was blocked by YAP siRNA. MiR-506 suppresses the proliferation of hepatoma cells by targeting YAP mRNA.

Inhibitory effect of 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo(b)pyran (K-1) on human primary endometrial hyperplasial cells mediated via combined suppression of Wnt/β-catenin signaling and PI3K/Akt survival pathway.

Endometrial hyperplasia is a precursor to the most common gynecologic cancer diagnosed in women. Apart from estrogenic induction, aberrant activation of the Wnt/β-catenin signal is well known to correlate with endometrial hyperplasia and its carcinoma. The benzopyran compound 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo (b) pyran(K-1), a potent antiestrogenic agent, has been shown to have apoptosis-inducing activity in rat uterine hyperplasia. The current study was undertaken to explore the effect of the benzopyran compound K-1 on growth and Wnt signaling in human endometrial hyperplasial cells. Primary culture of atypical endometrial hyperplasial cells was characterized by the epithelial cell marker cytokeratin-7. Results revealed that compound K-1 reduced the viability of primary endometrial hyperplasial cells and expression of ERα, PR, PCNA, Wnt7a, FZD6, pGsk3β and β-catenin without affecting the growth of the primary culture of normal endometrial cells. The β-catenin target genes CyclinD1 and c-myc were also found to be reduced, whereas the expression of axin2 and Wnt/β-catenin signaling inhibitor Dkk-1 was found to be upregulated, which caused the reduced interaction of Wnt7a and FZD6. Nuclear accumulation of β-catenin was found to be decreased by compound K-1. K-1 also suppressed the pPI3K/pAkt survival pathway and induced the cleavage of caspases and PARP, thus subsequently causing the apoptosis of endometrial hyperplasial cells. In conclusion, compound K-1 suppressed the growth of human primary endometrial hyperplasial cells through discontinued Wnt/β-catenin signaling and induced apoptosis via inhibiting the PI3K/Akt survival pathway.

The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer.

Colorectal cancer is the third most common cancer worldwide. Although the transcription factor c-MYC is misregulated in the majority of colorectal tumors, it is difficult to target directly. The deubiquitinase USP28 stabilizes oncogenic factors, including c-MYC; however, the contribution of USP28 in tumorigenesis, particularly in the intestine, is unknown. Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin-dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine. Mice lacking Usp28 had no apparent adverse phenotypes, but exhibited reduced intestinal proliferation and impaired differentiation of secretory lineage cells. In a murine model of colorectal cancer, Usp28 deletion resulted in fewer intestinal tumors, and importantly, in established tumors, Usp28 deletion reduced tumor size and dramatically increased lifespan. Moreover, we identified Usp28 as a c-MYC target gene highly expressed in murine and human intestinal cancers, which indicates that USP28 and c-MYC form a positive feedback loop that maintains high c-MYC protein levels in tumors. Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models. Hence, inhibition of the enzymatic activity of USP28 may be a potential target for cancer therapy.

Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer.

The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50-60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.

Development of resistance to BET bromodomain inhibitor JQ1 and demonstration of epithelial-mesenchymal transition in pancreatic cancer cells.

e15263 Background: Pancreatic ductal adenocarcinoma (PDAC) continues to be the leading cause of cancer related death in part due to limited efficacy of conventional chemotherapy. As such, there is increasing interest in developing inhibitors targeting epigenetic changes, including targeting BET proteins that bind acetylated histones to regulate gene transcription. We have found that the BET-inhibitor JQ1 is effective against pancreatic cancer cells. Since cancer cells can develop resistance to targeted therapies, we examined whether it was possible for PDAC cells to develop resistance to JQ1. Methods: The PDAC cell line CD18 cells (CD18-P) were treated with increasing concentration of JQ1 over a prolonged period of time to develop JQ1-resistent cells (CD18-JQ1r). We also generated CD18 cells resistant to 5-fluorouracil (CD18-CR) by treating CD18-P cells with increasing concentration of 5-fluorouracil. Results: Unlike CD18-P, CD18-JQ1r cells were resistant to the effects of JQ1 in 3D collagen. Moreover, JQ1 target genes c-MYC, FOSL1, and HMGA2, which are repressed in CD18-P cells treated with acute JQ1 treatment, were not repressed in CD18-JQ1R cells. When compared to the chemoresistant cells (CD18-CR) CD18-JQ1r showed less evidence of mesenchymal phenotype with lack of fibroblastic phenotype, repression of E-cadherin without expression of vimentin, repression of miR200 family of microRNA and upregulation of ZEB1. Additionally, CD18-JQ1r showed decreased cell matrix adhesion, lower levels of a2 and b1-integrin expression, and reduced FAK signaling. Transfection with ZEB1 siRNA restored cell-cell and cell-matrix adhesion defects seen in CD18-JQ1r cells. Moreover, ZEB1 siRNA partially repressed c-MYC, FOSL1 and HMGA2 in CD18-JQ1r cells. Conclusions: Pancreatic cancer cells can develop resistance to BET inhibitors. These resistant cells demonstrate defect in cell-cell and cell-matrix adhesion driven by ZEB1. Knockdown of ZEB1 can restore epithelial phenotype and enhance cell-cell and cell-matrix adhesion, and partly repress JQ1-target genes c-MYC, FOSL1 and HMGA2.