c-Myc Transcriptional Activity Research Articles

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis.

Background:Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear.Methods:si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role.Results:Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways.Conclusions:Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS.

Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells.

Leukemia remains life-threatening despite remarkable advances in chemotherapy. The poor prognosis and drug resistance are challenging treatment. Novel drugs are urgently needed. Shikonin, a natural naphthoquinone, has been previously shown by us to be particularly effective towards various leukemia cell lines compared to solid tumors. However, the underlying mechanisms are still poorly understood. Here, we investigated shikonin and 14 derivatives on U937 leukemia cells. Four derivatives (isobutyrylshikonin, 2-methylbutyrylshikonin, isovalerylshikonin and β,β-dimethylacrylshikonin) were more active than shikonin. AnnexinV-PI analysis revealed that shikonins induced apoptosis. Cell cycle G1/S check point regulation and the transcription factor c-MYC, which plays a vital role in cell cycle regulation and proliferation, were identified as the most commonly down-regulated mechanisms upon treatment with shikonins in mRNA microarray hybridizations. Western blotting and DNA-binding assays confirmed the inhibition of c-MYC expression and transcriptional activity by shikonins. Reduction of c-MYC expression was closely associated with deregulated ERK, JNK MAPK and AKT activity, indicating their involvement in shikonin-triggered c-MYC inactivation. Molecular docking studies revealed that shikonin and its derivatives bind to the same DNA-binding domain of c-MYC as the known c-MYC inhibitors 10058-F4 and 10074-G5. This finding indicates that shikonins bind to c-MYC. The effect of shikonin on U937 cells was confirmed in other leukemia cell lines (Jurkat, Molt4, CCRF-CEM, and multidrug-resistant CEM/ADR5000), where shikonin also inhibited c-MYC expression and influenced phosphorylation of AKT, ERK1/2, and SAPK/JNK. In summary, inhibition of c-MYC and related pathways represents a novel mechanism of shikonin and its derivatives to explain their anti-leukemic activity.

Regulation of c-Myc expression by the histone demethylase JMJD1A is essential for prostate cancer cell growth and survival.

The histone demethylase JMJD1A, which controls gene expression by epigenetic regulation of H3K9 methylation marks, functions in diverse activities, including spermatogenesis, metabolism, and stem cell self-renewal and differentiation. Here, we found that JMJD1A knockdown in prostate cancer cells antagonizes their proliferation and survival. Profiling array analyses revealed that JMJD1A-dependent genes function in cellular growth, proliferation and survival, and implicated that the c-Myc transcriptional network is de-regulated following JMJD1A inhibition. Biochemical analyses confirmed that JMJD1A enhances c-Myc transcriptional activity by upregulating c-Myc expression levels. Mechanistically, JMJD1A activity promoted recruitment of androgen receptor (AR) to the c-Myc gene enhancer and induced H3K9 demethylation, increasing AR-dependent transcription of c-Myc mRNA. In parallel, we found that JMJD1A regulated c-Myc stability, likely by inhibiting HUWE1, an E3 ubiquitin ligase known to target degradation of several substrates including c-Myc. JMJD1A (wild-type or mutant lacking histone demethylase activity) bound to HUWE1, attenuated HUWE1-dependent ubiquitination and subsequent degradation of c-Myc, increasing c-Myc protein levels. Furthermore, c-Myc knockdown in prostate cancer cells phenocopied effects of JMJD1A knockdown, and c-Myc re-expression in JMJD1A-knockdown cells partially rescued prostate cancer cell growth in vitro and in vivo. c-Myc protein levels were positively correlated with those of JMJD1A in a subset of human prostate cancer specimens. Collectively, our findings identify a critical role for JMJD1A in regulating proliferation and survival of prostate cancer cells by controlling c-Myc expression at transcriptional and post-translational levels.

Abstract 1246: c-Myc is critical for apoptosis in 4-hyroxynonenal-treated colorectal cancer cells

Abstract The transcription factor c-myc has an important role in the control of cell proliferation, differentiation and apoptosis. The dysregulation of c-myc activity is a feature of many cancers. Although it enhances proliferation, the overexpression of c-myc also sensitizes cells to a variety of pro-apoptotic stimuli. 4-hydroxynonenal (HNE) is a reactive lipid that is generated as a consequence of oxidative stress, and is found to be elevated in many solid lesions. HNE is known to promote apoptosis in various cancer cell types, including colorectal cancer cells. We therefore decided to investigate whether c-myc has a role in HNE-induced apoptosis in this setting. We began by investigating the effect of HNE on c-myc expression and function. Treatment of colorectal cancer cell lines RKO and HCT-116 with HNE caused a time- and concentration-dependent nuclear accumulation of c-myc protein. The increase in nuclear c-myc levels was not accompanied by an increase in either c-myc mRNA or total cellular levels, leading us to speculate that enhanced nuclear import or sequestration are involved. To evaluate whether c-myc activity was also increased in response to HNE, we used a luciferase reporter construct bearing a conserved c-myc binding element. These data reveal that in addition to causing its nuclear accumulation, HNE also enhances c-myc transcriptional activity. To examine the consequence of c-myc activation and explore its role in HNE-induced apoptosis, we silenced c-myc expression using 3 different siRNA sequences in both the RKO and HCT-116 cell lines. Silencing c-myc expression caused a significant attenuation of apoptosis in HNE-treated cells, as evidenced by a reduction in caspase 3 cleavage, PARP cleavage, and caspase 3 activity assays. To investigate the reason behind this effect, we examined known pathways for HNE-induced apoptosis. In accordance with published data, we find that HNE promotes apoptosis through the ASK1-JNK pathway, and that apoptosis can be blocked with an ASK1-specific inhibitor. Furthermore, our data show for the first time that silencing c-myc expression leads to a profound reduction in the phosphorylation and activity of JNK, which we propose explains the reduction in apoptosis in c-myc deficient cells. Since c-myc is a protein of interest and potential target for colorectal therapy, the consequence of its inhibition on cancer cell viability and cell death pathways is critical to understand. Together, our work reveals a novel pathway for c-myc mediated apoptosis and highlights its importance in HNE-treated cancer cells. Citation Format: Christina TK Wales, Rachel Gristock, Nadine So, Aaron T. Jacobs. c-Myc is critical for apoptosis in 4-hyroxynonenal-treated colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1246. doi:10.1158/1538-7445.AM2015-1246

Xenosensor CAR mediates down-regulation of miR-122 and up-regulation of miR-122 targets in the liver

MiR-122 is a major hepatic microRNA, accounting for more than 70% of the total liver miRNA population. It has been shown that miR-122 is associated with liver diseases, including hepatocellular carcinoma. Mir-122 is an intergenic miRNA with its own promoter. Pri-miR-122 expression is regulated by liver-enriched transcription factors, mainly by HNF4α, which mediates the expression via the interaction with a specific DR1 site. It has been shown that phenobarbital-mediated activation of constitutive androstane receptor (CAR), xenobiotic nuclear receptor, is associated with a decrease in miR-122 in the liver. In the present study, we investigated HNF4α–CAR cross-talk in the regulation of miR-122 levels and promitogenic signalling in mouse livers. The level of miR-122 was significantly repressed by treatment with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is an agonist of mouse CAR. ChIP assays demonstrated that TCPOBOP-activated CAR inhibited HNF4α transactivation by competing with HNF4α for binding to the DR1 site in the pri-miR-122 promoter. Such transcription factor replacement was strongly correlated with miR-122 down-regulation. Additionally, the decrease in miR-122 levels produced by CAR activation is accompanied by an increase in mRNA and cellular protein levels of E2f1 and its accumulation on the target cMyc gene promoter. The increase in accumulation of E2f1 on the target cMyc gene promoter is accompanied by an increase in cMyc levels and transcriptional activity. Thus, our results provide evidence to support the conclusion that CAR activation decreases miR-122 levels through suppression of HNF4α transcriptional activity and indirectly regulates the promitogenic protein cMyc. HNF4α–CAR cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments.

SCP1 regulates c-Myc stability and functions through dephosphorylating c-Myc Ser62.

Serine 62 (Ser62) phosphorylation affects the c-Myc protein stability in cancer cells. However, the mechanism for dephosphorylating c-Myc is not well understood. In this study, we identified carboxyl-terminal domain RNA polymerase II polypeptide A small phosphatase 1 (SCP1) as a novel phosphatase specifically dephosphorylating c-Myc Ser62. Ectopically expressed SCP1 strongly dephosphorylated c-Myc Ser62, destabilized c-Myc protein and suppressed c-Myc transcriptional activity. Knockdown of SCP1 increased the c-Myc protein levels in liver cancer cells. SCP1 interacted with c-Myc both in vivo and in vitro. In addition, Ser245 at the C-terminus of SCP1 was essential for its phosphatase activity towards c-Myc. Functionally, SCP1 negatively regulated the cancer cell proliferation. Collectively, our findings indicate that SCP1 is a potential tumor suppressor for liver cancers through dephosphorylating c-Myc Ser62.

Inauhzin(c) Inactivates c-Myc Independently of p53

Oncogene MYC is deregulated in many human cancers, especially in lymphoma. Previously, we showed that inauhzin (INZ) activates p53 and inhibits tumor growth. However, whether INZ could suppress cancer cell growth independently of p53 activity is still elusive. Here, we report that INZ(c), a second generation of INZ, suppresses c-Myc activity and thus inhibits growth of human lymphoma cells in a p53-independent manner. INZ(c) treatment decreased c-Myc expression at both mRNA and protein level, and suppressed c-Myc transcriptional activity in human Burkitt's lymphoma Raji cells with mutant p53. Also, we showed that overexpressing ectopic c-Myc rescues the inhibition of cell proliferation by INZ(c) in Raji cells, implicating c-Myc activity is targeted by INZ(c). Interestingly, the effect of INZ(c) on c-Myc expression was impaired by disrupting the targeting of c-Myc mRNA by miRNAs via knockdown of ribosomal protein (RP) L5, RPL11, or Ago2, a subunit of RISC complex, indicating that INZ(c) targets c-Myc via miRNA pathways. These results reveal a new mechanism that INZ(c) targets c-Myc activity in human lymphoma cells.

Attacking c-Myc: targeted and combined therapies for cancer.

The onset of cancer is a complex process that is driven by the accumulation of multiple genetic mutations. However, the fact that inhibition of a single oncogene can impair the proliferation and survival of cancer cells due to their "oncogene addiction" provides implications for the so-called "molecular targeted therapy" in cancer treatment. The oncogenic transcription factor c-Myc is overexpressed in many types of cancers, and as a typical oncogene to which many cancers are addicted, c-Myc is necessary for the rapid proliferation of cancer cells. Strategies aimed at targeting c-Myc, including interfering with c-Myc synthesis, stability and transcriptional activity, have emerged as effective cancer treatments. We have recently shown that a natural agent, oridonin, promotes the Fbw7-mediated proteasomal degradation of c-Myc, leading to subsequent cell growth inhibition and apoptosis and demonstrating a new c-Myc-targeting strategy. Despite the effectiveness of molecular targeting in cancer treatment, failure to achieve long-lasting efficacy with a single agent is observed because cancer cells can recover from oncogene addiction as a result of their genomic instability and heterogeneity. Combined cancer therapies were therefore developed and showed better efficacies than single-agent therapy in cancer cell lines and mouse models. Combined therapy based on c-Myc targeting can be achieved through various strategies. Agents that also target c-Myc but use different mechanisms, or agents that act on other genes in the c-Myc pathway, can be selected for combination. In addition, the targeting of genes involved in different cellular processes in other pathways might also be a successful strategy. Regardless of the therapy adopted, it is important to first determine the molecular mechanisms underlying the agents to inform the therapy design. Among the various targets of therapeutic agents is a family of noncoding small RNAs, called microRNAs, that have been implicated in the anti-cancer activity of many therapeutic agents. c-Myc, as a transcription factor, regulates the expression of many microRNAs and is in turn regulated by microRNAs. Combining c-Myc-targeting agents with those that target microRNAs might provide a novel approach for cancer therapy.

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

Critical role of SHP2 (PTPN11) signaling in germinal center-derived lymphoma.

Germinal center lymphoma is a heterogeneous human lymphoma entity. Here we report that constitutive activity of SHP2 (PTPN11) and its downstream kinase ERK is essential for the viability of germinal center lymphoma cells and disease progression. Mechanistically, SHP2/ERK inhibition impedes c-Myc transcriptional activity, which results in the repression of proliferative phenotype signatures of germinal center lymphoma. Furthermore, SHP2/ERK signaling is required to maintain the CD19/c-Myc loop, which preferentially promotes survival of a distinct subtype of germinal center lymphoma cells carrying the MYC/IGH translocation. These findings demonstrate a critical function for SHP2/ERK signaling upstream of c-Myc in germinal center lymphoma cells and provide a rationale for targeting SHP2 in the therapy of germinal center lymphoma.

E2F1 acts as a negative feedback regulator of c-Myc‑induced hTERT transcription during tumorigenesis.

Since induction of hTERT expression and subsequent telomerase activation play a critical role in the multistep process of tumorigenesis, a better understanding of hTERT regulation may provide not only a rationale for the molecular basis of cancer progression but also a path to the development of cancer prevention. The c-Myc oncoprotein can function effectively in activating the transcriptional expression of hTERT through E-box elements on its promoter. E2F transcription factor1(E2F1) was found to be a repressor of hTERT transcription by directly binding to its promoter, thereby inhibiting hTERT protein expression. For the extensively crosstalk between c-Myc and E2F1 signals, which is now known to be vital to cell fate, we speculated that E2F1 may play a negative regulatory role in c-Myc-induced hTERT transcription. In the present study, we chose to use human embryonic fibroblast cells as an experimental model system, and present evidence that the E2F1 transcription factor constitutes a negative regulatory system to limit c-Myc transcriptional activation of hTERT in normal cells. Furthermore, we demonstrated that upregulation of the miR-17-92 cluster (miR-20a/miR-17-5p) is involved in the regulation of E2F1-mediated negative feedback of the c-Myc/hTERT pathway. Our results not only reveal novel insights into how normal cells control the transmission of c-Myc-mediated oncogenic signals, but also further establish E2F1 as an important molecular target for cancer therapy.

Expression of androgen receptor target genes in skeletal muscle

We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR)-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (ARΔZF2) versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in ARΔZF2 muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7, p57Kip2, Igf2 and calcineurin Aa, was increased in ARΔZF2 muscle, and the expression of all but p57Kip2 was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

Changes of c-Myc and DNMT1 mRNA and protein levels in the rat livers induced by dibutyl phthalate treatment

We investigated the relationship between dibutyl phthalate (DBP)-induced hypomethylation of the c-Myc promoter region (as evident in our early study) and the expression of c-Myc and DNMT1 genes (at messenger RNA (mRNA) and protein level) in the rat liver. Male Wistar rats received DBP in 1, 3, or 14 daily doses of 1800 mg kg(-1) body weight. Levels of DNMT1, c-Myc mRNA, and proteins were detected using real-time polymerase chain reaction and Western blot analysis, respectively. Our findings indicate that DBP caused an increase in mRNA levels of c-Myc at all time points. The results showed that protein levels of c-Myc in rat liver also increased significantly by DBP treatment, which were more pronounced at last time point (after 14 doses). Furthermore, overexpression of DNMT1gene have been found after one dose of DBP, which was confirmed at the protein level by Western blot analysis. Reduced levels of DNMT1mRNA and proteins (3 and 14 doses) were coordinated with depletion DNA synthesis (reported previously). Based on our previous results and those presented here, the following conclusion could be drawn: (1) DBP exerted biological activity through epigenetic modulation of c-Myc gene expression; (2) it seems possible that DBP-induced active demethylation of c-Myc gene through mechanism(s) linked to generation of reactive oxygen species by activated c-Myc; and (3) control of DNA replication was not directly dependent on c-Myc transcriptional activity and we attribute this finding to DNMT1gene expression which was tightly coordinated with DNA synthesis.

USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

Bromodichloromethane induces cell proliferation in different tissues of male F344 rats by suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1

The aim of this study was to investigate the mechanism of bromodichloromethane (BDCM) – induced cell proliferation in different tissues of male F344 rats. Rats were administered at doses of 0 and 100mg/kg/day BDCM dissolved in corn oil by gavage for 5 days/week for 1, 4, 8 and 12 weeks. Then the colon, kidney and liver were collected. No histologic lesions were observed in the colon of rats exposed to BDCM, while there were mild nephrotoxicity and marginal hepatotoxicity related to BDCM treatment. Moreover, BDCM enhanced cell proliferation in the colon and kidney but not in the liver. In colons, hypermethylation in E-cadherin promoter might be associated with inhibition of mRNA and protein expression after 12 weeks of BDCM exposure. In kidneys, BDCM decreased E-cadherin mRNA expression, accompanying with transcriptional activation of c-myc and cyclin D1. However, suppression of E-cadherin mRNA and protein expression occurred in the absence of significant changes in DNA methylation. Therefore, suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1 may be involved in BDCM-induced cell proliferation in different tissues of male F344 rats.

Ribosomal Protein S14 Negatively Regulates c-Myc Activity

The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA.

Genome Wide Proteomics of ERBB2 and EGFR and Other Oncogenic Pathways in Inflammatory Breast Cancer

In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with reads per kilobase per million mapped reads (RPKM) values for ERBB2 (14.4, 400, and 300 for SUM149, SUM190, and SKBR3, respectively) and for EGFR (60.1, not detected, and 1.4 for the same 3 cell lines). We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g., 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used bioinformatics sites GeneGo, PathwayCommons and NCI receptor signaling networks to identify pathways that contained the four main oncogenes and had good coverage in the transcriptomic and proteomic data sets as well as a significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling; caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings; branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 subpathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.

Abstract 2918: Docosahexaenoic acid-induced oxidative stress increases cellular apoptosis through a c-MYC-dependent mechanism in breast cancer.

Abstract MYC is a potent oncogene that transcriptionally regulates 15 percent of the genome and is found to be deregulated in approximately 70 percent of cancers (c-MYC), including breast cancer. Activation of c-MYC induces association between its required partners, MYC-associated factor X (MAX) or MAX dimerization protein (MAD), regulating pro-survival and pro-apoptotic pathways, respectively. c-MYC has proved to be a difficult molecular target of inhibition. Only recently have there been strategies providing a way to inhibit c-MYC-dependent tumors and its downstream targets. However, dietary factors like, long-chain fatty acids (LCFA), have been shown to change activity and expression levels of c-MYC in cell culture and animal models. LCFAs have also been reported to induce apoptosis through c-MYC. The present study evaluated whether changes in c-MYC expression or activity were responsible for induction of oxidative stress and apoptosis in response to docosahexaenoic acid (DHA, C22:6) supplementation in breast cancer. c-MYC mRNA and protein levels were determined in vitro in the breast cancer BT-474, MDA-MB-231, and the non-transformed MCF-10A cell lines. qPCR results showed increases in c-MYC mRNA expression upon PUFA treatment in both BT-474 and MDA-MB-231 cancer lines, but not in the MCF-10A non-transformed line. Protein analysis revealed no changes in total c-MYC expression, however total phosphorylation at Thr58 and Ser62 are significantly upregulated in a dose-response manner upon DHA supplementation in the BT-474 cell line. Remarkably, transcriptional activity of c-MYC is highly upregulated as well, suggesting that c-MYC activity increases in response to DHA treatment. Using dihydrodichlorofluorescein diacetate (H2-DCF-DA) assay, which measures intracellular reactive oxygen species (ROS) and oxidative stress, we found increases in response to DHA treatment in all cell lines. Finally, the increase in oxidative stress also resulted in an increase in apoptosis, which we suggest is through c-MYC activation. Further analysis of the c-MYC isoform status, association between MYC-MAX and MYC-MAD complexes, as well as inducers and inhibitors of c-MYC are currently underway to define specific targets where DHA is applying some of its anti-cancer effects. Ultimately, with a goal that this research will reveal dietary targets of DHA that could be used to enhance efficacy to current therapies. Citation Format: Michael Mouradian, Haviva Kobany, Christopher R. Douglas, Nam D. Hoang, Keith D. Kikawa, Ronald S. Pardini. Docosahexaenoic acid-induced oxidative stress increases cellular apoptosis through a c-MYC-dependent mechanism in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2918. doi:10.1158/1538-7445.AM2013-2918

Abstract 771: Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis.

Abstract The c-Myc oncoprotein is generally considered to be a master regulator of gene transcription by virtue of its ability to activate or repress the expression of a large percentage of all genes. However, mechanisms that direct c-Myc's recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl-isomerase regulates c-Myc transcriptional activity by altering c-Myc's association with target gene promoters. Specifically, we show that Pin1 enhances the recruitment of Serine62-phosphorylated c-Myc along with its co-activators to selected target gene promoters during gene activation, followed by promoting c-Myc's release associated with its degradation, resulting in cyclic c-Myc DNA binding. This facilitates c-Myc's activation of target genes associated with an increase in elongating RNA Polymerase II in the gene body. Genes impacted by this mechanism are involved in cell growth and metabolism, resulting in enhanced pro-proliferative activity, even while controlling c-Myc expression. In cancer cells with impaired c-Myc degradation, Pin1 still regulates cyclic c-Myc DNA binding, even though it no longer enhances c-Myc degradation. This allows for simultaneous high expression of Pin1 and c-Myc in cancer, which can drive a gene expression pattern that we show is enriched in poor outcome subtypes of breast cancer. This study provides new insight into mechanisms regulating c-Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in transcription factor regulation, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and c-Myc. Citation Format: Amy Farrell, Carl Pelz, Xiaoyan Wang, Colin Daniel, Yulong Su, Mahnaz Janghorban, Soren Impey, Rosalie C. Sears. Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2013-771

C-Myc phosphorylation by PKCζ represses prostate tumorigenesis

Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.