Expression Of C-myc Oncogene Research Articles

Pharmacological activation of FOXO3 suppresses triple-negative breast cancer in vitro and in vivo.

Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. Lacking effective therapeutic options hinders treatment of TNBC. Here, we show that bepridil (BPD) and trifluoperazine (TFP), which are FDA-approved drugs for treatment of schizophrenia and angina respectively, inhibit Akt-pS473 phosphorylation and promote FOXO3 nuclear localization and activation in TNBC cells. BPD and TFP inhibit survival and proliferation in TNBC cells and suppress the growth of TNBC tumors, whereas silencing FOXO3 reduces the BPD- and TFP-mediated suppression of survival in TNBC cells. While BPD and TFP decrease the expression of oncogenic c-Myc, KLF5, and dopamine receptor DRD2 in TNBC cells, silencing FOXO3 diminishes BPD- and TFP-mediated repression of the expression of these proteins in TNBC cells. Since c-Myc, KLF5, and DRD2 have been suggested to increase cancer stem cell-like populations in various tumors, reducing these proteins in response to BPD and TFP suggests a novel FOXO3-dependent mechanism underlying BPD- and TFP-induced apoptosis in TNBC cells.

Abstract 2280: A novel c-Myc transgenic model for human cancers

Abstract Androgen-deprivation therapy (ADT) is a standard therapy for advanced and metastatic prostate cancer (PCa). However, most PCa will relapse and become the lethal castration-resistant PCa (CRPC). Chemotherapy such as Docetaxel, only modestly extends patient survival. To overcome castration resistance and chemoresistance, it is crucial to delineate the underlying molecular mechanisms. c-Myc is the most significantly amplified oncogene in human PCa, and its overexpression is very common in PCa as early as PIN. The Hi-Myc model uses an enhanced probasin promoter to drive c-Myc overexpression in prostate epithelia. Hi-Myc mice develop invasive prostate carcinomas that share molecular features with human PCa. However, since the probasin promoter activity is androgen-dependent, Hi-Myc tumors lose c-Myc expression upon castration. Therefore, the tumor regression represents the mixed effects of both an artificial direct effect from loss of c-Myc oncogene expression and a potential real effect from tumor cellular response to androgen-ablation that is relevant to human PCa. Finally, Hi-Myc mice do not develop CRPC tumors. To study CRPC and chemoresistance of CRPC, we have developed a novel transgenic model to express the c-Myc oncogene in Any tissue in an inducible manner (Ai-Myc model). After crossing with PB-Cre4 mice expressing Cre in prostate epithelia, this model allows us to specifically turn on the expression of c-Myc along with Luc2 (an enhanced luciferase for BLI imaging) in prostate. In addition, once turned on, the expression of c-Myc and Luc2 are no longer dependent on androgen. The PB-Cre4/Ai-Myc mice developed early onset of mPIN, but with much delayed prostate tumor progression. Only a fraction of mice developed significant prostate tumors after one-year of age. Since c-Myc oncogene may induce p53 tumor suppressor activation, and loss or mutation of p53 is a frequent event in late-stage human PCa, we further crossed these mice with p53loxP/loxP mice to generate the PB-Cre4/Ai-Myc/p53loxP/loxP model. Loss of p53 greatly enhanced prostate tumor progression; about 40% of mice developed significant prostate tumors within 4-5 months when all mice have to be sacrificed due to the unexpected growth of large tumors of the epididymis, a rare cancer in human. These epididymis tumors are positive for c-Myc and Luc2, confirming the previously reported off-target activity of PB-Cre4 mice. The nature of these epididymis tumors remains to be determined. We are exploring using several approaches, including early castration together with implantation of androgen pellets, to overcome this problem, which will allow us to concisely study the c-Myc signaling pathway in castration response, castration and chemo-resistance of prostate tumors. Finally, different tissue / organ-specific Cre transgenic mouse line will allow our Ai-Myc model to be used to study different types of human cancers and/or other diseases. Citation Format: Wei Wang, Jiangong Ren, Bingning Dong, Michael M. Ittmann, David D. Moore, Feng Yang. A novel c-Myc transgenic model for human cancers. [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 2280. doi:10.1158/1538-7445.AM2015-2280

Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model.

Growth arrest and DNA damage-inducible protein (GADD34/Ppp1r15a) is induced by various stimuli including DNA damage and ER stress. DNA damage and oncogene activation, accompanied by tumor-specific DNA repair defects and a failure to stall the cell cycle, are early markers of hepatocellular carcinoma (HCC). However, whether GADD34 accounts for regulating HCC tumorigenesis remains elusive. Here, we demonstrated that GADD34 expression was upregulated in the liver of mice after exposure to a carcinogen, diethylnitrosamine (DEN). In both acute and chronic DEN treatment models, GADD34 deficiency not only decreased oncogene expression, but also reduced hepatic damage. Moreover, loss of GADD34 attenuated immune cell infiltration, pro-inflammatory cytokine expression and hepatic compensatory proliferation. Finally, GADD34-deficient mice showed impaired hepatocarcinogenesis. Thus, the process of DEN-induced HCC proceeded as follows. First, DEN treatment induced DNA damage in hepatocytes, resulting in elevated expression of GADD34 in the liver. The increased expression of GADD34 augmented hepatic necrosis followed by elevated expression of interleukin (IL)-1β and monocyte chemoattractant protein 1. This process promoted immune cell infiltration and Kupffer cell/macrophage activation followed by production of reactive oxygen species and pro-tumorigenic cytokines such as IL-6 and tumor necrosis factor-α. The pro-tumorigenic cytokines stimulated compensatory proliferation of surviving and mutant hepatocytes. Together with oncogene c-Myc expression, these processes led to HCC. Our results suggest therapeutic opportunities for HCC by targeting GADD34-related pathways.

Inhibition of 5-Lipoxygenase Selectively Triggers Disruption of c-Myc Signaling in Prostate Cancer Cells

Myc is up-regulated in almost all cancer types and is the subject of intense investigation because of its pleiotropic effects controlling a broad spectrum of cell functions. However, despite its recognition as a stand-alone molecular target, development of suitable strategies to block its function is hindered because of its nonenzymatic nature. We reported earlier that arachidonate 5-lipoxygenase (5-Lox) plays an important role in the survival and growth of prostate cancer cells, although details of the underlying mechanisms have yet to be characterized. By whole genome gene expression array, we observed that inhibition of 5-Lox severely down-regulates the expression of c-Myc oncogene in prostate cancer cells. Moreover, inhibition of 5-Lox dramatically decreases the protein level, nuclear accumulation, DNA binding, and transcriptional activities of c-Myc. Both the 5-Lox inhibition-induced down-regulation of c-Myc and induction of apoptosis are mitigated when the cells are treated with 5-oxoeicosatetraenoic acid, a metabolite of 5-Lox, confirming a role of 5-Lox in these processes. c-Myc is a transforming oncogene widely expressed in prostate cancer cells and maintains their transformed phenotype. Interestingly, MK591, a specific 5-Lox inhibitor, strongly affects the viability of Myc-overactivated prostate cancer cells and completely blocks their invasive and soft agar colony-forming abilities, but it spares nontransformed cells where expression of 5-Lox is undetectable. These findings indicate that the oncogenic function of c-Myc in prostate cancer cells is regulated by 5-Lox activity, revealing a novel mechanism of 5-Lox action and suggesting that the oncogenic function of c-Myc can be suppressed by suitable inhibitors of 5-Lox.

Eosinophil MBP Extract Modulates Oncogene Expression in Prostate Tumor Cells: A Preliminary Study with Monolayer Cultures

Prostate cancer is the second leading cause of cancer deaths in the United States and remains a significant health concern for men throughout the world. Despite the discovery of promising immunotherapeutic strategies, curative outcomes remain elusive. We have investigated eosinophils as potential anti-cancer effector cells, and have reported the ability of their toxic granular proteins (MBP, EPO, ECP, EDN) to inhibit prostate tumor cell growth in vitro. This study investigates the effect of eosinophil MBP extract on the expression of oncogenes p53, bcl-xl, bax, and c-myc, which modulate tumor growth, proliferation, and apoptosis. Briefly, granular proteins were differentially extracted from GRC.014.22 and GRC.014.24, eosinophilic cell lines established in our laboratory from a patient with moderate asthma. Protein extracts were fractionated on Sephadex G-50 columns, and prostate tumor cell lines DU-145, LNCaP, PC-3, and HPC8L (established in our laboratory from a tumor resected from an African American patient) were treated with MBP extracts from the pooled third peaks. Colony formation and monolayer cell growth inhibition assays were used to evaluate the protein’s growth inhibitory activity against prostate tumor cells; and gene expression analyses, to determine p53, bcl-xl, bax, and c-myc oncogene expression. We show that the granular proteins were potent in their action on HPC8L, inhibiting colony formation in a dose-dependent manner. Treated prostate tumor cell lines trended toward apoptosis-induction, as evident in bcl-xl/bax ratios < 1, increased p53 expression, and up or downregulation of c-myc. These preliminary results demonstrate the growth inhibitory potential of eosinophil granular proteins and strongly support the hypothesis that eosinophils modulate the expression of oncogenes associated with prostate tumor proliferation and apoptosis. More importantly, this study offers insights into possible applications of eosinophilic mediators in oncogenic-targeted prostate cancer treatment strategies and demonstrates the potential therapeutic implications of enhancing eosinophilic activity in prostate cancer.

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

High-Throughput Fluorescence Anisotropy Screen for Inhibitors of the Oncogenic mRNA Binding Protein, IMP-1

Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z′ factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1–positive cancer cells with very little or no effect on proliferation of IMP-1–negative cells.

The Effects of Combination Treatment Using Phenoxodiol and Docetaxel, and Phenoxodiol and Secreted Frizzled-related Protein 4 on Prostate Cancer Cell Lines

Although much progress has been made for the treatment of prostate cancer, patients with advanced prostate cancer still have a poor 5 year survival rate. Current practices for hormone-refractory/castrate resistant, metastatic prostate cancer involve the use of taxanes. Docetaxel, in particular, is being incorporated in numerous current clinical trials either as a single or combination agent against androgen-independent prostate cancer. Combination therapies have the potential to increase the effectiveness of drug treatments while simultaneously increasing quality of life by reducing side effects, lowering effective dosage rates, or by increasing effectiveness of one compound once combined with another. Using three diverse human prostate cancer cell lines, LNCap, DU145, and PC3, we studied the effect of the novel prostate cancer drug phenoxodiol in combination with docetaxel by utilizing isobolograms, and found that docetaxelinduced cell death was attenuated by co-treatment or pre-treatment of cells with phenoxodiol. This attenuation is associated with the prevention of cells from entering the G2/M phase of the cell cycle where docetaxel is functional in damaging the spindle fibers, and potentially due to p21WAF1 mediated cell survival after docetaxel treatment. We also investigated the use of the Wnt signaling pathway antagonist secreted frizzled-related protein 4 (sFRP4) to increase the effectiveness of phenoxodiol treatment. We found that, through stabilization of the GSK3β molecule, sFRP4 induces degradation of active β-catenin, which causes an increased sensitivity to isoflavone cytotoxic induction by increasing p21WAF1 expression and decreasing expression of c-Myc, Cyclin-D1, and other potent oncogenes. Phenoxodiol induces significant cytotoxicity when combined with a Wnt/β-catenin receptor blocker such as sFRP4. This promotes the concept that combination therapy of a Wnt inhibitor with phenoxodiol might increase the effectiveness of phenoxodiol and give a subset population of prostate cancer sufferers a more effective treatment regime.

HIV-Associated Burkitt Lymphoma: Case Report and Literature Review

IntroductionHIV associated Burkitt lymphoma (BL) is a highly aggressive lymphoma. Compared to other AIDS related non-Hodgkin lymphoma's its clinical presentation is less predictable and characterized by rapid clinical changes. The paradigm shift to treat HIV associated disease with highly effective intensive chemotherapy regimens while managing immunosuppression has made it possible to achieve remission and long-term survival. Southern Louisiana has one the of the highest incidence rates of HIV and cancers in the US. The authors present a challenging case of a 33-year old HIV+ male from southern Louisiana with advanced stage BL. The patient presented with advanced disease and a poor performance status. His initial providers recommended palliation only and significant education was needed to allow aggressive treatment. The patient tolerated induction chemotherapy and remains in remission. This is a case based review of local and nationwide epidemiology and the trial data supporting different treatment options for HIV associated Burkitt lymphoma. CaseOur patient is a 33 year-old male who presented with severe cachexia, poor performance status and clinical symptomatic cord compression. He was diagnosed with advanced stage Burkitt lymphoma including bone marrow involvement. His CD4 count was 277 with an HIV viral load of 571,000 copies/mL. Although he had a poor performance status and his primary team favored palliation only he underwent induction chemotherapy with 4 cycles of infusional dose adjusted R-EPOCH with IT CNS chemoprophylaxis. HAART therapy with efavirenz/emtricitabine/tenofovir was started during his initial admission. He tolerated this regimen well, clinically dramatically improved being independent in his ADL/IADLs, and remains in remission. DiscussionThe pathogenesis of HIV-associated Burkitt lymphoma is linked to oncogenic virus co-infection (40% EBV association) and chronic HIV antigen stimulation that provoke expansion of polyclonal B-cells. This leads to oncogenic chromosomal translocations - most notably t(8;14) causing constitutive expression of oncogene c-Myc. HIV patients have a 60 to 200-fold increased incidence of NHL. Relative risk for BL in HIV patients is 261-fold higher compared to the general population. Unlike the nationwide reported 19 per 100,000 person-years, data from a large HIV clinic in southern Louisiana continues to show an incidence rate of 23 per 100,000 person-years. Louisiana continues to have comparatively high incidence rates in HIV (4th in US) and cancer (11th in US) with the highest rates in southern Louisiana parishes. It is important for community physicians, especially in these areas, to feel more confident when treating these HIV associated malignancies.Historically highly immunosuppressive chemotherapy has been avoided in HIV patients with lymphoma as studies in the 1990's showed increased toxicity. The addition of the monoclonal C20 antibody rituximabin the treatment of AIDS associated NHLs was also controversial, as trial results showed increase infectious death rates. However, these clinical trials confirmed similar death rates from infections in individuals with a CD4 count >50. In addition concomitant use of HAART with chemotherapy has improved overall survival. Although the more dose intense Burkitt regimens have not been studied extensively in HIV-associated BL, CODOX-M/IVAC and DA-EPOCH +/- Rituximab (depending on CD4 count) have shown good efficacy and tolerance and are treatment regimens of choice this disease. A recent trial with 14 patients with HIV associated BL receiving RD-CODOXM/IVAC had 86% progression free survival rate at 1 year; patients receiving concomitant HAART had 100% PFS rate at 1 year. In another trial, 10 HIV associated BL patients receiving DA-EPOCH-R showed a 100% overall survival rate at 57 months. ConclusionHIV associated Burkitt lymphoma should be treated with HAART and high dose chemotherapy. Long-term remission has been shown with regimens including CODOX-M/IVAC and DA-EPOCH +/-Rituximab (R use discouraged in low CD4 counts). This case based report outlines the relatively high prevalence of disease in Southern Louisiana and emphasizes that remission is possible in patients with poor performance status and advanced disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acidosis Decreases c-Myc Oncogene Expression in Human Lymphoma Cells: A Role for the Proton-Sensing G Protein-Coupled Receptor TDAG8

Acidosis is a biochemical hallmark of the tumor microenvironment. Here, we report that acute acidosis decreases c-Myc oncogene expression in U937 human lymphoma cells. The level of c-Myc transcripts, but not mRNA or protein stability, contributes to c-Myc protein reduction under acidosis. The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation. TDAG8 is expressed in U937 lymphoma cells, and the overexpression or knockdown of TDAG8 further decreases or partially rescues c-Myc expression, respectively. Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs). Instead, c-Myc is slightly increased by acidosis in H1299 cells, but this increase is completely inhibited by ectopic overexpression of TDAG8. Interestingly, TDAG8 expression is decreased by more than 50% in human lymphoma samples in comparison to non-tumorous lymph nodes and spleens, suggesting a potential tumor suppressor function of TDAG8 in lymphoma. Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

Abstract 440: The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene.

Abstract The sublethal dose radiation may induce cellular senescence and promote secondary maligancy during radiotherapy. However, the molecular mechanisms are unclear. It has been reported that a variety of early response oncogenes can be up-regulated by the low dose radiation. Because oncogene-induced senescence (OIS) has been considered important for tumor development, we are interested in investigating whether the sublethal dose radiation also raises OIS to affect the malignancy. To this end, human diploid fibroblasts WI-38 cells and human non-small lung cancer H1299 cells were exposed to less than 2Gy of X-rays delivered by the RS2000 X-rays machine. Both cell types exhibited senescent phenotypes within a dose-dependent manner using the senescence-associated β-galactosidase (SA-β-gal) staining. Subsequently, we investigated the expression of c-Myc and c-Ras oncogenes, and the results showed that sublethal dose radiation could induce c-Myc, but not c-Ras, in both transcriptional and translational levels. Additionally, up-regulated c-Myc exhibited transcriptional activity as demonstrated by the promoter-based reporter gene assys. Although the sublethal dose radiation caused cellular senescence and growth delay in exposed cells, the collected culture medium could increase the motility of non-irradiated H1299 cancer cells using the wound healing assay. Interestingly, the culture medium obtained from irradiated H1299 cancer cells caused higher motility in non-irradiated cells than that from irradiated WI-38 fibroblasts. Furthermore, the sublethal dose radiation induced senescent phenotype was suppressed in cells treated with siRNA against c-Myc. Meanwhile, the motility of non-irradiated cells was not increased by the cultured medium obtained from irradiated c-Myc silenced cells. Taken together, the current data suggest that the sublethal dose radiation causes a c-Myc oncogene induced senescence, and this event may promote the bystandard effects that influence the motility of non-irradiated cancer cells. Citation Format: Chia-Chien Lo, Yen-Ting Chou, Su-Jun Chiu, Jeng-Jung Hwang, Yi-Jang Lee. The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene. [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 440. doi:10.1158/1538-7445.AM2013-440 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Leptin induces cell proliferation and reduces cell apoptosis by activating c-myc in cervical cancer

Leptin may be involved in the pathogenesis of numerous cancer types by activation of cellular signal-transduction pathways. In this study, we analyzed the role of leptin and the mechanism(s) underlying its action in cervical carcinoma cells. Firstly, we examined the expression of leptin in 80cases of cervical carcinoma using immunohistochemical staining. The results showed that the levels of leptin correlated significantly with the grades of cervical carcinoma. At the same time, the expression of leptin correlated positively with c-myc and its downstream gene, bcl-2. The expression of c-myc and bcl-2 was evaluated in leptin-treated HeLa cells by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Recombinant leptin significantly activated the expression of bcl-2 and c-myc in HeLa cells. Finally, the apoptotic index, the proliferative activity and the expression levels of c-myc and bcl-2 were determined in the HeLa cells treated with silencing of leptin. We found that silencing of leptin inhibited the proliferation of HeLa cells and reduced the expression of bcl-2 and c-myc. Our data demonstrated that leptin interferes with the expression of oncogenic c-myc and anti-apoptotic bcl-2, and regulates cell turnover and facilitates the progression of cervical cancer.

C-Myc oncogene expression in selected odontogenic cysts and tumors: An immunohistochemical study

Aim:To investigate the role of c-Myc oncogene in selected odontogenic cysts and tumors.Materials and Methods:Ten cases each of ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst, and radicular cyst were selected and primary monoclonal mouse anti-human c-Myc antibody was used in a dilution of 1: 50. Statistical Analysis was performed using Mann Whitney U test.Results:80% positivity was observed in ameloblastoma, AOT and OKC; 50% positivity in radicular cyst and 20% positivity in dentigerous cyst. Comparison of c-Myc expression between ameloblastoma and AOT did not reveal significant results. Similarly, no statistical significance was observed when results of OKC were compared with ameloblastoma and AOT. In contrast, significant differences were seen on comparison of dentigerous cyst with ameloblastoma and AOT and radicular cyst with AOT.Conclusion:From the above data we conclude that (1) Ameloblastoma and AOT have similar proliferative potential and their biologic behavior cannot possibly be attributed to it. (2) OKC has an intrinsic growth potential which is absent in other cysts and reinforces its classification as keratocystic odontogenic tumor.

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

SOX2 Gene Regulates the Transcriptional Network of Oncogenes and Affects Tumorigenesis of Human Lung Cancer Cells

Recent studies demonstrated that cancer stem cells (CSCs) have higher tumorigenesis properties than those of differentiated cancer cells and that transcriptional factor-SOX2 plays a vital role in maintaining the unique properties of CSCs; however, the function and underlying mechanism of SOX2 in carcinogenesis of lung cancer are still elusive. This study applied immunohistochemistry to analyze the expression of SOX2 in human lung tissues of normal individuals as well as patients with adenocarcinoma, squamous cell carcinoma, and large cell and small cell carcinoma and demonstrated specific overexpression of SOX2 in all types of lung cancer tissues. This finding supports the notion that SOX2 contributes to the tumorigenesis of lung cancer cells and can be used as a diagnostic probe. In addition, obviously higher expression of oncogenes c-MYC, WNT1, WNT2, and NOTCH1 was detected in side population (SP) cells than in non-side population (NSP) cells of human lung adenocarcinoma cell line-A549, revealing a possible mechanism for the tenacious tumorigenic potential of CSCs. To further elucidate the function of SOX2 in tumorigenesis of cancer cells, A549 cells were established with expression of luciferase and doxycycline-inducible shRNA targeting SOX2. We found silencing of SOX2 gene reduces the tumorigenic property of A549 cells with attenuated expression of c-MYC, WNT1, WNT2, and NOTCH1 in xenografted NOD/SCID mice. By using the RNA-Seq method, an additional 246 target cancer genes of SOX2 were revealed. These results present evidence that SOX2 may regulate the expression of oncogenes in CSCs to promote the development of human lung cancer.

Abstract 3281: Development of a novel transgenic mouse model for prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death in American men. Androgen-deprivation therapy (ADT) is a common and effective therapy for advanced / metastatic PCa. However, most initially regressed PCa will relapse and become castration-resistant PCa (CRPC). Currently, there is no cure for CRPC. Chemotherapy, including agents such as Docetaxel, is a standard therapy for CRPC. However, it only modestly extends patient survival. Therefore, castration resistance and chemoresistance of PCa are huge medical problems for PCa treatment. To overcome castration resistance and chemoresistance, it is crucial to delineate the underlying molecular mechanisms. c-Myc is the most significantly amplified oncogene in human PCa, and its overexpression is very common in PCa as early as prostatic intraepithelial neoplasia (PIN). These indicate its critical roles in PCa progression and the development of therapy-resistance. Transgenic models are widely used in cancer research. Accordingly, Hi-MYC transgenic model has been created using an enhanced probasin promoter to drive c-Myc overexpression in prostate epithelia. These mice develop invasive prostate carcinomas that share molecular features with human PCa. However, since the probasin promoter activity is androgen-dependent, these MYC prostate tumors lose c-Myc oncogene expression upon castration. As a result, the tumor regression in these androgen-depleted Hi-MYC mice represents the mixed effects of both an artificial direct effect from loss of oncogene expression and a potential real effect from tumor cellular responses to androgen-ablation that are relevant to human PCa. Finally, Hi-MYC mice do not develop CRPC tumors after castration. Therefore, the Hi-MYC model, along with other similarly created Myc-based transgenic models for PCa, cannot be used to study CRPC, the lethal form of PCa. To study CRPC and chemoresistance of CRPC, we have developed a novel transgenic model (CAG-SMIL) for human PCa. This model allows us to specifically turn on c-Myc oncogene expression in prostate epithelia after crossing CAG-SMIL mice with PB-Cre4 mice. In addition, once turned on, the expression of c-Myc transgene will be driven by an enhanced beta-actin promoter that is androgen-independent. Therefore, after castration of the prostate tumor bearing PB-Cre4/CAG-SMIL males, we will be able to, for the first time, concisely study c-Myc signaling pathway 1) in castration induced prostate tumor regression, 2) in the recurrence of CRPC tumors, and 3) in the development of chemoresistance in CRPC tumors. Finally, we have incorporated an enhanced luciferase reporter into our transgenic model. This will allow a real-time in vivo bioluminescence imaging (BLI imaging) for prostate tumor progression, tumor response to various therapeutic agents, as well as tumor relapse after the development of therapy (castration and chemotherapy) resistance. 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 3281. doi:1538-7445.AM2012-3281

MicroRNA-34a Modulates c-Myc Transcriptional Complexes to Suppress Malignancy in Human Prostate Cancer Cells

MicroRNA-34a (miR-34a), a potent mediator of tumor suppressor p53, has been reported to function as a tumor suppressor and miR-34a was found to be downregulated in prostate cancer tissues. We studied the functional effects of miR-34a on c-Myc transcriptional complexes in PC-3 prostate cancer cells. Transfection of miR-34a into PC-3 cells strongly inhibited in vitro cell proliferation, cell invasion and promoted apoptosis. Transfection of miR-34a into PC-3 cells also significantly inhibited in vivo xenograft tumor growth in nude mice. miR-34a downregulated expression of c-Myc oncogene by targeting its 3′ UTR as shown by luciferase reporter assays. miR-34a was found to repress RhoA, a regulator of cell migration and invasion, by suppressing c-Myc–Skp2–Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a was also found to repress c-Myc-pTEFB transcription elongation complex, indicating one of the mechanisms by which miR-34a has profound effects on cellular function. This is the first report to document that miR-34a suppresses assembly and function of the c-Myc–Skp2–Miz1 complex that activates RhoA and the c-Myc-pTEFB complex that elongates transcription of various genes, suggesting a novel role of miR-34a in the regulation of transcription by c-Myc complex.

Induction of Apoptosis by 3-Amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via Modulation of MAPKs (p38 and c-Jun N-terminal Kinase) and c-Myc in HL-60 Human Leukemia Cells

Recently, we reported that 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (AM6-36), sharing structural similarity with naturally occurring isoquinolines, induced activities mediated by retinoid X receptor (RXR) response element accompanied by antiproliferative effects on breast cancer cells. To further characterize the biologic potential of AM6-36, we currently report studies conducted with HL-60 human leukemia cells. AM6-36 significantly inhibited cellular proliferation in a dose- and time-dependent manner with an IC(50) value of 86 nM. When evaluated at low test concentrations (≤0.25 μM), AM6-36 induced arrest in the G2/M phase of the cell cycle. At higher concentrations (1 and 2 μM), the response shifted to apoptosis, which was consistent with the effect of AM6-36 on other apoptotic signatures including an increase of apoptotic annexin V(+) 7-AAD(-) cells, loss of mitochondrial membrane potential, induction of poly(ADP-ribose) polymerase cleavage, and activation of several caspases. These apoptotic effects are potentially due to up-regulation of p38 MAPK and JNK phosphorylation and down-regulation of c-Myc oncogene expression. Taken together, AM6-36 might serve as an effective anticancer agent by inducing G2/M cell cycle arrest and apoptosis through the activation of MAPKs and inhibition of c-Myc.

Prognostic classification of pediatric medulloblastoma based on chromosome 17p loss, expression of MYCC and MYCN, and Wnt pathway activation

Pediatric medulloblastoma is considered a highly heterogeneous disease and a new strategy of risk stratification to optimize therapeutic outcomes is required. We aimed to investigate a new risk-stratification approach based on expression profiles of medulloblastoma cohorts. We analyzed gene expression profiles of 30 primary medulloblastomas and detected strong evidence that poor survival outcome was significantly associated with mRNA expression profiles of 17p loss. However, it was not supported in independent cohorts from previously published data (n = 100). We speculated that this discrepancy might come from complex conditions of two important prognostic determinants: loss of tumor suppressors (chromosome 17p) and high expression of oncogenes c-myc (MYCC) or N-myc (MYCN). When patients were stratified into 5 or 7 subgroups based on simultaneous consideration of these 2 factors while defining the Wnt group as independent, obviously different survival expectancies were detected between the subgroups. For instance, predicted 5-year survival probabilities ranged from 19% to 81% in the 5 subgroups. We also found that age became a significant prognostic marker after adjusting for 17p, MYCC, and MYCN status. Diminished survival in age <3 years was more substantial in subgroups with high expression of MYCC, MYCN, or 17p loss but not in other subgroups, indicating that poor survival outcome might be synergistically affected by these 3 factors. Here we suggest a more tailored subgrouping system based on expression profiles of chromosome 17p, MYCC, and MYCN, which could provide the basis for a novel risk-stratification strategy in pediatric medulloblastoma.

Expression of vascular endothelial growth factor in glioblastoma cells after silencing oncogene c-myc through RNA interference

Objective To explore the expression of vascular endothelial growth factor (VEGF) in glioblastoma cells after silencing the oncogene c-myc through RNA interference (RNAi). Methods The shRNA interference plasmid named pCMYC targeting c-myc gene and the pHK plasmid as negative control were constructed. The expression of c-myc and VEGF mRNA and protein was detected by using reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry and Western blotting among different groups. Results ( 1 ) The mRNA expression of c-myc and VEGF in IN500Δ-pCMYC cells (0. 273 ±0. 028, 0. 443 ±0. 048) was lower than that of IN500Δ ( 1. 185 ±0. 145, 1. 116 ±0. 072) and IN500Δ-pHK cells ( 1. 098 t0. 128, 1. 208 ±0. 133) (P ＜0. 01 ) ; (2) Immunocytochemical staining revealed that the expression of c-myc and VEGF protein in IN500Δ-pCMYC cells was lower than in IN500Δ cells and IN500Δ-pHK cells (P＜0. 05); (3) Western blotting indicated that the expression of c-myc and VEGF protein in IN500Δ-pCMYC cells (0. 221 ± 0. 041, 0. 284 ± 0. 025 ) was lower than that in IN500Δ cells (1.821±0.191, 1.531 ±0.213) and IN500A-pHK cells (1.650±0.110, 1.820±0.122) (P＜0. 01 ). Conclusion Sliencing the expression of c-myc oncogene could effectively inhibit the expression of VEGF. Key words: c-myc gene; RNA interference; Vascular endothelial growth factor; Glioma