Regulation Of C-myc Expression Research Articles

IFITM3 upregulates c-myc expression to promote hepatocellular carcinoma proliferation via the ERK1/2 signalling pathway.

Interferon-induced transmembrane protein 3 (IFITM3) is associated with cancer development. Proto-oncogene c-myc can promote tumor proliferation. However, collections of IFITM3 and c-myc in hepatocellular carcinoma (HCC) and the potential role and mechanisms of IFITM3 in c-myc-mediated tumor proliferation remain unclear. In this study, we investigated a positive correlation between the expression of IFITM3 and c-myc in HCC. The down-regulation of IFITM3 significantly reduced c-myc expression and inhibited the proliferation of HCC in vitro and in vivo. In addition, upregulated c-myc expression restored the decrease in cell proliferation caused by the downregulation of IFITM3, while downregulation of c-myc reduced the proliferation of HCC enhanced by IFITM3. Mechanistically, IFITM3 regulates c-myc expression via the ERK1/2 signalling pathway. In conclusion, a novel path of IFITM3-ERK1/2-c-myc regulatory circuitry was identified, and its dysfunction may lead to HCC tumorigenesis.

LncRNA ROR facilitates myocardial fibrosis in rats with viral myocarditis through regulating C-Myc expression.

The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) ROR on myocardial fibrosis in rats with viral myocarditis by regulating c-myc expression. A total of 120 healthy male rats were randomly divided into three groups, including: blank control group (group N, healthy rats, n=40), model control group (group M, viral myocarditis, n=40), and group P (lncRNA ROR + viral myocarditis, n=40). At 5, 10, and 15 d after modeling, the rats were sacrificed. Meanwhile, the left ventricular mass index (LVMI), the left ventricular weight/tibial length (LVW/TL) and the heart mass index (HMI) were measured, respectively. The myocardial tissues of rats were observed via staining. The protein and mRNA expressions of the transforming growth factor-β (TGF-β) and c-myc in myocardial tissues were detected via Western blotting and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. Moreover, the level of the serum interleukin-6 (IL-6) was detected via enzyme-linked immunosorbent assay (ELISA). Compared with group N, HMI, LVMI, and LVW/TL in group M significantly increased at each time point (p<0.01). Meanwhile, they were remarkably elevated at each time point in group P when compared with those in group M (p<0.01). According to the results of hematoxylin-eosin staining (HE) staining, the myocardial fibers were normal and orderly arranged in group N. Meanwhile, no inflammatory cells were observed in group N. In group M, the proliferation of a large number of inflammatory cells was observed, and the myocardial cells were disorderly arranged and became enlarged. In group P, the myocardial fibrosis was significantly severer than that of the other two groups (p<0.05). The results of the Masson staining revealed that there was significantly less accumulation of collagen fibers in myocardium in group N. There was a little accumulation of collagen fibers in the myocardium and fibrosis declined in group M. The myocardial fibrosis in group P remarkably increased when compared with group M (p<0.05). According to the results of the Western blotting, the protein expressions of c-myc and TGF-β were significantly up-regulated in group P, compared with those in group M and N (p<0.05). However, they were remarkably higher in group M than in group N (p<0.05). QRT-PCR showed that the relative messenger ribonucleic acid (mRNA) expressions of c-myc and TGF-β were remarkably up-regulated in group P when compared with those in group M and N, showing statistically significant differences (p<0.05). Moreover, they were significantly higher in group M than those in group N (p<0.05). Besides, the expression level of serum IL-6 remained highest in group P (p<0.01). LncRNA ROR up-regulates the expression of c-myc and increases the level of serum IL-6, thereby facilitating the proliferation and differentiation of cardiac fibroblasts.

Histone deacetylase inhibitors differentially regulate c-Myc expression in retinoblastoma cells.

Retinoblastoma (RB) is the most prevalent childhood intraocular cancer type. Previous studies have demonstrated that c-myc (a proto-oncogene) is associated with tumorigenesis. However, at present, the influence of the expression profile and bioactivity of c-Myc on RB occurrence and progression is yet to be characterised. Notably, the present study demonstrated that c-myc is downregulated in the RB cell line WERI-Rb1. However, treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) was revealed to significantly upregulate the expression of c-Myc mRNA and protein in WERI-Rb1 cells. Moreover, TSA increased the activity of the c-myc promoter in WERI-Rb1 cells, and the expression of c-Myc was also regulated by other HDAC inhibitors, including vorinostat (SAHA), valproic acid sodium salt (VPA) and entinostat. Notably, although c-myc was silenced in the Y79 cell line, the HDAC inhibitor TSA did not induce upregulation of mRNA and protein in Y79 cells. By contrast, certain HDAC inhibitors (TSA, VPA and SAHA) were discovered to significantly decrease the activity of the c-myc promoter in Y79 cells. Furthermore, the current data indicated that exogenous c-myc expression has a mild inhibitory effect on WERI-Rb1 and Y79 cell viability. Therefore, the present study revealed novel insights into the expression mechanism and bioactivity of c-Myc in RB cells.

Effect of different expression levels of USP28 on apoptosis and related protein expression in esophageal cancer cell ECA109 after irradiation

Objective To observe the effect of different expression levels of USP28 on the radiosensitivity of ECA109 cells by gene transfection method, aiming to provide theoretical basis for comprehensive treatment of esophageal cancer. Methods The expression levels of USP28 and c-Myc in the esophageal epithelial cells Het-1A, ECA109 and ECA109R were quantitatively measured by qRT-PCR. The specific siRNA sequences were designed according to the USP28 and c-Myc genes. The pcDNA-USP28 and pcDNA-c-Myc plasmids were constructed. The esophageal cancer cell ECA109 was transfected with Lipofectamine 2000 to observe the transfection effect and related protein expression. ECA109 and ECA109R cells were exposed to 6 Gy X-ray radiation. The cell apoptosis in each group was detected by flow cytometry. The radiosensitivity was evaluated by clone formation assay. Results The expression levels of USP28 and c-Myc in ECA109 were significantly higher than those in Het-1A (both P<0.05), and the expression levels of USP28 and c-Myc in ECA109R were remarkably higher than those in ECA109(both P<0.05). The pcDNA-USP28 and pcDNA-c-Myc recombinant plasmids were successfully constructed. Compared with the negative control group, the expression of USP28 at the protein and mRNA levels in the si-USP28 group was significantly down-regulated, whereas those in the pcDNA-USP28 group were remarkably up-regulated. Similar results were obtained in terms of c-Myc. Compared with the control group, the expression level of c-Myc protein was significantly up-regulated in the pcDNA-USP28 group, whereas considerably down-regulated in the si-USP28 group. After 6 Gy irradiation, the apoptosis rate and radiosensitivity of ECA109 cells were significantly declined. The apoptosis rate and radiosensitivity of ECA109R cells were increased in the si-USP28 group. Conclusions The expression of USP28 protein is closely correlated with the radiosensitivity of esophageal cancer cells. The underlying mechanism may be related to the regulation of c-Myc expression by USP28. Key words: USP28 gene; c-Myc gene; Esophageal carcinoma cell line; Radiosensitivity

PTTG1 is involved in TNF-α-related hepatocellular carcinoma via the induction of c-myc.

Hepatocellular carcinoma (HCC) is a malignant disease caused by a variety of factors. However, the genomic and molecular aberrations in HCC are largely unknown. Herein, pituitary tumor transforming gene 1 (PTTG1) was discovered as a potential inflammation‐related oncogene in HCC, and its functions and molecular mechanisms were investigated. mRNA expression microarray, real‐time polymerase chain reaction (PCR), immunohistochemistry, and western blotting analyses revealed that PTTG1 is upregulated in HCC. Further in vitro and in vivo studies indicated that the proinflammatory cytokine tumor necrosis factor‐α (TNF‐α) induces PTTG1 expression, and PTTG1 was found to upregulate c‐myc, a well‐known oncogene. Downregulation of PTTG1 reduced c‐myc and proliferating cell nuclear antigen (PCNA) expression and inhibited cell proliferation. Interestingly, inhibition of c‐myc by 10058‐F4 did not affect PTTG1, which suggests that PTTG1 regulates c‐myc expression. Furthermore, PTTG1 expression levels are inversely correlated with HCC patient survival, indicating an independent prognostic biomarker for patients with HCC. Our data demonstrate that PTTG1 is involved in TNF‐α‐related HCC via c‐myc induction and that PTTG1 may be a potential therapeutic target for HCC.

LncRNA KCNQ1OT1 controls cell proliferation, differentiation and apoptosis by sponging miR-326 to regulate c-Myc expression in acute myeloid leukemia.

Long noncoding RNAs (lncRNAs) have been reported to play essential roles in development and treatment of acute myeloid leukemia (AML). However, the role of lncRNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in AML progression and its mechanism remain largely unknown. The expressions of KCNQ1OT1, microRNA-326 (miR-326) and c-Myc were measured by quantitative real-time polymerase chain reaction and western blot, respectively. Phorbol myristate acetate (PMA) was used for cell differentiation. Cell proliferation, apoptosis and differentiation were measured by MTT assay, flow cytometry and qRT-PCR, respectively. The interaction between miR-326 and KCNQ1OT1 or c-Myc was explored by luciferase activity, RNA immunoprecipitation or RNA pull-down assay. We found that the expression of KCNQ1OT1 was enhanced in AML samples compared with control. KCNQ1OT1 knockdown inhibited cell proliferation but promoted apoptosis and cell differentiation. KCNQ1OT1 was a decoy of miR-326 and c-Myc was a target of miR-326. KCNQ1OT1 regulated AML cell proliferation, apoptosis and differentiation by sponging miR-326. Moreover, overexpression of miR-326 suppressed proliferation but promoted apoptosis and PMA-induced differentiation by targeting c-Myc in AML cells. Besides, c-Myc protein level was suppressed by KCNQ1OT1 interference and rescued by miR-326 abrogation. Our data showed that KCNQ1OT1 regulates proliferation, differentiation and apoptosis in AML cells by acting as a competing endogenous RNA (ceRNA) for miR-326 to regulate c-Myc, providing a novel avenue for AML treatment.

ZNFX1 anti-sense RNA 1 promotes the tumorigenesis of prostate cancer by regulating c-Myc expression via a regulatory network of competing endogenous RNAs.

ZNFX1 anti-sense RNA 1 (ZFAS1) has been indicated in the tumorigenesis of various human cancers. However, the role of ZFAS1 in prostate cancer (PCa) progression and the underlying mechanisms remain incompletely understood. In the present study, we discovered that ZFAS1 is upregulated in PCa and that ZFAS1 overexpression predicted poor clinical outcomes. ZFAS1 overexpression notably promoted the proliferation, invasion, and epithelial-mesenchymal transition of PCa cells. Furthermore, we not only discovered that miR-27a/15a/16 are targeted by ZFAS1, which binds to their miRNA-response elements, but also revealed their tumor suppressor roles in PCa. We also identified that the Hippo pathway transducer YAP1, as well as its cooperator, TEAD1, are common downstream targets of miR-27a/15a/16. In addition, H3K9 demethylase KDM3A was found to be another target gene of miR-27a. Importantly, YAP1, TEAD1, and KDM3A all act as strong c-Myc inducers in an androgen-independent manner. Taken together, we suggest a regulatory network in which ZFAS1 is capable of enhancing c-Myc expression by inducing the expression of YAP1, TEAD1, and KDM3A through crosstalk with their upstream miRNAs, thereby globally promoting prostate cancer tumorigenesis.

HMGA1 promoting gastric cancer oncogenic and glycolytic phenotypes by regulating c-myc expression

The high mobility group A1 (HMGA1) protein, an architectural transcription factor, is profoundly implicated in the pathogenesis and progression of multiple malignant tumors. Reprogrammed energy metabolism is a hallmark of diverse types of cancer cells. However, little is known about the regulatory role of HMGA1 in aerobic glycolysis. In this study, we found that HMGA1 was highly expressed in many types of human cancers including gastric cancer and predicted a poor prognosis. However, high HMGA1 expression was not correlated with TNM stages. Gene set enrichment analysis result suggested a link between HMGA1 expression and glycolytic phenotype in gastric cancer. Genetic silencing of HMGA1 significantly inhibited gastric cancer glycolytic activity as revealed by reduced glucose uptake, lactate release, and extracellular acidification ratio. In addition, cell proliferation and invasive capacity of gastric cancer cells were also suppressed by HMGA1 knockdown. Mechanistically, the key glycolysis regulator c-Myc was identified as a downstream target of HMGA1. In gastric cancer patients, HMGA1 and c-Myc expression were closely associated with the glycolysis gene signature. Taken together, our findings identify a novel function of HMGA1 in regulating aerobic glycolysis in gastric cancer.

Leflunomide regulates c-Myc expression in myeloma cells through PIM targeting

Key Points Teriflunomide, the active metabolite of leflunomide, downregulates c-Myc expression through inhibition of PIM kinases. Leflunomide together with lenalidomide significantly extended survival in an in vivo MM model.

The Tumor Suppressor FBW7 and the Vitamin D Receptor Are Mutual Cofactors in Protein Turnover and Transcriptional Regulation

The E3 ligase and tumor suppressor FBW7 targets drivers of cell-cycle progression such as the oncogenic transcription factor c-MYC, for proteasomal degradation. Vitamin D signaling regulates c-MYC expression and turnover in vitro and in vivo, which is highly significant as epidemiologic data link vitamin D deficiency to increased cancer incidence. We hypothesized that FBW7 and the vitamin D receptor (VDR) controlled each other's function as regulators of protein turnover and gene transcription, respectively. We found that hormonal 1,25-dihydroxyvitamin D3 (1,25D) rapidly enhanced the interaction of FBW7 with VDR and with c-MYC, whereas it blocked FBW7 binding to c-MYC antagonist MXD1. 1,25D stimulated the recruitment of FBW7, SCF complex subunits, and ubiquitin to DNA-bound c-MYC, consistent with 1,25D-regulated c-MYC degradation on DNA. 1,25D also accelerated the turnover of other FBW7 target proteins such as Cyclin E, c-JUN, MCL1, and AIB1, and, importantly, FBW7 depletion attenuated the 1,25D-induced cell-cycle arrest. Although the VDR contains a consensus FBW7 recognition motif in a VDR-specific insertion domain, its mutation did not affect FBW7-VDR interactions, and FBW7 ablation did not stabilize the VDR. Remarkably, however, FBW7 is essential for optimal VDR gene expression. In addition, the FBW7 and SCF complex subunits are recruited to 1,25D-induced genes and FBW7 depletion inhibited the 1,25D-dependent transactivation. Collectively, these data show that the VDR and FBW7 are mutual cofactors, and provide a mechanistic basis for the cancer-preventive actions of vitamin D. IMPLICATIONS: The key findings show that the VDR and the E3 ligase FBW7 regulate each other's functions in transcriptional regulation and control of protein turnover, respectively, and provide a molecular basis for cancer-preventive actions of vitamin D.Visual Overview: http://mcr.aacrjournals.org/content/17/3/709/F1.large.jpg.

MiR-140-3p functions as a tumor suppressor in squamous cell lung cancer by regulating BRD9

Squamous cell lung cancer (SqCLC) is among the most malignant lung cancers worldwide, lacking biomarkers for diagnostic and targets for treatment. In this study, we observed that miR-140-3p was expressed at low levels both in SqCLC cell lines and patient samples, while overexpression of miR-140-3p dramatically reduced the cell proliferation and invasion in SqCLC cells and Patient derived xenograft (PDX) models. Our further investigation indicated miR-140-3p negatively affected the tumorigenesis of SqCLC by down-regulating the expression of BRD9, an oncogene in SqCLC. Inhibition of BRD9 repressed SqCLC tumorigenesis by regulating c-myc expression. Meanwhile, BRD9 expression is up-regulated and negatively correlated with miR-140-3p in clinical samples; a meta-analysis of survival data indicates that SqCLC patients with high levels of BRD9 in their tumors have a worse prognosis. Collectively, our study suggests the prognostic and therapeutic roles of miR-140-3p and BRD9 axis in squamous cell lung cancer.

Heat Shock Protein 90α-Dependent B-Cell-2-Associated Transcription Factor 1 Promotes Hepatocellular Carcinoma Proliferation by Regulating MYC Proto-Oncogene c-MYC mRNA Stability.

B‐cell lymphoma 2 (Bcl‐2)‐associated transcription factor 1 (Bclaf1) is known to be involved in diverse biological processes, but, to date, there has been no evidence for any functional role of Bclaf1 in hepatocellular carcinoma (HCC) progression. Here, we demonstrate that Bclaf1 is frequently up‐regulated in HCC and that Bclaf1 up‐regulation is associated with Edmondson grade, lower overall survival rates, and poor prognosis. Overexpression of Bclaf1 in HCC cell lines HepG2 and Huh7 promoted proliferation considerably, whereas Bclaf1 knockdown had the opposite effect. Xenograft tumors grown from Bclaf1 knockdown Huh7 cells had smaller tumor volumes than tumors grown from control cells. Furthermore, our study describes MYC proto‐oncogene (c‐Myc) as a downstream target of Bclaf1, given that Bclaf1 regulates c‐MYC expression posttranscriptionally by its RS domain. To exert this function, Bclaf1 must interact with the molecular chaperone, heat shock protein 90 alpha (Hsp90α). In HCC tissue samples, Hsp90α levels were also increased significantly and Hsp90α‐Bclaf1 interaction was enhanced. Bclaf1 interacts with the C‐terminal domain of Hsp90α, and this interaction is disrupted by the C‐terminal domain inhibitor, novobiocin (NB), resulting in proteasome‐dependent degradation of Bclaf1. Moreover, NB‐induced disruption of Hsp90α‐Bclaf1 interaction dampened the production of mature c‐MYC mRNA and attenuated tumor cell growth in vitro and in vivo. Conclusion: Our findings suggest that Bclaf1 affects HCC progression by manipulating c‐MYC mRNA stability and that the Hsp90α/Bclaf1/c‐Myc axis might be a potential target for therapeutic intervention in HCC.

Camptothecin enhances c-Myc-mediated endoplasmic reticulum stress and leads to autophagy by activating Ca2+-mediated AMPK

Camptothecin (CPT) from Camptotheca acuminate was discovered for anticancer drugs, which targets topoisomease I. However, whether CPT regulates c-Myc expression has not been understood in endoplasmic reticulum (ER) stress and autophagy. In this study, we found that CPT enhanced c-Myc expression and that the transient knockdown of c-Myc abrogated reactive oxygen species (ROS) generation, which resulted in the accumulation of ER stress-regulating proteins, such as PERK, eIF2α, ATF4, and CHOP. Moreover, the transfection of eIF2α-targeted siRNA attenuated CPT-induced autophagy and decreased the levels of Beclin-1 and Atg7, which indicated that CPT upregulated ER stress-mediated autophagy. In addition, CPT phosphorylated AMPK in response to intracellular Ca2+ release. Ca2+ chelators, ethylene glycol tetraacetic acid and a CaMKII inhibitor, K252a, decreased CPT-induced Beclin-1 and Atg7, and downregulated AMPK phosphorylation, which suggested that CPT-induced Ca2+ release leads to the activation of autophagy through CaMKII-mediated AMPK phosphorylation. CPT also phosphorylated JNK and activated the DNA-binding activity of AP-1; furthermore, knockdown of JNK abolished the expression level of Beclin-1 and Atg7, which implied that the JNK-AP-1 pathway was a potent mediator of CPT-induced autophagy. Our findings indicated that CPT promoted c-Myc-mediated ER stress and ROS generation, which enhances autophagy via the Ca2+-AMPK and JNK-AP-1 pathways.

PO-348 Two novel synthetic analogues of miR-1207–3 p, NB5 and NB1207, target AR-V7 and C-MYC and demonstrate in vivo therapeutic efficacy in metastatic castrate-resistant prostate cancer (mCRPC)

IntroductionChromosome 8q24 prostate cancer (PCa) susceptibility region encodes miR-1207–3 p which directly targets fibronectin type III domain containing 1 (FNDC1) to regulate fibronectin (FN1) and subsequently the androgen receptor (AR), and FNDC1/FN1/AR is overexpressed in metastatic PCa (Das et al, Exp Cell Res, 2016).Material and methodsExpression of miR-1207–3 p and c-MYC in histologically confirmed normal prostate, benign prostatic hyperplasia (BPH) and PCa from prostatectomy or transrectal ultrasound-guided biopsies was analysed. Molecular mechanisms were elucidated using mCRPC cell lines. Effects of NB1207 and NB5, two novel synthetic analogues of miR-1207–3 p, on AR-V7 (implicated in PCa therapeutic resistance), mCRPC tumour growth in mice were studied.Results and discussions miR-1207–3 p is underexpressed in PCa (0.10±0.02, 95% CI [0.1, 0.2], p=0.000) in comparison to normal prostate (1.02±0.17, 95% CI [0.7, 1.4], p=0.000) and BPH (0.74±0.15, 95% CI [0.4, 1.1] p=0.004). c-MYC was overexpressed in PCa (34.76±8.11, 95% CI [18.1, 51.4], p=0.000; normal: 1.99±0.34, 95% CI [1.3, 2.7]; BPH: 1.01±0.21, 95% CI [0.6, 1.4], p=0.000). miR-1207–3 p was underexpressed by nearly 3-fold in PCa with Gleason score ≥8 versus those with Gleason score <8, while c-MYC was overexpressed by nearly 5-fold in PCa with Gleason score ≥8 versus those with Gleason score <8. NB1207 significantly inhibited c-MYC expression in mCRPC cell lines: PC-3, E006AA-hT and C4-2B. miR-1207–3 p regulates c-MYC expression via the miR-1207–3 p/FNDC1/FN1/AR pathway: c-MYC protein expression is inhibited in mCRPC cell lines by overexpression of miR-1207–3 p, and by inhibition of expression of FNDC1, FN1, and AR. In AR-V7-expressing mCRPC cell lines, NB1207 and NB5 significantly reduced AR-V7 expression, and NB1207 and NB5 more effectively inhibited AR-V7 compared to abiraterone, enzalutamide and apalutamide. In mCRPC cell lines, abiraterone, enzalutamide, and apalutamide failed to significantly inhibit cell proliferation or induce apoptosis. However, NB1207 and NB5 significantly inhibited proliferation and induced apoptosis. In mCRPC 22RV1 tumor-bearing mice, NB5 and NB1207 significantly inhibited tumour growth and metastatic spread. While enzalutamide minimally inhibited growth of 22RV1 tumours in mice, apalutamide and abiraterone had no inhibitory effects on tumour growth.ConclusionThis study shows potential diagnostic and prognostic value of miR-1207–3 p in mCRPC, and that NB5 and NB1207, novel analogues of miR-1207–3 p, may be candidate therapeutics for mCRPC.

Abstract B61: c-Myc is regulated and therapeutically targetable via the miR-1207-3p/FNDC1/FN1/AR pathway in aggressive prostate cancer in men of African ancestry

Abstract Prostate cancer is the 2nd most common cancer in the world for men. For reasons still unclear, aggressive PCa disproportionately affects males of African ancestry (MoAA). Incidence and mortality rates are highest in MoAA as they have consistently shown a 2.3-3.0-fold higher risk of mortality compared to Caucasian men (CM). This aggressiveness of PCa may be due to specific biologic factors. Located downstream of c-Myc at chromosome 8q24 is PVT1, which encodes miR-1207-3p. Studies have shown that PVT1/MYC cooperation is a fundamental feature in all cancers with 8q24 amplification, and 98% of the 8q24 amplicons contained concurrent amplification of the MYC and PVT1 loci. Moreover, MYC has been linked to PCa aggressiveness and has been reported to be downstream of AR in some PCa. However, the mechanisms regulating c-MYC have never been studied in MoAA. We recently demonstrated that miR-1207-3p directly binds to FNDC1 to regulate a novel FNDC1/FN1/AR pathway upregulated in metastatic prostate cancer (PCa). However, the mechanisms regulating c-Myc in PCa remain unclear, and the relevance of our novel and clinically significant miR-1207-3p molecular pathway in PCa in MoAA is unknown. The aim of this study was to determine if c-Myc is regulated and therapeutically targetable via the miR-1207-3p/FNDC1/FN1/AR pathway in aggressive PCa in MoAA. We used qPCR, immunoblotting, RNA pulldown, proliferation, migration, and apoptosis assays to evaluate miR-1207-3p regulation of c-Myc in aggressive PCa in MoAA. Also, miR-1207-3p, FNDC1, FN1, AR, and c-Myc expression was analyzed in prostate tissues (normal = 21; benign = 41; tumor = 26) of patients who received prostatectomy or transrectal ultrasound-guided biopsies at the University College Hospital, Ibadan, Nigeria, a sub-Saharan Black African population. Seventeen patients had tumor tissues with Gleason score ≥ 8. Tissues were collected in compliance with Institutional Review Board-approved protocols. ANOVA, student's t-test, and Tukey post-hoc tests were used for statistical analysis. Prostate tissue analysis revealed that underexpression of miR-1207-3p and the overexpression of FNDC1, FN1, AR, and c-Myc is significantly associated with aggressive PCa in MoAA. Also, miR-1207-3p was underexpressed while FNDC1 and c-MYC were overexpressed in tumors with Gleason score ≥8 in comparison to those with Gleason score &amp;lt;8. Thus, miR-1207-3p and FNDC1 may be useful for risk stratification in PCa in MoAA. Next, we observed that our novel synthetic biotinylated miR-1207-3p duplex (NB1207, patent pending) significantly inhibited c-Myc protein expression in 7 PCa cell lines when compared with a biotinylated scramble duplex (NB-1, patent pending). RNA pulldown assay determined that c-Myc is not a direct molecular target of miR-1207-3p. However, siRNAs against FNDC1, FN1, and AR revealed significant suppression of c-Myc expression by &amp;gt;75% in the MoAA-derived indolent E006AA PCa cell line and the MoAA-derived aggressive/castration-resistant E006AA-hT PCa cell line, indicating that c-Myc is downstream of AR. c-Myc expression is higher in the E006AA-hT PCa cell line when compared to the E006AA PCa cell line, suggesting that c-Myc is associated with aggressive PCa. Moreover, NB1207 significantly inhibited migration and induced apoptosis in E006AA and E006AA-hT PCa cell lines. Next, we compared the efficacy of NB1207 in inhibiting proliferation to the commercially available drugs for treatment of CPRC (enzalutamide and abiraterone). NB1207 inhibited proliferation in the CRPC cell line E006AA-hT by nearly 50% while enzalutamide and abiraterone had no effect. In conclusion, miR-1207-3p regulates c-Myc expression via the miR-1207-3p/FNDC1/FN1/AR pathway in aggressive PCa in MoAA. miR-1207-3p may be a biomarker for risk stratification in PCa in MoAA. NB1207 has potential for therapeutic targeting of c-Myc for treatment of aggressive PCa in MoAA. Citation Format: Dibash K. Das, Akintunde T. Orunmuyi, Gabriel Olabiyi Ogun, S. Adekola Adebayo, A. Ayo Salako, Adeodat Ilboudo, Cuong Bach, E. O. Olapade-Olaopa, Olorunseun O. Ogunwobi. c-Myc is regulated and therapeutically targetable via the miR-1207-3p/FNDC1/FN1/AR pathway in aggressive prostate cancer in men of African ancestry [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B61.

PO-006 The MAPK/c-Myc axis in CRC: new pathogenic mechanisms and therapeutic approaches

Introduction c-Myc plays a central role in cellular proliferation, differentiation, and apoptosis. Therefore its deregulation represents a powerful trigger of tumorigenesis, particularly in colorectal cancer (CRC). It has been shown that the MEK/ERK pathway phosphorylates c-Myc on serine 62, which stabilises c-Myc by preventing ubiquitin/proteasomal degradation. We recently reported that MEK/ERK inhibition is counteracted by over-activation of p38α MAPK. Here, we identified cellular mechanisms that lead to c-Myc deregulation, which is a crucial issue for improving CRC treatment and survival. Material and methods The cross-talk between p38α and ERK was assessed in CRC cell lines and in APC Min/+ mice, a murine model of familial adenomatous polyposis. To this aim, animals were treated with the p38α inhibitor 4-(4-Fluorophenyl)−2-(4-hydroxyphenyl)−5-(4-pyridyl)−1H-imidazole (SB202190) alone or in combination with the MEK1 inhibitor N-[(2R)−2,3-Dihydroxypropoxy]−3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901). In order to evaluate the role of p38α and ERK in c-Myc regulation, we used pharmacological inhibitors of these two kinases alone or in combination with inhibitors of the transcriptional mechanism, translational process and proteasome in CRC cell lines. Moreover, the function of p38α and ERK in Myc stabilisation was assessed by genetic ablation. Results and discussions Here we show that concomitant inhibition of the p38α and MEK/ERK pathways significantly increases the survival of APC Min/+ mice in which tumorigenesis is driven by c-Myc deregulation. Genetic ablation of p38α and ERK revealed that these two MAPKs do not regulate c-Myc expression, nor do they affect c-Myc protein translational process. We found that p38α and ERK collaborate in c-Myc stabilisation by inhibiting its proteasomal degradation in CRC cell lines. These results were also confirmed by using the p38α and ERK pharmacological inhibitors LY2228820 (Ralimetinib) and GSK1120212 (Mekinist), respectively, which are currently in clinical trials for inflammatory diseases and cancer. Conclusion Since c-MYC supports the processes required for normal growth and homeostasis, its ablation is less attractive than modulation of its expression or function. Our results confirmed the essential role of the MAPK/c-Myc axis in intestinal tumorigenesis regulation, suggesting MAPK manipulation as a potential therapeutic approach to counteract c-Myc dependent carcinogenesis.

MicroRNA-451-modulated hnRNP A1 takes a part in granulocytic differentiation regulation and acute myeloid leukemia.

Myelopoiesis is under the control of a complex network containing various regulation factors. Deregulation of any important regulation factors may result in serious consequences including acute myeloid leukemia (AML). In order to find out the genes that may take a part in AML development, we analyzed data from AML cDNA microarray (GSE2191) in the NCBI data pool and noticed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is abnormally over-expressed in AML patients. Then we investigated the function and mechanisms of hnRNP A1 in myeloid development. A gradually decreased hnRNP A1 expression was detected during granulocytic differentiation in ATRA-induced-NB4 and HL-60 cells and cytokines-induced hematopoietic stem and progenitor cells. By function-loss and winning experiments we demonstrated hnRNP A1's inhibition role via inhibiting expression of C/EBPα, a key regulator of granulocytic differentiation, in the granulocytic differentiation. During granulocytic differentiation the decrease of hnRNP A1 reduces inhibition on C/EBPα expression, and the increased C/EBPα promotes the differentiation. We also demonstrated that miR-451 promotes granulocytic differentiation via targeting to and down-regulating hnRNP A1, and hnRNP A1 positively regulates c-Myc expression. Summarily, our results revealed new function and mechanisms of hnRNP A1 in normal granulocytiesis and the involvement of a feed-back loop comprising c-Myc, miR-451 and hnRNP A1 in AML development.

The heterogeneity of cancer stem-like cells at the invasive front

Cancer stem-like cells exhibit the multi-functional roles to survive and persist for a long period in the minimal residual disease after the conventional anti-cancer treatments. Cancer stem-like cells of solid malignant tumors which highly express CD44v8-10, the variant isoform of CD44 generated by alternative splicing, has a resistance to redox stress by the robust production of glutathione mediated by ESRP1-CD44v-xCT (cystine/glutamate antiporter) axis. It has been reported that CD44v and c-Myc tend to show the inversed expression pattern at the invasive front of the aggressive tumors. Given that the accumulation of reactive oxygen species triggers the activation of Wnt/β-catenin signal pathway, it is hypothesized that CD44v causes the negative feedback machinery in the regulation of c-Myc expression via the attenuated ROS-induced Wnt signal pathway. To address the fundamental question whether and how both proliferative and quiescent cancer stem-like cells heterogeneously exist at the invasive/metastatic edge, researchers need to investigate into the E3-ubiquitin ligase activity essential for c-Myc degradation. CSCs heterogeneity at the invasive/metastatic front is expected to demonstrate the dynamic tumor evolution with the selective pressure of anti-cancer treatments. Furthermore, the novel molecular targeting therapeutic strategies would be established to disrupt the finely-regulated c-Myc expression in the heterogeneous CSC population in combination with the typical drug-repositioning with xCT inhibitor.

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

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

Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression.

G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.