c-Myc Target Genes Research Articles

TMEPAI/PMEPA1 inhibits Wnt signaling by regulating β-catenin stability and nuclear accumulation in triple negative breast cancer cells

Transmembrane prostate androgen-induced protein (TMEPAI) is a type I transmembrane protein induced by several intracellular signaling pathways such as androgen, TGF-β, EGF, and Wnt signaling. It has been reported that TMEPAI functions to suppress TGF-β and androgen signaling but here, we report a novel function of TMEPAI in Wnt signaling suppression. First, we show that TMEPAI significantly inhibits TCF/LEF transcriptional activity stimulated by Wnt3A, LiCl, and β-catenin. Mechanistically, TMEPAI overexpression prevented β-catenin accumulation in the nucleus and TMEPAI knockout in triple negative breast cancer cell lines promoted β-catenin stability and nuclear accumulation together with increased mRNA levels of Wnt target genes AXIN2 and c-MYC. The presence of TGF-β type I receptor kinase inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells. These data suggest that TMEPAI suppresses Wnt signaling by interfering with β-catenin stability and nuclear translocation in a TGF-β signaling-independent manner.

Lysine-Specific Demethylase 1 (LSD1/KDM1A) Is a Novel Target Gene of c-Myc.

Lysine-specific demethylase 1 (LSD1/KDM1A) is a histone demethylase and specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 (H3K4). The LSD1-mediated demethylation of H3K4 promotes the assembly of the c-Myc-induced transcription initiation complex. Although LSD1 and c-Myc are both strongly expressed in human cancers, the mechanisms by which their activities are coordinated remain unclear. We herein demonstrated that LSD1 is a direct target gene of c-Myc. The knockdown of c-Myc decreased the expression of LSD1 in several cancer cell lines. We identified two non-canonical E-boxes in the proximal promoter region of the LSD1 gene. A chromatin immunoprecipitation assay showed that c-Myc bound to these E-boxes in the LSD1 promoter. Importantly, LSD1 mRNA expression correlated with c-Myc expression in human acute myeloid leukemia (AML), glioblastoma, stomach adenocarcinoma, and prostate adenocarcinoma. The present results suggest that LSD1 is induced by c-Myc and forms a positive feedback mechanism in transcription reactions by c-Myc.

Luteolin inhibits cell cycle progression and induces apoptosis of breast cancer cells through downregulation of human telomerase reverse transcriptase.

Luteolin is a flavonoid, which has been extensively investigated due to its antitumor effects; however, the underlying mechanisms of its action remain largely unknown. The present study aimed to investigate the role of luteolin in breast cancer (BC), and explored how luteolin suppresses the growth and induces the apoptosis of BC cells. The MTS assay was used to determine the anticancer activity of luteolin. Colony formation and Transwell assays were performed to evaluate the effects of luteolin on cell growth and invasion. Cell cycle progression and apoptosis were analyzed by flow cytometry. In addition, western blotting was performed to analyze cellular apoptosis and signaling pathways elicited by luteolin. The present study revealed that the proliferation of the BC cell line MDA-MB-231 was effectively suppressed by luteolin in a dose-dependent manner. Additionally, luteolin was revealed to increase apoptotic rates in BC cells. Dose-dependent cell cycle arrest in S phase was observed following treatment with luteolin in MDA-MB-231 cells. Mechanistically, luteolin reduced telomerase levels in a dose-dependent manner. Additionally, luteolin inhibited phosphorylation of the nuclear factor-κB inhibitor α and its target gene c-Myc, to suppress human telomerase reverse transcriptase (hTERT) expression, which encodes the catalytic subunit of telomerase. Collectively, the results of the present study indicated that luteolin may inhibit BC cell growth by targeting hTERT, suggesting that the mechanism of hTERT regulation by luteolin may justify further study regarding its potential as a therapeutic target for BC treatment.

SPAG5 upregulation contributes to enhanced c-MYC transcriptional activity via interaction with c-MYC binding protein in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks effective therapeutic targets. Sperm-associated antigen 5 (SPAG5) is a mitotic spindle-associated protein that is involved in various biological processes in cervical cancer and bladder urothelial carcinoma. However, the role of SPAG5 in TNBC remains undefined.MethodsThe expression of SPAG5 was examined in TNBC patients via quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry (IHC). The biological functions of SPAG5 in TNBC and the underlying mechanisms were investigated in vitro and in vivo.ResultsSPAG5 expression was significantly upregulated in TNBC tissues compared with that in paired adjacent noncancerous tissues (ANTs). High SPAG5 expression was associated with increased lymph node metastasis and high risk of local recurrence. SPAG5 protein expression was significantly associated with poor disease-free survival in TNBC. Gene set enrichment analysis of TNBC data from The Cancer Genome Atlas (TCGA) indicated that high SPAG5 expression was significantly associated with cell cycle and the ATR-BRCA pathway. Functional assays demonstrated that SPAG5 expression promoted tumor growth in vitro and in vivo. In addition, SPAG5-silenced cells were more sensitive to the PARP inhibitor (PARPi) olaparib. Mechanistically, SPAG5 interacted with c-MYC binding protein (MYCBP), thereby increasing MYCBP protein levels and leading to increased c-MYC transcriptional activity, which promoted the expression of the c-MYC target genes: CDC20, CDC25C, BRCA1, BRCA2, and RAD51.Knockdown of MYCBP or c-MYC abolished the SPAG5-induced cell-cycle progression and cell proliferation of TNBC.ConclusionsCollectively, our results indict that SPAG5 is an efficient prognostic factor in TNBC, and that SPAG5 knockdown increases the sensitivity of TNBC to the PARPi olaparib. SPAG5 promotes tumor growth and DNA repair by increasing c-MYC transcriptional activity via interaction with MYCBP. The SPAG5/MYCBP/c-MYC axis may represent a potential therapeutic target for TNBC treatment.

HJC0152, a novel STAT3 inhibitor with promising anti-tumor effect in gastric cancer.

BackgroundAberrant activation of the signal transducer and activator of transcription 3 (STAT3) is frequently seen in patients with gastric cancer (GC), and is generally associated with worse prognosis. HJC0152, a novel STAT3 inhibitor, has shown significant anti-tumor effects in several cancers, although its role in GC remains to be clarified.MethodsThe effect of HJC0152 on STAT3 signaling pathway and the biological behaviors of GC cells were evaluated through in vitro and/or in vivo experiments. Meanwhile, RNA sequence analysis was used to further explore its potential anti-tumor mechanisms.ResultsHJC0152 inhibited the expression of activated STAT3 and its downstream target genes (c-Myc and clyclinD1) in GC cells, and restrained tumor growth in vivo. HJC0152 treatment induced apoptosis in the STAT3 hyper-activated AGS and MKN45 cell lines, along with down-regulation of survivin and Mcl1, and up-regulation of cleaved-poly(ADP-ribose) polymerase. Moreover, HJC0152 markedly inhibited migration and invasion of these cells. Finally, RNA sequence analysis and protein expression analyses showed that in addition to STAT3 suppression, HJC0152 also exerts its anti-tumor effects at least partly via the mitogen-activated protein kinases pathway.ConclusionOur findings highlight that HJC0152 is a promising therapeutic agent for GC.

Paris Saponin II inhibits colorectal carcinogenesis by regulating mitochondrial fission and NF-κB pathway.

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Accumulating evidence suggests that mitochondrial dynamics are closely implicated in carcinogenesis including CRC. Paris Saponin II (PSII), a major steroidal saponin extracted from Rhizoma Paris polyphylla, has emerged as a potential anticancer agent. However, the effects of PSII on CRC and its underlying mechanisms remain unknown. In the present study, we found PSII induced apoptosis and inhibited colony formation in HT 29 and HCT 116 cells, and cell cycle arrest in G1 phase. PSII inhibited the phosphorylation of ERK1/2 and mitochondrial translocation of dynamin-related protein 1 (Drp1) by dephosphorylating Drp1 at Ser616, leading to the suppression of mitochondrial fission. PSII also suppressed NF-κB activation as a result of the inhibition of IKKβ and p65 translocation. Drp1 knockdown remarkably downregulated the nuclear expression of p65 and its target genes cyclin D1 and c-Myc in HCT 116 cell, confirming the link between mitochondrial fission and NF-κB pathway. Silencing of Drp 1 enhanced the inhibitory effects of PSII on p65 phosphorylation and the expressions of cyclin D1 and c-Myc, revealing that the inhibitory effects of PSII on cyclin D1 and c-Myc were relevant in the suppression of Drp1 and NF-κB activation. An in vivo study demonstrated PSII remarkably decreased the xenograft tumor size and suppressed the phosphorylation of ERK1/2 and Drp1 at Ser616. Taken together, our results suggested that PSII could inhibit colorectal carcinogenesis, at least in part, by regulating mitochondrial fission and NF-κB pathway.

SNIP1 Recruits TET2 to Regulate c-MYC Target Genes and Cellular DNA Damage Response

SUMMARYThe TET2 DNA dioxygenase regulates gene expression by catalyzing demethylation of 5-methylcytosine, thus epigenetically modulating the genome. TET2 does not contain a sequence-specific DNA-binding domain, and how it is recruited to specific genomic sites is not fully understood. Here we carried out a mammalian two-hybrid screen and identified multiple transcriptional regulators potentially interacting with TET2. The SMAD nuclear interacting protein 1 (SNIP1) physically interacts with TET2 and bridges TET2 to bind several transcription factors, including c-MYC. SNIP1 recruits TET2 to the promoters of c-MYC target genes, including those involved in DNA damage response and cell viability. TET2 protects cells from DNA damage-induced apoptosis dependending on SNIP1. Our observations uncover a mechanism for targeting TET2 to specific promoters through a ternary interaction with a co-activator and many sequence-specific DNA-binding factors. This study also reveals a TET2-SNIP1-c-MYC pathway in mediating DNA damage response, thereby connecting epigenetic control to maintenance of genome stability.

Cell division cycle associated 7 like predicts unfavorable prognosis and promotes invasion in glioma

BackgroundCell division cycle associated 7 like (CDCA7L) belongs to the JPO protein family, which is recently identified as a target gene of c-Myc and is frequently dysregulated in multiple cancers. This study aimed to explore the clinicpathological value and biological role of CDCA7L in glioma. MethodsCDCA7L expression in glioma patients was determined using the Oncomine database, and the prognostic role of CDCA7L expression was assessed in a retrospective cohort study. Moreover, the relationship of CDCA7L expression with the clinicopathological characteristics in glioma patients, including age, gender, tumor size, cystic change, Karnofsky performance scale (KPS) score, tumor location, extent of resection, WHO grade, adjuvant therapy and tumor recurrence, was analyzed in this study. In addition, the CDCA7L small interfering (si) RNA was constructed and transfected into the glioma U251 cells, so as to examine the role of CDCA7L in glioma patients. Besides, the changes in U251 cell invasion after transfection with CDCA7L siRNA were also monitored through Transwell assay. ResultsOur results suggested that CDCA7L expression was up-regulated in different glioma types, including glioblastoma, oligodendroglioma, diffuse astrocytoma and anaplastic astrocytoma. Moreover, the current retrospective cohort study indicated that high CDCA7L expression was associated with tumor size, WHO grade, adjuvant therapy and recurrence, as well as the poor overall survival (OS) and recurrence-free survival (RFS) in glioma patients. Lastly, CDCA7L expression was knocked down using CDCA7L siRNA, which could block the invasion abilities of glioma U251 cells. ConclusionsCDCA7L is highly expressed in human glioma tissues and a high CDCA7L expression level predicts the dismal prognosis for glioma patients. Moreover, CDCA7L can promote glioma invasion, which can serve as an independent potential prognostic biomarker for glioma patients.

Physciosporin suppresses the proliferation, motility and tumourigenesis of colorectal cancer cells

BackgroundLichens, which represent symbiotic associations of fungi and algae, are potential sources of numerous natural products. Physciosporin (PHY) is a potent secondary metabolite found in lichens and was recently reported to inhibit the motility of lung cancer cells via novel mechanisms. PurposeThe present study investigated the anticancer potential of PHY on colorectal cancer (CRC) cells. MethodsPHY was isolated from lichen extract by preparative TLC. The effect of PHY on cell viability, motility and tumourigenicity was elucidated by MTT assay, hoechst staining, flow cytometric analysis, transwell invasion and migration assay, soft agar colony formation assay, Western blotting, qRT-PCR and PCR array in vitro as well as tumorigenicity study in vivo. ResultsPHY decreased the viability of various CRC cell lines (Caco2, CT26, DLD1, HCT116 and SW620). Moreover, PHY elicited cytotoxic effects by inducing apoptosis at toxic concentrations. At non-toxic concentrations, PHY dose-dependently suppressed the invasion, migration and colony formation of CRC cells. PHY inhibited the motility of CRC cells by suppressing epithelial-mesenchymal transition and downregulating actin-based motility markers. In addition, PHY downregulated β-catenin and its downstream target genes cyclin-D1 and c-Myc. Moreover, PHY modulated KAI1 C-terminal-interacting tetraspanin and KAI1 expression, and downregulated the downstream transcription factors c-jun and c-fos. Finally, PHY administration showed considerable bioavailability and effectively decreased the growth of CRC xenografts in mice without causing toxicity. ConclusionPHY suppresses the growth and motility of CRC cells via novel mechanisms.

Abstract B04: Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules

Abstract Acute myeloid leukemia (AML) is a disease that can affect both children and adults and is characterized by complex and heterogeneous genomic changes, including MYC overexpression driven in part by the bromodomain (BRD) and extraterminal (BET) protein family of epigenetic readers. Current limitations in treatment approaches, including protein overexpression and mutations that confer resistance to therapy, lead to a wide range of response variability to conventional therapy, excessive treatment-related toxicity, and an overall poor outcome. A new therapeutic strategy involves stabilizing the MYC promoter G-quadruplex to downregulate expression. We hypothesize that direct downregulation of MYC in AML cells using MYC-promoter G-quadruplex interacting drugs (GQID) is a feasible novel approach in targeted therapy of AML. Treatment of AML cell lines KG-1a and UT-7epo with the MYC-promoter GQID, GQC-05 resulted in decreased MYC expression and induced a more significant decrease in c-Myc protein than treatment with the BET inhibitor (+)-JQ1. GQC-05 also decreased cell viability and increased apoptosis. RNA-seq of GQC-05-treated KG-1a cells identified 947 significantly differentially expressed genes compared to DMSO. The expression of several c-MYC target genes (including E2F1, GNL3, HSPA8, MAT2A, RCC1, SHMT1, and TFRC) was significantly decreased concomitant with MYC downregulation. The 947 genes were compared with genes with putative quadruplex sequences (PQS) from the literature. Of the 17 genes in common, MYC, NOTCH1, PIM1, and RHOU are significantly downregulated following GQC-05 treatment. These findings were validated by Q-RT-PCR in KG-1a and several other AML cell lines. Drug sensitivity and resistance (DSR) screening on a panel of AML cell lines using a library of 55 anticancer and targeted agents identified a range of sensitivities to GQC-05 (AUC range: 1.65-2.99; DMSO AUC ~ 4). These were compared to microarray gene-expression profiles and, as expected, GQC-05 sensitivity was not correlated with MYC expression. Nevertheless, ANOVA identified 1,049 significantly differentially expressed probe sets between the most sensitive vs. the least sensitive cell lines. Functional annotation revealed over-representation of cadherin, Wnt, and p53 signaling pathways as well over genes involved in hemostasis and hematopoietic cell lineage. In conclusion, directly targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of GQID for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. Citation Format: Megan A. Turnidge, Daniel H. Wai, Apurvi Patel, Justin J. Montoya, David W. Lee, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B04.

Butyl benzyl phthalate promotes prostate cancer cell proliferation through miR-34a downregulation

Prostate cancer is the most common malignancy in men. Phthalate esters are a class of environmental endocrine disruptors and were reported to be cancer promoting agents, however the potential role of phthalate esters in prostate cancer has been rarely reported. Mounting evidence has shown that miR-34a is a master tumor suppressor miRNA in cancer. The aim of this study was to investigate the role of butyl benzyl phthalate (BBP), one of the typical phthalate esters, in cell proliferation of prostate cancer cells. Human prostate cancer LNCaP and PC-3 cells were exposed to low dose of BBP for 6 days. The results showed that 10−6 and 10−7 mol/L BBP increased the expression of cyclinD1 and PCNA, decreased p21 expression, and induced cell growth in both LNCaP and PC-3 cells. Furthermore, we found that BBP significantly downregulated the expression of miR-34a, along with upregulation of miR-34a target gene c-myc. Using cell tranfection of miR-34a mimic and inhibitor, we demonstrated that BBP promoted cell proliferation through miR-34a/c-myc axis in prostate cancer cells. Findings from this study could provide new insight into the involvement and the molecular mechanism of phthalate esters on prostate cancer.

Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo.

MYC target 1 (MYCT1), a direct target gene of c-Myc, is a novel candidate tumor suppressor gene first cloned from laryngeal squamous cell carcinoma. The downregulation of MYCT1 has been reported to be associated with carcinogenesis. However, the role of MYCT1 in the development and progress of acute myeloid leukemia (AML) remains unknown and requires further investigation. In this study, we first found that the expression level of MYCT1 was significantly lower in the bone marrow (BM) derived from AML patients than that from healthy individuals. The low expression of MYCT1 in AML BM may be due to the hypermethylation in its promoter. MYCT1 expression was strongly associated with French–American–British classifications of AML. The low expression level of MYCT1 was more often observed in patients of M1, M5 and M6 types. In vitro, lentiviral particles carrying the complete CDS of MYCT1 gene were used to mediate the forced overexpression of MYCT1 in two AML cell lines, HL-60 and KG-1a. MYCT1 overexpression significantly inhibited cell proliferation, arrested cell cycle at G0/G1 phase, and downregulated the expression of cyclins D and E. Moreover, MYCT1 overexpression triggered apoptosis in AML cells, which was accompanied by enhanced cleavage of caspase-3 and -9, upregulated expression of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), and downregulated Bcl-2. Finally, in BALB/c nude mice bearing xenograft tumors generated by HL-60 and KG-1a cells, we noted that the intratumoral injection of MYCT1 lentivirus repressed tumor growth and led to massive apoptosis. In summary, our results reveal that MYCT1’s promoter is hypermethylated and its expression is downregulated in the BM of AML patients. MYCT1 plays a tumor-suppressive role, and it may serve as a promising target for the genetic therapeutic strategy in treating AML.

STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small Cell Lung Cancer.

Constitutively activated STAT3 plays a critical role in non-small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T (P < 0.007) and H1975 models (P < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. Mol Cancer Ther; 17(9); 1917-26. ©2018 AACR.

Pimozide suppresses colorectal cancer via inhibition of Wnt/β-catenin signaling pathway

ObjectiveWnt/β‑catenin signaling pathway plays important role in colorectal cancer (CRC) and acts as a potential therapeutic target. Pimozide is a FDA-approved clinical drug used to treat psychotic diseases and it has shown anticancer effect in some tumors partially via inhibition of Wnt/β‑catenin signaling pathway. This study aimed to investigate whether pimozide exerts anticancer effect on CRC and explore underlying mechanism. Methods and resultsPimozide was administrated to treat HCT116 and SW480 cells. Quantitative real-time polymerase chain reaction and western blot were used to detect the expression of epithelial-to-mesenchymal transition markers and Wnt/β‑catenin signaling pathway-related proteins. Cell proliferation and migration were measured by Cell Counting Kit-8 and Transwell assays respectively. HCT116 and SW480 cells were subcutaneously injected into nude mice and when the volume of tumor grown measureable (approximately 100 mm3) animals were treated with vehicle saline or pimozide at a dose of 25 mg/kg·d by oral gavage and then tumor size was measured at 7, 14, 21 and 28 days post treatment. Pimozide dose-dependently inhibited cell proliferation and migration in both HCT116 and SW480 cells, increased expression of E-cadherin and decreased expression of N‑cadherin, vimentin and Snail. In addition, tumor growth was inhibited by pimozide in both HCT116 and SW480 xenografts in vivo. Expression of β‑catenin and Wnt target genes c-Myc, cyclin D1, Axin 2 and survivin was reduced by pimozide treatment in both HCT 116 and SW480 cells. ConclusionPimozide exerts anticancer effect in CRC via inhibition of wnt/β‑catenin signaling pathway, suggesting it as a potential therapeutic drug for CRC.

Abstract 3557: Beta-hydroxybutyrate inhibits oncogenic signaling and cellular motility in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma is a devastating malignancy with a five-year survival rate less than 7%. It is characterized by a plethora of metabolic and signaling pathways, which plays a critical role in the pathogenesis of the disease. Caloric restriction and ketogenic diets have been shown to be beneficial in the management of several kinds of malignancies including pancreatic cancer. During caloric restriction or ketogenic diet supplementation, blood level of ketone bodies (beta-hydroxy butyrate or BHB, acetoacetate, acetone) gets elevated in the body. Among, these ketone bodies, BHB, the most abundant ketone body in mammals has been shown to act as the modulator of several signaling and inflammatory pathways. We have evaluated the effect of BHB on oncogenic signaling pathways and motility of pancreatic cancer cells. We observed reduced activation of key oncogenic signaling pathways, including mTOR. We also observed inhibition of cellular motility in a dose-dependent manner. Furthermore, we observed reduced expression of c-Myc, a key oncogene and a downstream effector molecule of mTOR. Correspondingly, we also observed reduced expression of several c-Myc target genes. We have also evaluated the effect of BHB on epithelial-mesenchymal transition (EMT) markers and observed altered expression of EMT markers like N-cadherin and E-cadherin. We also evaluated the effect of BHB on the expression of inflammatory cytokines in the pancreatic cancer cells and observed that BHB treatment leads to the reduction of expression of several inflammatory cytokines including IL6 and IL1-beta. Overall, our findings suggest that BHB inhibits inflammatory signaling in pancreatic cancer cells and reduces the cellular motility by modulating the expression of EMT regulators. Citation Format: Surendra K. Shukla, Nina V. Chaika, Pankaj K. Singh. Beta-hydroxybutyrate inhibits oncogenic signaling and cellular motility in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3557.

Abstract 1273: Effect of a Western diet on colitis-associated colon tumor formation in BALB/c mice

Abstract Patients with inflammatory bowel disease (IBD) are at increased risk for inflammation associated colorectal cancer (CAC). The incidence of IBD is increasing in the Western industrialized world. Epidemiological studies suggest that a diet, high in fat but low in vitamin D, calcium, fibre and methyl donors is a risk factor for both, IBD and colorectal cancer (CRC). It has been shown previously that a ”New Western Diet”, caused development of sporadic colonic tumours in mice. Our study was performed to evaluate whether a Western Diet (WD) aggravates azoxymethane (AOM) /dextran sodium sulfate salt (DSS)-induced colitis-associated carcinogenesis in mice, and if a switch to the standard purified mouse diet AIN93G after cancer initiation is still able to ameliorate or delay the onset of the disease. Female BALB/c mice received either the WD (0.5 mg/g calcium, 2% fibre, 21 % butter fat, 0.23 μg/g folic acid) or AIN93G standard diet for 5 weeks until AOM was administered at a dose of 12.5 μg/kg intraperitoneally, followed by 3 cycles of DSS (2.5%). In one group the WD was switched to AIN93G diet one day before first administration of DSS. The mice were euthanized 80 days after AOM injection. One part of the colon, small intestine, kidney and liver were frozen in liquid nitrogen, the rest formalin fixed and paraffin embedded for mRNA and immunhistochemical analysis. Feeding constantly the WD shortened the colon (p&amp;lt;0.05) and increased the number (p&amp;lt;0.05) and size (p&amp;lt;0.01) of aberrant colonic crypt foci. The switch to the AIN93G diet ameliorated this effect, leading to reduced colitis and tumor promotion comparable with the AIN93G group. In colon ascendens of the WD group, Ki67 protein levels (p&amp;lt;0.001) and relative mRNA levels of the Wnt target genes c-myc (p&amp;lt;0.05) and axin2 (p&amp;lt;0.001) were reduced while mRNA levels of the vitamin D catabolizing enzyme cyp24a1 (p&amp;lt;0.001) and inos (p&amp;lt;0.05) were increased when compared with the AIN93G group. A healthy diet protects colonic mucosa against AOM/DSS-treatment and is an effective chemopreventive strategy to reduce chronic colonic inflammation and tumorigenesis. This project is funded by the Vienna Science and Technology Fund and the Austrian Research Fund. Citation Format: Charlotte Groeschel, Samawansha Tennakoon, Abhishek Aggarwal, Enikoe Kallay. Effect of a Western diet on colitis-associated colon tumor formation in BALB/c mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1273.

MAP3K7 is recurrently deleted in pediatric T-lymphoblastic leukemia and affects cell proliferation independently of NF-\u03baB

BackgroundDeletions of 6q15–16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4.MethodsMAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR.ResultsMAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α.ConclusionsThis study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts.

The suppressive effect of soluble Klotho on fibroblastic growth factor 23 synthesis in UMR-106 osteoblast-like cells.

Fibroblastic growth factor 23 (FGF23) is a hormone secreted primarily by bone. FGF23 is elevated in the serum of chronic kidney disease (CKD) patients, but the exact mechanism is not well known. Klotho is identified as an aging suppressor, which is mainly expressed in the kidney, and the level of soluble Klotho is negatively associated with FGF23 in CKD. The aim of this study was to investigate the effect and possible mechanism of Klotho on FGF23 synthesis in osteoblast-like UMR-106 cells. UMR-106 cells were divided into five groups: (i) control group; (ii) β-glycerophosphate (β-GP) group; (iii) β-GP + Klotho group; (iv) β-GP+ lithium chloride (LiCl, a Wnt/β-catenin pathway agonist) group; and (v) β-GP + Klotho + LiCl group. Subsequently, UMR-106 cells were cultured for 72 h, and the expression of FGF23, P-glycogen synthase kinase-3β (P-GSK-3β), and glycogen synthase kinase-3β(GSK-3β) were measured with Western blot analysis. The mRNA levels of FGF23 and the Wnt/β-catenin pathway target gene c-myc were determined with RT-qPCR. The results showed that β-GP induced increased expression of FGF23 mRNA and protein. Compared with the β-GP group, expression of FGF23 mRNA and protein expression were downregulated in the β-GP + Klotho group. In addition, β-GP induced increased expression of P-GSK-3β/GSK-3β and c-myc, which were all downregulated in the β-GP + Klotho group. Moreover, the expression of FGF23, P-GSK-3β/GSK-3β, and c-myc mRNA were upregulated when treated with LiCl. These results demonstrate that soluble Klotho suppresses FGF23 synthesis in osteoblast-like UMR-106 cells. The mechanism of this suppression may be partially through the inhibition of the Wnt/β-catenin pathway.

Transcription factor 7 functions as an unfavorable prognostic marker of glioblastoma multiforme by promoting proliferation by upregulating c-Myc.

Transcription factor 7 (TCF7) is an oncogenic transcription factor in several kinds of cancers. However, the clinical significance of TCF7 in glioblastoma multiforme (GBM) has not been well elucidated. A total of 107 patients with surgical resection of GBM were enrolled in our study. TCF7 expression in these cases was detected by immunohistochemistry and the difference in TCF7 mRNA levels between tumor tissues and adjacent tissues was compared with a real-time PCR. The correlation between TCF7 expression and the clinicopathologic factors was analyzed using the χ-test. Moreover, the prognostic value of TCF7 was evaluated by univariate and multivariate analyses. In addition, the influence of TCF7 on the proliferation of the GBM cell line U251 was detected using an MTT assay after regulating TCF7 and its target gene c-Myc. The high and low expressions of TCF7 accounted for 54.21 and 45.79%, respectively, in all cases. The mRNA level of TCF7 in GBM tissues was markedly higher than that in adjacent tissues, indicating the oncogenic role of TCF7 in GBM. High expression of TCF7 was associated significantly with high Ki67 percentage and the sex of the patients, and it was identified as an independent prognostic factor for patients with GBM. With experiments in vitro, TCF7 was shown to promote cell proliferation by increasing c-Myc expression in GBM. TCF7 could be considered an independent prognostic factor in GBM, and could enhance GBM cell proliferation by upregulating c-Myc, indicating that it may be a potential and promising molecular drug target for GBM.

JAK2 is dispensable for maintenance of JAK2 mutant B-cell acute lymphoblastic leukemias.

Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.