c-Myc Signaling Pathways Research Articles

Inhibition of TMEM16A impedes embryo implantation and decidualization in mice.

Recent studies revealed that TMEM16A is involved in several reproductive processes, including ovarian estrogen secretion and ovulation, sperm motility and acrosome reaction, fertilization and myometrium contraction. However, little is known about the expression and function of TMEM16A in embryo implantation and decidualization. In this study, we focused on the expression and regulation of TMEM16A in mouse uterus during early pregnancy. We found that TMEM16A is upregulated in uterine endometrium in response to embryo implantation and decidualization. Progesterone treatment could induce TMEM16A expression in endometrial stromal cells through progesterone receptor/c-Myc pathway, which is blocked by progesterone receptor antagonist or the inhibitor of c-Myc signaling pathway. Inhibition of TMEM16A by small molecule inhibitor (T16Ainh-A01) resulted in impaired embryo implantation and decidualization in mice. Treatment with either specific siRNA of Tmem16a or T16Ainh-A01 inhibited the decidualization and proliferation of mouse endometrial stromal cells. In conclusion, our results revealed that TMEM16A is involved in embryo implantation and decidualization in mice, compromised function of TMEM16A may lead to impaired embryo implantation and decidualization.

Maclurin exerts anti-cancer effects on PC3 human prostate cancer cells via activation of p38 and inhibitions of JNK, FAK, AKT, and c-Myc signaling pathways

Maclurin is a phenolic compound extracted from purple mangosteen and mulberry twigs. Earlier reports indicated that it exerts antioxidant activity. We hypothesized that maclurin exerts antioxidant activity and anti-cancer effects in small cell neuroendocrine carcinomas (SCNCs), a very aggressive type of human prostate cancer. To verify our hypothesis, we selected PC3 cells as a model system and investigated the antioxidant activity and anti-cancer effects of maclurin. In the reactive oxygen species (ROS) detection assay for the verification of antioxidant activity, we observed the unexpected prooxidant activity of maclurin in PC3 cells. For the anti-cancer activities, we investigated the effects of maclurin on induction of apoptosis and inhibition of metastatic characteristics of PC3 cells. In the apoptosis assay, maclurin significantly induced apoptosis of PC3 cells. Maclurin also showed significant anti-metastatic effects. Maclurin inhibited cell migration in a dosage-dependent manner. In addition, the gelatin zymography assay indicated that maclurin inhibited activities of matrix metalloproteinase-2 and 9 (MMP-2 and MMP-9) that affect cell migration and extracellular matrix (ECM) degradation. Then, we investigated the effects of maclurin on the cancer-related signaling molecules. Maclurin activated p38 signaling and inhibited c-Jun N-terminal kinase (JNK), focal-adhesion kinase (FAK), AKT, and c-Myc signalings in PC3 cells. Finally, we observed prooxidant activity and anti-SCNC effects of maclurin in DU145 cells. This suggests that the effects of maclurin may not be specifically limited to PC3 cells. Our findings suggest that maclurin exerts anti-cancer effects on SCNC cells via activation of p38 and inhibitions of JNK, FAK, AKT and c-Myc signalings.

Abstract 5002: KRT13 promotes stemness and drives metastasis in breast cancer through direct interaction with plakoglobin-desmoplakin complexes regulating c-Myc signaling pathway

Abstract Introduction and Objective: Keratins (KRTs) were reported to interact with multiple cellular proteins and initiate signaling cascades that promote cell proliferation, migration and metastasis in cancers. Keratin13 (KRT13), a member of the intermediate filament proteins, plays important role in breast cancer progression and metastasis. The objectives of this study are to determine the role and molecular mechanism of KRT13 in promoting breast cancer growth and metastatic progression. Methods and Results: We evaluated KRT13 protein expression in 58 primary breast cancer tissues by immunohistochesmistry and found that KRT13 is overexpressed in metastatic breast cancer specimens (12/18) (p<0.05). KRT13 expression in primary breast cancer is associated with decreased patient overall survival based on publicly available Oncomine gene expression datasets. To understand the functional roles of KRT13 in breast cancer progression and metastasis, we performed studies by overexpressing and knocking down KRT13 and investigating alterations of breast cancer cell behaviors in vitro and tumor growth and metastasis in mice. KRT13-overexpressing MCF7 cells promote and KRT13-knocked down HCC1954 and MDA-MB-468 cells inhibit in vitro cell proliferation, invasion and migration and in vivo tumor growth and metastasis to bone and lung. KRT13 overexpression increased the stemness and mammosphere-forming ability of MCF7 cells compared to vehicle control. KRT13 directly interacted with plakoglobin(γ-catenin) to form complexes with desmoplakin as assessed by mass spectrometry and confirmed by Western blot. KRT13 decreased the expression and nuclear translocation of plakoglobin, abrogated plakoglobin-mediated c-Myc gene suppression, promoted cancer stemness and tumor metastasis. Conversely, genetic silencing of KRT13 in HCC1954 and MDA-MB-468 cells increased nuclear translocation of plakoglobin and enhanced c-Myc suppression, and decreased breast cancer cell growth and stemness in vitro and xenograft tumor formation and metastasis in vivo. Conclusion: KRT13 exerts a novel function in the regulation of breast cancer cell growth, progression and metastasis via alterations of nuclear translocation of plakoglobin that modulates downstream c-Myc-dependent signaling, inducing stem cell-like phenotype and metastasis of breast cancer. Our findings could reveal potential novel targeting of breast cancer progression and metastasis. Funded by an NCI P01 CA098912 and a Board of Governors Chair of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung. Citation Format: Lijuan Yin, Gina Chia-Yi Chu, Qinlong Li, Liyuan Yin, Michael Lewis, Mourad Tighiouart, Leland W. k. Chung, Haiyen E. Zhau. KRT13 promotes stemness and drives metastasis in breast cancer through direct interaction with plakoglobin-desmoplakin complexes regulating c-Myc signaling pathway [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 5002.

Reciprocal Regulation among miR-181d/CRY2/FBXL3/c-myc Signaling Axis Modulates Metabolism in Colorectal Cancer

Patients with CRC (colorectal cancer) usually have a poor prognosis and the cure rate of CRC remained unsatisfied due to unfavorable curative effect. It is well known that microRNAs (miRNAs) and energy metabolism have pivotal roles in CRC progression. In a recent article in Cell Death & Disease by Xiaofeng Guo. et al. 2017, we have reported an oncogenic role of miR-181d in CRC by promoting glycolysis, and its underlying molecular mechanism about a new feedback loop among miR-181d/CRY2/FBXL3/c-myc signaling axis. Among these, we have identified the level of miR-181d was upregulated in CRC and the inhibition of miR-181d decreased glycolysis in CRC cells. We also found that c-myc played a central role in regulating cell glycolysis, which is required for the metabolic shift induced by miR-181d. Besides, we have demonstrated FBXL3 and CRY2 were direct targets of miR-181d and c-myc promoted miR-181d upregulation while inhibiting the expression of CRY2 and FBXL3 in CRC cells. The data from our recent article strongly suggest a new light onto the oncogenic function of the miR-181d in CRC. Furthermore, these findings represent a novel potential approach for silencing miR-181d/c-myc signaling pathway in CRC treatment.

H2-P, a honokiol derivative, exerts anti-angiogenesis effects via c-MYC signaling pathway in glioblastoma.

H2-P, a derivative of honokiol, was first synthesized in our laboratory. Compared with honokiol, H2-P has even high anti-tumor activity. In the present study, we evaluated the ability of H2-P to inhibit the survival rate in four gliomas cell lines. The result showed that H2-P could significantly inhibit proliferation of gliomas cells in a dose-dependent manner (IC50U251 = 9.03, IC50SHG-44 = 10.74, IC50U78 = 19.87, and IC50c6 = 22.56 nM). Furthermore, to determine the mechanism underlying the anti-gliomas effects of H2-P, six kinase activities was detected by Z'-LYTE™ system. The high-throughput screening shown that effect targets of H2-P were MEK and VEGFR2. We also studied the inhibition of H2-P vascular endothelial cells (EA.HY926). The data shown that H2-P could increase endothelial cells apoptosis rate, while inhibiting endothelial cell proliferation (IC50EA.hy926 = 16.11 nM) and migration. Besides, we investigated anti-angiogenesis of H2-P in the rat thoracic aorta rings, chicken chorioallantoic membrane (CAM), and capillary tube formation models. H2-P showed strong inhibition of angiogenesis. Moreover, we found that H2-P also could reduce tumor volume in mice significantly (P < 0.01), and downregulate gene expression level of VEGFR2, MEK, and c-MYC in tumor. These data suggest that H2-P have an excellent anti-tumor activity by exerting anti-angiogenesis effects via c-MYC signaling pathway in glioblastoma (GBM).

Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma.

Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual "first-in-class" PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. Mol Cancer Ther; 15(11); 2553-62. ©2016 AACR.

Non-immunosuppressive triazole-based small molecule induces anticancer activity against human hormone-refractory prostate cancers: the role in inhibition of PI3K/AKT/mTOR and c-Myc signaling pathways.

A series of triazole-based small molecules that mimic FTY720-mediated anticancer activity but minimize its immunosuppressive effect have been produced. SPS-7 is the most effective derivative displaying higher activity than FTY720 in anti-proliferation against human hormone-refractory prostate cancer (HRPC). It induced G1 arrest of cell cycle and subsequent apoptosis in thymidine block-mediated synchronization model. The data were supported by a decrease of cyclin D1 expression, a dramatic increase of p21 expression and an associated decrease in RB phosphorylation. c-Myc overexpression replenished protein levels of cyclin D1 indicating that c-Myc was responsible for cell cycle regulation. PI3K/Akt/mTOR signaling pathways through p70S6K- and 4EBP1-mediated translational regulation are critical to cell proliferation and survival. SPS-7 significantly inhibited this translational pathway. Overexpression of Myr-Akt (constitutively active Akt) completely abolished SPS-7-induced inhibitory effect on mTOR/p70S6K/4EBP1 signaling and c-Myc protein expression, suggesting that PI3K/Akt serves as a key upstream regulator. SPS-7 also demonstrated substantial anti-tumor efficacy in an in vivo xenograft study using PC-3 mouse model. Notably, FTY720 but not SPS-7 induced a significant immunosuppressive effect as evidenced by depletion of marginal zone B cells, down-regulation of sphingosine-1-phosphate receptors and a decrease in peripheral blood lymphocytes. In conclusion, the data suggest that SPS-7 is not an immunosuppressant while induces anticancer effect against HRPC through inhibition of Akt/mTOR/p70S6K pathwaysthat down-regulate protein levels of both c-Myc and cyclin D1, leading to G1 arrest of cell cycle and subsequent apoptosis. The data also indicate the potential of SPS-7 since PI3K/Akt signalingis responsive for the genomic alterations in prostate cancer.

Abstract 1258: Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is now the fastest growing cancer in the western world and most EAC patients present with widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. The transcription factor proto-oncogene c-Myc is a potent activator of tumorigenesis. Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype. c-Myc amplification has been shown to be frequent in esophageal adenocarcinoma and has been implicated in Barrett's carcinogenesis. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. In this study, we therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a cyclin-dependent kinase (CDK) inhibitor flavopiridol. Methods: Western blot analysis was done to see the expression of c-Myc and apoptotic signaling pathways in a panel of nine esophageal cancer cell lines. c-Myc overexpression and knockdown were performed using both genetic and novel chemical approaches. Cell viability assays were performed in 96-well plates using the colorimetric WST-1 reagent. Esophageal cancer tumors growth was measured in xenograft and a novel peritoneal disseminated metastatic survival model of immunodeficient mice. Results: Western blot analysis revealed frequent overexpression of c-Myc in EAC cell lines. In this panel of esophageal cancer cell lines tested more than 70% of EAC cell lines showed overexpression of c-Myc. When we tested these cell lines for their ability to form xenograft tumor and peritoneal dissemination, c-Myc overexpression correlated with accelerated EAC tumor growth in xenograft and peritoneal disseminated metastatic survival model of NOD/SCID mice. The xenograft tumor growth rate and formation rate of peritoneal cancer after injection of 5 million cells were highest in OE19 EAC cell line which showed the highest c-Myc expression. In addition, median animal survival with peritoneal dissemination was lowest for OE19 (55 days) whereas OACM5.1 C EAC cell line which had the lowest c-Myc expression didn't form any peritoneal tumor. EAC cell lines with elevated c-Myc expression are preferentially more sensitive to induction of apoptosis by CDK inhibitor flavopiridol compared to EAC cell lines with lower c-Myc expression. When we tested the role of c-Myc expression by upregulation/downregulation in this apoptotic effect we found that this effect is very much dependent on c-Myc expression. Conclusion: These results indicate that CDK inhibitor alone or in combination with other cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing EAC. Citation Format: Sazzad Hassan, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs V. Holzen. Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1258.

Abstract 2944: Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer

Abstract Purpose: Expression of astrocyte-elevated gene-1 (AEG-1), a novel oncoprotein, is elevated in multiple cancers. Thymidylate synthase (TS) is an important enzyme in purine synthesis and DNA replication. In this study, we investigated the AEG-1 expression characteristics in non-small cell lung cancer (NSCLC) to determine whether its expression is correlated with TS regulation of NSCLC. Materials and Methods: A549, H157, CL1-0, CL1-5, PC9, H1975, H520 and H292 human lung cancer cell-lines were cultured in medium with RPMI1640 + 10% FBS. Immunohistochemical stain and Western blot of AEG-1 and TS were studied in all cell-lines. The primary antibodies used were anti-AEG-1 (1:1,000; chicken polyclonal), anti-actin (1:1,000; rabbit polyclonal; Santa Cruz), and anti-TS (1:1,000; mouse monoclonal; Abcam). Total RNA was extracted using a Qiagen mRNA easy mini kit (Qiagen). Real-time PCR was performed using ABI 7900 fast real-time PCR system to detect expression of AEG-1 and TS. Cell viability was determined by standard MTS assays to recognize IC50 of Pemetrexed in lung cancer cell-lines. Transfection of siRNA for AEG-1 was carried out to knock down AEG-1. AEG-1 pcDNA (Life technologies, CA, USA) were used to quantitatively examine AEG-1 activity. Expressive change of AEG-1associated with TS and signaling pathways were analyzed by Western blot and PCR. Results: TS and AEG-1 in H157, Cl1-0, CL1-5, H520 and H292 were higher detected by IHC stain, Western blot and RT-PCR mRNA level. The expression of TS was positive correlated with AEG-1 in lung cancer cell-lines. The IC50 values of Pemetrexed for cells were lower in the lung cancer cell-lines with lower TS gene expression. After knockdown of AEG-1 by siRNA in Pemetrexed-resistance PC-9 and A549 cell-lines, the expression of TS decreased associated with decreasing change IC50 of Pemetrexed. In contrast, transfection with AEG-1 pcDNA in PC-9 and A549 cell-lines overexpressed AEG-1 gene, TS level also enhanced associated with increasing change IC50 of Pemetrexed. AEG-1 expression can also activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway that augments binding of c-Myc thereby regulating AEG-1 transcription. Conclusions: Our data indicated a significant correlation between TS and AEG-1 gene expression in NSCLC cell-lines. TS expression might be regulated by AEG-1 through activation of PI3K/AKT and c-Myc signaling pathways associated with development of chemoresistance to pemetrexed in NSCLC treatment Citation Format: Chung-Yu Chen, Ying-Yin Chen, Kuan-Yu Chen, Jin-Yuan Shih, Yih-Leong Change, Chong-Jen Yu, Pan-Chyr Yang. Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2944.

Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines.

Aristolochic acid I (AAI) existing in plant drugs from Aristolochia species is an environmental human carcinogen associated with urothelial cancer. Although gene association network analysis demonstrated gene expression profile changes in the liver of human TP53 knock-in mice after acute AAI exposure, to date, whether AAI causes hepatic tumorigenesis is still not confirmed. Here, we show that hepatic premalignant alterations appeared in canines after a 10-day AAI oral administration (3 mg/kg/day). We observed c-Myc oncoprotein and oncofetal RNA-binding protein Lin28B overexpressions accompanied by cancer progenitor-like cell formation in the liver by AAI exposure. Meanwhile, we found that forkhead box O1 (FOXO1) was robustly phosphorylated, thereby shuttling into the cytoplasm of hepatocytes. Furthermore, utilizing microarray and qRT-PCR analysis, we confirmed that microRNA expression significantly dysregulated in the liver treated with AAI. Among them, we particularly focused on the members in let-7 miRNAs and miR-23a clusters, the downstream of c-Myc and IL6 receptor (IL6R) signaling pathway linking the premalignant alteration. Strikingly, when IL6 was added in vitro, IL6R/NF-κB signaling activation contributed to the increase of FOXO1 phosphorylation by the let-7b inhibitor. Therefore, it highlights the new insight into the interplay of the network in hepatic tumorigenesis by AAI exposure, and also suggests that anti-premalignant therapy may be crucial for preventing AAI-induced hepatocarcinogenesis.

Bis-cyclopropane analog of disorazole C1 is a microtubule-destabilizing agent active in ABCB1-overexpressing human colon cancer cells.

The novel, chemically stabilized disorazole analog, (-)-CP2-disorazole C1 (1) displayed potent anti-proliferative activity against a broad-spectrum of human colorectal cancer cells. HCT15 and H630R1 cell lines expressing high basal levels of the ABCB1 protein, known to cause multi-drug resistance, were also sensitive to growth inhibition by 1 but were resistant to both vincristine and docetaxel, two commonly used microtubule inhibitors. Compound 1 exhibited strong inhibition of tubulin polymerization at a level comparable to vincristine. In addition, treatment with 1 resulted in decreased protein levels of β-tubulin but not α-tubulin. An analysis of cellular proteins known to interact with microtubules showed that 1 caused decreased expression of c-Myc, APC, Rb, and additional key cellular signaling pathways in CRC cells. Treatment with compound 1 also resulted in G2/M cell cycle arrest and induction of apoptosis, but not senescence. Furthermore, endothelial spheroid sprouting assays demonstrated that 1 suppressed angiogenesis and can, therefore, potentially prevent cancer cells from spreading and metastasizing. Taken together, these findings suggest that the microtubule disruptor 1 may be a potential drug candidate for the treatment of mCRC.

Abstract B22: Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine

Abstract Erufosine is a third generation alkylphosphocholine, which has been shown to inhibit the proliferation of oral squamous cell carcinoma (OSCC) cells. It simultaneously induces apoptosis and autophagy by modulating the Akt–mTOR signaling pathway and is known to down regulate the expression of cell cycle related proteins, but the exact mechanism of action of this membrane seeking compound is still unknown. For investigating the mechanism of action in a systematic way, we studied the effects of erufosine in a panel of OSCC cell lines (Cal-27, FaDu and HN-5). The concentration effecting the 50% inhibition in cell proliferation (IC50) in response to erufosine was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using concentrations ranging from 0.6µM – 100µM at various time points (24h, 48h and 72h) after exposure. Based on these results, FaDu cells were exposed to erufosine at concentrations effecting IC25, IC50 and IC75, and then the mRNA of respectively treated cells was isolated and subjected to microarray analysis by Illumina Chip array. Selected genes with highly modulated mRNA expression were further investigated by quantitative RT-PCR and Western Blot. IC50 values in response to erufosine, following exposure for 24h, 48h and 72h exposure ranged from 20µM to 9.7µM for Cal-27 -, 27µM to 14µM for FaDu - , and 43µM to 37µM for HN-5 cells. Analysis of microarray data by the Ingenuity Pathways Analysis (IPA; Ingenuity® Systems, www.ingenuity.com) software revealed that 725 genes were modulated at 16h, averaged for all concentrations, with 47 genes being modulated more than 3 fold. The respective 24h gene expression analysis revealed that 1215 genes were significantly modulated, with 77 genes deregulated over 3 fold, whereas a total of 1305 genes were altered at the end of 48h, with 93 genes being deregulated over 3 fold. In a first approach, we concentrated on genes related to the c-MYC signaling pathway. The cyclin-dependent kinase inhibitor 1A (p21, Cip1) was found to be upregulated 9.2, 7.9 and 9.9 fold, averaged for all concentrations, at 16h, 24h and 48h, respectively. The Max dimerization protein 1 (MDX1) was upregulated 3.7 fold and 3.3 fold over the untreated control at 24h and 48h post treatment. The combined modulation of the two mRNA species hints at the involvement of c-MYC as a target of erufosine treatment. These results were further validated by qRT-PCR, which also confirmed the modulation of these genes across the OSCC cell panel. In line with this, the Western blots also showed a clear decrease in c-MYC protein levels and a corresponding increase in p21 in a concentration- and time-dependent manner. In summary, the known effect of erufosine on cell cycle related proteins as well as the increased mRNA (p21, MDX-1) and protein (p21) expression reported here is in accordance with the decreased expression of c-MYC at protein level. These findings support the hypothesis that c-MYC is an indirect target of erufosine in OSCC cells. Citation Format: Shariq S. Ansari, Martin R. Berger. Inhibition of c-MYC expression in oral squamous carcinoma cells contributes to the antiproliferative activity of the alkylphosphocholine erufosine. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B22.

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

CIP2A mediates prostate cancer progression via the c-Myc signaling pathway.

Recent evidence suggests that cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that acts as a novel therapeutic target in a variety of tumors. In this study, we investigated the clinical significance of CIP2A and its function in our large collection of prostate samples. Between August 2000 and December 2013, 126 patients with histologically confirmed prostate cancer (PCa) and 92 with benign prostate hyperplasia (BPH) were recruited into the study. Quantitative real-time PCR (RT-PCR), Western blot, and immunohistochemistry analyses were used to quantify CIP2A expression in PCa clinical samples and cell lines. The relationships between CIP2A expression and clinicopathological features were analyzed. The functional role of CIP2A in PCa cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation and invasion. High expression of CIP2A staining was 86.51 % (109/126) in 126 cases of PCa and 17.39 % (16/92) in 92 cases of BPH; the difference of CIP2A expression between PCa and BPH was statistically significant. CIP2A was significantly elevated in all five PCa cell lines when compared to the RWPE-1 cells at both the messenger RNA (mRNA) and protein levels. Silencing of CIP2A inhibited the proliferation of DU-145 cells which have a relatively high level of CIP2A in a time- and concentration-dependent manner, and the invasion and migration of DU-145 cells were distinctly suppressed. Furthermore, CIP2A knockdown led to substantial reductions in c-Myc levels in DU-145 cells, but no significant change in phosphorylated Akt expression after CIP2A knockdown in DU-145 cells. Our data suggest that the pathogenesis of human PCa maybe mediated by CIP2A, and CIP2A inhibition treatment may provide a promising strategy for the antitumor therapy of PCa, and thus, CIP2A could represent selective targets for the molecularly targeted treatments of PCa.

CIP2A mediates prostate cancer progression via the c-MYC signaling pathway.

Recent evidence suggests that cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that acts as a novel therapeutic target in a variety of tumors. In this study, we investigated the clinical significance of CIP2A and its function in our large collection of prostate samples. Between August 2000 and December 2013, 126 patients with histologically confirmed PCa and 92 with benign prostate hyperplasia (BPH) were recruited into the study. Quantitative RT-PCR, Western blot, and immunohistochemistry analyses were used to quantify CIP2A expression in PCa clinical samples and cell lines. The relationships between CIP2A expression and clinicopathological features were analyzed. The functional role of CIP2A in PCa cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation and invasion. High expression of CIP2A staining was 86.51 % (109/126) in 126 cases of PCa and 17.39 % (16/92) in 92 cases of BPH, and the difference of CIP2A expression between PCa and BPH was statistically significant. CIP2A was significantly elevated in all five PCa cell lines when compared to the RWPE-1 cells at both the messenger RNA (mRNA) and protein levels. Silencing of CIP2A inhibited the proliferation of DU-145 cells which have a relatively high level of CIP2A in a time- and concentration-dependent manner, and the invasion and migration of DU-145 cells were distinctly suppressed. Furthermore, CIP2A knockdown led to substantial reductions in c-Myc levels in PCa cell lines, but no significant change in phosphorylated Akt expression after CIP2A knockdown in DU-145 cells. Our data suggest that the pathogenesis of human PCa maybe mediated by CIP2A, and CIP2A inhibition treatment may provide a promising strategy for the antitumor therapy of PCa, and thus CIP2A could represent selective targets for the molecularly targeted treatments of PCa.

An Intensive Mind and Body Therapeutic Program Leads to Alteration in Gene Expression Critical to Aging Process in Peripheral Blood Stem Cells

Objective: Waiting to look young is not a new idea; the search for effective treatments prolonging youthfulness has been going on over many decades. Many scientific evidences have been suggestive of intensive or prolonged mind and body therapies (MBT) improving overall wellness and have anti-aging effects. However, the genetic basis of MBT-induced anti-aging and youthfulness are largely unknown. It is also known that aging adversely affects hematopoiesis in human through controlling compromised hematopoietic stem cells (HSC) and peripheral blood mononuclear cells (PBMNC’s). In this paper, we focus on evaluating changes in the expression levels of a critical panel of genes that regulates aging in PBMNC’s isolated from participants from MBT program. Design: Here, we have investigated the effects of a short intensive MBT program on aging related gene expression changes in the peripheral blood stem cells using affymetrix DNA microarray platform. A total of 108 people selected form many ethnicities were enrolled in the study; 38 men and 70 women (aged 18 - 90) randomly assigned for the study. PBMNC’s were collected from the volunteers before and after the completion of the MBT program and evaluated for meditation by examining gene expression patterns in peripheral blood stem cells. Results: Critical pathways known to regulate aging process such as pro-inflammatory TNF alpha/NF-kB, IL-12 signaling pathway, hypoxic HIF-1-alpha, key regulator of programmed cell death, C-MYC, and P38 MAPK (mitogen-activated protein kinase) signaling pathway found to be dysregulated in the cohorts compared to subjects prior to MBT program. Furthermore, GATA-2 and Bmi1, key regulators of hemtopoiesis and adult stem cells numbers, went up in the mediated group. Additionally, key pro-inflammatory mediators IFN? and STAT-2 went down in the mediated group. Conclusion: MBT augments critical genes in PMBC which upregulate hematopoiesis and stem cell numbers and also controls genes that regulate age-related complications.

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

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

CELLFOOD™ induces apoptosis in human mesothelioma and colorectal cancer cells by modulating p53, c-myc and pAkt signaling pathways

BackgroundCELLFOOD™ (CF) is a nutraceutical non-addictive, non-invasive, and completely non-toxic unique proprietary colloidal-ionic formula. Little is known about its effect on cancer cells in solid tumors. The aim of this study was to evaluate the effect that CF has on different cancer cell lines and the mechanism by which the nutraceutical works.MethodsThe effect of CF on HFF (normal fibroblasts), Met5A (mesothelium), MSTO-211H, NCI-2452, Ist-Mes1, MPP89, Ist-Mes2 (mesothelioma), M14 (melanoma), H1650, H1975 (lung cancer), SKRB3 (breast cancer), and HCT-116 (colorectal cancer) cell growth was tested by cell proliferation and clonogenic assay. Among all of them, MSTO-211 and HCT-116 were analyzed for cell cycle by flow cytometry and western blot.ResultsAll human cancer lines were suppressed on cell growth upon 1:200 CF treatment for 24 and 48 hours. Death was not observed in HFF and Met5A cell lines. Cell cycle analysis showed an increased sub-G1 with reduction of G1 in MSTO-211 and a cell cycle arrest of in G1 in HCT116. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for both cell lines. Increased expression levels of p53, p21, and p27, downregulation of c-myc and Bcl-2, and inhibition of Akt activation were also found in CF-treated MSTO-211 and HCT-116 cells.ConclusionsThese findings ascertained an interaction between p53, c-myc, p21, p27, Bcl-2, PI3K/Akt pathway, and CF-induced apoptosis in MSTO-211H and HCT-116 cells, suggesting that CF acts as an important regulator of cell growth in human cancer cell lines. CF could be a useful nutraceutical intervention for prevention in colon cancer and mesothelioma.

Proteomic analysis of perfluorooctane sulfonate‐induced apoptosis in human hepatic cells using the iTRAQ technique

Perfluorooctane sulfonate (PFOS) is one of the most commonly used perfluorinated compounds, whose environmental exposure has been associated with a number of adverse health outcomes. However, the molecular mechanisms involved in PFOS toxicity are still not well elucidated. In the present study, we applied iTRAQ labeling quantitative proteomic technology to investigate the differential protein expression profiles of non-tumor human hepatic cells (L-02) exposed to PFOS. A total of 18 proteins were differentially expressed in a dose-dependent manner in PFOS-treated cells versus the control. Among these, 11 proteins were up-regulated and 7 were down-regulated. Gene ontology analysis indicated that PFOS would exert toxic effects on L-02 cells by affecting multiple biological processes, including protein biosynthesis and degradation, mRNA processing and splicing, transcription, signal transduction and transport. Furthermore, the proteomic results especially proposed that the inhibition of HNRNPC, HUWE1 and UBQLN1, as well as the induction of PAF1 is involved in the activation of the p53 and c-myc signaling pathways, which then trigger the apoptotic process in L-02 cells exposed to PFOS. Overall, these data will aid our understanding of the mechanisms responsible for PFOS-mediated hepatotoxicity, and develop useful biomarkers for monitoring and evaluating PFOS contamination in the environment.

MicroRNA-17/20a/106a modulate macrophage inflammatory responses through targeting signal regulatory protein α. (P1303)

Abstract Myeloid cell-specific signal regulatory protein α (SIRPα) is an essential signaling molecule that modulates leukocyte inflammatory responses. The regulation of SIRPα expression and its dynamic changes in leukocytes, however, remains incompletely understood. Here we study the role of microRNAs in regulating SIRPα expression at posttranscriptional level and macrophage inflammatory responses. We identified SIRPα as a common target gene of miR-17, miR-20a and miR-106a. During SIRPα induction in promyelocytic cells by retinoic acid or phorbol-12-myristate-13-acetate, the levels of these three miRNAs were all reduced and their downreulation occurred through suppression of the c-Myc signaling pathway. All miR-17, miR-20a and miR-106a specifically bound to the same seed sequence within the SIRPα 3’-untranslated region and correlate inversely with SIRPα protein level in various cells. In macrophages, up-regulation of miR-17, miR-20a and miR-106a by lipopolysaccharide (LPS) served as the mechanism underlying LPS-induced SIRPα reduction and macrophage activation. Both in vitro and in vivo assays demonstrate that miR-17, miR-20a and miR-106a regulate macrophage infiltration, phagocytosis and proinflammatory cytokine secretion through targeting SIRPα. In summary, these findings demonstrate for the first time that miR-17, miR-20a and miR-106a regulate SIRPα production and SIRPα-mediated macrophage inflammatory responses in a redundant fashion.