Proliferation Of Human Cancer Research Articles

Development of Bexarotene Analogs for Treating Cutaneous T-Cell Lymphomas.

Bexarotene, a drug approved for treatment of cutaneous T-cell lymphoma (CTCL), is classified as a rexinoid by its ability to act as a retinoid X receptor (RXR) agonist with high specificity. Rexinoids are capable of inducing RXR homodimerization leading to the induction of apoptosis and inhibition of proliferation in human cancers. Numerous studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, many treated patients present with cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor (RAR), thyroid hormone receptor (TR), and liver X receptor (LXR) signaling, respectively. In this study, 10 novel analogs and three standard compounds were evaluated side-by-side with bexarotene for their ability to drive RXR homodimerization and subsequent binding to the RXR response element (RXRE). In addition, these analogs were assessed for proliferation inhibition of CTCL cells, cytotoxicity, and mutagenicity. Furthermore, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Our results suggest that these new compounds may possess similar or enhanced therapeutic potential since they display enhanced RXR activation with equivalent or greater reduction in CTCL cell proliferation, as well as the ability to induce ATF3 and EGR3. This work broadens our understanding of RXR-ligand relationships and permits development of possibly more efficacious pharmaceutical drugs. Modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent compound, potentially leading to improved patient outcomes.

Molecular pathway and structural mechanism of human oncochannel TRPV6 inhibition by the phytocannabinoid tetrahydrocannabivarin

The calcium-selective oncochannel TRPV6 is an important driver of cell proliferation in human cancers. Despite increasing interest of pharmacological research in developing synthetic inhibitors of TRPV6, natural compounds acting at this channel have been largely neglected. On the other hand, pharmacokinetics of natural small-molecule antagonists optimized by nature throughout evolution endows these compounds with a medicinal potential to serve as potent and safe next-generation anti-cancer drugs. Here we report the structure of human TRPV6 in complex with tetrahydrocannabivarin (THCV), a natural cannabinoid inhibitor extracted from Cannabis sativa. We use cryo-electron microscopy combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to identify THCV binding sites in the portals that connect the membrane environment surrounding the protein to the central cavity of the channel pore and to characterize the allosteric mechanism of TRPV6 inhibition. We also propose the molecular pathway taken by THCV to reach its binding site. Our study provides a foundation for the development of new TRPV6-targeting drugs.

Identification of candidate target genes of oral squamous cell carcinoma using high-throughput RNA-Seq data and in silico studies of their interaction with naturally occurring bioactive compounds

Oral Squamous Cell Carcinoma (OSCC) accounts for more than 90% of all kinds of oral neoplasms that develop in the oral cavity. It is a type of malignancy that shows high morbidity and recurrence rate, but data on the disease’s target genes and biomarkers is still insufficient. In this study, in silico studies have been performed to find out the novel target genes and their potential therapeutic inhibitors for the effective and efficient treatment of OSCC. The DESeq2 package of RStudio was used in the current investigation to screen and identify differentially expressed genes for OSCC. As a result of gene expression analysis, the top 10 novel genes were identified using the Cytohubba plugin of Cytoscape, and among them, the ubiquitin-conjugating enzyme (UBE2D1) was found to be upregulated and playing a significant role in the progression of human oral cancers. Following this, naturally occurring compounds were virtually evaluated and simulated against the discovered novel target as prospective drugs utilizing the Maestro, Schrodinger, and Gromacs software. In a simulated screening of naturally occurring potential inhibitors against the novel target UBE2D1, Epigallocatechin 3-gallate, Quercetin, Luteoline, Curcumin, and Baicalein were identified as potent inhibitors. Novel identified gene UBE2D1 has a significant role in the proliferation of human cancers through suppression of ‘guardian of genome’ p53 via ubiquitination dependent pathway. Therefore, the treatment of OSCC may benefit significantly from targeting this gene and its discovered naturally occurring inhibitors. Communicated by Ramaswamy H. Sarma

Structural mechanism of human oncochannel TRPV6 inhibition by the natural phytoestrogen genistein

Calcium-selective oncochannel TRPV6 is the major driver of cell proliferation in human cancers. While significant effort has been invested in the development of synthetic TRPV6 inhibitors, natural channel blockers have been largely neglected. Here we report the structure of human TRPV6 in complex with the plant-derived phytoestrogen genistein, extracted from Styphnolobium japonicum, that was shown to inhibit cell invasion and metastasis in cancer clinical trials. Despite the pharmacological value, the molecular mechanism of TRPV6 inhibition by genistein has remained enigmatic. We use cryo-EM combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to show that genistein binds in the intracellular half of the TRPV6 pore and acts as an ion channel blocker and gating modifier. Genistein binding to the open channel causes pore closure and a two-fold symmetrical conformational rearrangement in the S4–S5 and S6-TRP helix regions. The unprecedented mechanism of TRPV6 inhibition by genistein uncovers new possibilities in structure-based drug design.

Efficient Identification of the MYC Regulator with the Use of the CRISPR Library and Context-Matched Database Screenings.

MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring “Similar” and “Not exactly similar” contexts. INTS14 and ERI2 were identified using datasets featuring the “Similar” context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes.

Development of Novel Bexarotene Analogs for Treating Cutaneous T‐Cell Lymphomas

Agonists are chemical compounds that bind to and activate their cognate receptors allowing for the propagation of a downstream signal. Receptor agonists exhibit varying pharmacodynamic properties, including binding affinity and receptor specificity. Bexarotene, a drug approved by the FDA in 1999 for the treatment of cutaneous T-cell lymphoma (CTCL), is classified as a rexinoid due to its ability to act as a retinoid X receptor (RXR) agonist with high specificity. RXR is known to associate with other receptors in the nuclear receptor superfamily as either permissive or nonpermissive heterodimers that then modulate gene expression of numerous target genes. In a similar fashion, RXR-selective rexinoids, such as bexarotene, also act as RXR agonists and influence RXR homodimerization leading to the induction of apoptosis and inhibition of cell proliferation in human cancers. In fact, several studies have shown that bexarotene is effective in reducing viability and proliferation in CTCL cell lines. However, the side effects of bexarotene therapy are significant and can include cutaneous toxicity, hypothyroidism, and hyperlipidemia due to crossover activity with retinoic acid receptor, thyroid hormone receptor, and liver X receptor signaling, respectively. Thus, the discovery of compounds with greater RXR specificity is an important goal in cancer therapeutics and drug development. In the current study, we have evaluated 14 novel analogs for their potential to bind RXR through modeling and via an RXR-RXR mammalian-2-hybrid system. In addition, RXR response element-mediated luciferase reporter assays were employed to determine the degree of transcriptional activation induced by each analog. Moreover, using a cell proliferation assay, these analogs were tested for their effectiveness to inhibit proliferation in CTCL cells relative to bexarotene. Finally, the most effective analogs were analyzed via qPCR to determine efficacy in modulating expression of two critical tumor suppressor genes, ATF3 and EGR3. Current results suggest that these new compounds possess similar or even enhanced therapeutic potential, as several of our novel rexinoids display more selective RXR activation with equivalent or greater reduction in CTCL cell proliferation, as well as the ability to induce ATF3 and EGR3. This work broadens our understanding of RXR-ligand relationships and permits the development of more highly efficacious pharmaceutical drugs. Our results reveal that modifications of RXR agonists can yield agents with enhanced biological selectivity and potency when compared to the parent bexarotene compound, potentially leading to improved patient outcomes.

LncRNA-uc003opf.1 rs11752942 A>G polymorphism decreases neuroblastoma risk in Chinese children

ABSTRACT Recent studies have revealed that long non-coding RNAs (lncRNAs) play critical roles in the tumorigenesis and proliferation of human cancer. Several polymorphisms of lncRNAs have been found to be involved in the risk of neuroblastoma (NB). However, studies on the relationship between polymorphisms in lncRNA exons and NB are infrequent. We evaluated the association between rs11752942 A > G polymorphism in lnc-RNA-uc003opf.1 exon and neuroblastoma susceptibility by performing a hospital-based study with 275 patients and 531 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) assessed by using logistic regression models were used to determine the strength of the association. We found that the rs11752942 G allele is significantly associated with decreased neuroblastoma risk (AG vs. AA: adjusted OR = 0.72, 95% CI = 0.53–0.98, P = 0.038; and AG/GG vs. AA: adjusted OR = 0.74, 95% CI = 0.55–0.99, P = 0.045) after adjusting for age and gender. This association was more prominent in females, subjects with tumor in the mediastinum or early-stage. Furthermore, the expression quantitative trait locus analysis indicated that rs11752942 G was associated with decreased expression of its neighboring gene LRFN2 mRNA. These results indicate that lncRNA-uc003opf.1 may be a novel potentially functional lncRNA that may be used as a predictive marker, for it might contribute to decreased neuroblastoma risk.

CCEPR is a novel clinical biomarker for prognosis and regulates cell proliferation through PCNA in osteosarcoma.

CCEPR (cervical carcinoma expressed PCNA regulatory lncRNA) has been found to be upregulated and enhance cell proliferation in human cancers. However, the role of CCEPR in osteosarcoma remains to be discovered. In this study, we found CCEPR expression was elevated in osteosarcoma tissue specimens and cell lines compared with adjacent normal tissue specimens and osteoblast cell line, respectively. Meanwhile, osteosarcoma patients with advanced stage or tumor size greater than 8 cm had higher expression of CCEPR than patients with early stage or tumor size less than or equal to 8 cm, respectively. Survival analysis suggested that osteosarcoma patients with high CCEPR expression had a worse overall survival rate than those with low CCEPR expression. The in vitro study indicated that CCEPR positively regulated proliferating cell nuclear antigen (PCNA) expression in osteosarcoma cells and silencing of CCEPR inhibited osteosarcoma cell proliferation through decreasing PCNA expression. In conclusion, CCEPR is a potential prognostic predictor and functions as oncogenic long non-coding RNA (lncRNA) to regulate cell proliferation via PCNA in osteosarcoma.

<em>HOXA11-AS</em>: a novel regulator in human cancer proliferation and metastasis

Multiple studies have demonstrated that lncRNAs extensively participate in human cancer proliferation and metastasis. Epigenetic modification, transcriptional and posttranscriptional regulatory mechanisms are involved in lncRNA-led tumorigenesis and transfer. Recently, a novel identified homeobox (HOX) A11 antisense lncRNA, HOXA11-AS, 1,628 bp in length, has been excessively highlighted to be an essential initiator and facilitator in the process of malignant tumor proliferation and metastasis. As found in many reports, HOXA11-AS can not only act as a molecular scaffold of PRC2, LSD1 and DNMT1 to epigenetically modify chromosomes in the nucleus but also occur as ceRNA competitively sponging miRNAs in the cytoplasm. Furthermore, HOXA11-AS may function as a potential biomarker for cancer diagnosis and prognosis. In this review, we summarize the evolvement and mechanisms of HOXA11-AS in proliferation and metastasis of various human cancers.

Current clinical status of 18F-FLT PET or PET/CT in digestive and abdominal organ oncology.

Positron emission tomography (PET) or PET/computed tomography (CT) using 18F-3'-fluoro-3'-deoxythymidine (18F-FLT) offers noninvasive assessment of cell proliferation in human cancers in vivo. The present review discusses the current status on clinical applications of 18F-FLT-PET (or PET/CT) in digestive and abdominal oncology by comparing with 18F-fluorodeoxyglucose (18F-FDG)-PET (or PET/CT). The results of this review show that although 18F-FLT uptake is lower in most cases of digestive and abdominal malignancies compared with 18F-FDG uptake, 18F-FLT-PET can be used to detect primary tumors. 18F-FLT-PET has shown greater specificity for N staging than 18F-FDG-PET which can show false-positive uptake in areas of inflammation. However, because of the high background uptake in the liver and bone marrow, it has a limited role of assessing liver and bone metastases. Instead, 18F-FLT-PET will be a powerful tool for monitoring response to treatment and provide prognostic information in digestive and abdominal oncology.

Give it or take it: the flux of one-carbon in cancer cells.

The sequence of the human genome together with sequence similarity analyses has advanced the discovery of missing steps in the mitochondrial one-carbon metabolism pathway. That together with the revived interest in cancer metabolism has brought the research on one-carbon metabolism back under the spotlight. Here, we present a brief review of recent advances in the field of one-carbon metabolism, with a bias towards its relevance to cell growth and proliferation in human cancers. We will address the requirements of one-carbon metabolism for biosynthesis and the major sources to satisfy that demand. We will also discuss some recent discoveries indicating a role of one-carbon metabolism beyond biosynthesis. We conclude with a concise enumeration of some fundamental questions that remain unanswered.

Abstract A44: Inhibition of Foxm1 transcription factor diminished Kras-mutated tumor development

Abstract Increased expression level of the Forkhead Box M1 (FOXM1) transcription factor is found in multiple types of cancers, and its expression is correlated with poor prognosis in patients with cancers, including non-small cell lung cancers and pancreatic cancers. We and others have shown that FoxM1 transcriptionally regulates a variety of genes essential for cell cycle progression, inflammation, angiogenesis, tumor cell invasiveness and survival. Published studies demonstrated that genetic deletion of Foxm1 gene from SPC-rtTA/tetO-KrasG12D/Foxm1 fl/fl mouse lung epithelial cells conferred resistance to tumorigenesis caused by oncogenic KrasG12D, indicating Foxm1 is a critical regulator of Kras signaling pathway during lung cancer initiation. When treated SPC-rtTA/tetO-KrasG12D/tetO-FOXM1 mice with Doxycycline, lung epithelial cell-specific expressed transgenic FOXM1 and Kras cooperate to promote tumor growth in mouse lungs. In the present study, we determine whether expression of FoxM1 affects proliferation of human cancer cells harboring oncogenic Kras. Here, we show that depletion of FOXM1 expression using inducible shFOXM1 diminished proliferation of Kras-mutated human non-small cell adenocarcinoma (A549 and NCI-H23) and pancreatic cancer (PANC-1 and AsPC-1) cell lines. In contrast, increased expression of FOXM1 caused increased proliferation of Kras mutated cancer cells. Scratch wound healing assay demonstrated that FOXM1 stimulates the migration of these cells, which is associated with elevated epithelial-mesenchymal transition (EMT) markers. Our studies demonstrate that FOXM1 transcription factor is required for Kras mutated human cancer cells proliferation during tumor progression. Citation Format: I-Ching Wang, Hsin Chih Chien, Sheng-Yang Chao, Chien-Cheng Li. Inhibition of Foxm1 transcription factor diminished Kras-mutated tumor development. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A44.

Abstract 1133: Synthetic lethal screen identifies candidates that modulate cancer cell sensitivity in response to HSP90 inhibition.

Abstract Heat shock protein 90 (HSP90) is an important cancer therapeutic target. HSP90 chaperone activity maintains the conformation, stability and function of mutated variants and over-expressed oncogenic client proteins responsible for driving the proliferation of human cancer. To further increase the efficacy and specificity of HSP90 inhibitor treatment, cancer cell response to HSP90 inhibition can be modulated through the use of combinatorial therapeutics. An siRNA screen targeting 7,593 genes encoding proteins considered to be druggable was used to identify candidates that modulate the cellular response to HSP90 inhibition by 17-AAG. This approach may provide information on the potential mechanisms of cancer cell resistance, allow selection of responsive patient populations based on the identification of novel biomarkers, and potentially provide targets for combinatorial therapy. Screening produced robust data with an average plate z-factor of 0.6 and results were validated with an alternative chemotype of HSP90 inhibitor. As anticipated, significant sensitisation to HSP90 inhibition was observed with HSF1 depletion, and conversely resistance was observed with NQO1 depletion. Sensitisation candidates were the main focus of analysis as they potentially represent the more amenable drug targets. There was an enrichment of mitochondrial constituents, cell cycle regulators and post-translational protein modifiers that when depleted produced significant sensitisation to HSP90 inhibitors in human carcinoma cells. Future work aims to reveal the molecular mechanisms underpinning the sensitisation phenotypes observed and to identify points of intervention for clinical combinatorial therapy with HSP90 inhibitors. Citation Format: Jennifer Howes, Bing-Feng Lu, Marissa Powers, Paul Clarke, Paul Workman. Synthetic lethal screen identifies candidates that modulate cancer cell sensitivity in response to HSP90 inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1133. doi:10.1158/1538-7445.AM2013-1133

Calyculin A from Discodermia Calyx Is a Dual Action Toxin that Blocks Calcium Influx and Inhibits Protein Ser/Thr Phosphatases

Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor.

Conjugated linoleic acid 황갈색의 인체암세포와 인체정상세포에 대한 세포독성

체지방감소 효과가 있는 시판 conjugated linoleic acid (CLA)의 색깔을 제거하고 이 색깔의 세포독성에 관한 연구를 하였다. 황갈색의 시판 CLA 제품을 구입하여 crude CLA (c-CLA) 시료로 하였다. c-CLA 시료를 감압증류(10 mmHg-<TEX>$220^{\circ}C$</TEX>, 10 mmHg-<TEX>$235^{\circ}C$</TEX>, 10 mmHg-<TEX>$240^{\circ}C$</TEX>, 20 mmHg-<TEX>$260^{\circ}C$</TEX>; 30분)하여 증류된 CLA (distilled CLA; d-CLA) 시료와 증류되지 않고 남아있는 황갈색 CLA (residual CLA; r-CLA) 시료로 분리하였다. 10 mmHg-<TEX>$220^{\circ}C$</TEX>에서 증류하여 얻은 d-CLA 시료의 색깔은 L (brightness), a (red/blue), b (yellow/green)로 분석한 결과 무색에 가까웠고 r-CLA 시료는 황갈색이었고, 이들 두 CLA 시료의 CLA 이성체 조성은 변하지 않았다. 따라서 10 mmHg-<TEX>$220^{\circ}C$</TEX>에서 얻은 r-CLA 시료의 인체암세포(유방암 MCF-7. 폐암 A-549, 직장암 HT-29, 전립선암 PC-3)와 인체 정상세포(신경모세포 SK-N-SH)에 대한 세포독성을 d-CLA 시료와 비교하였다. 이들 암세포와 정상세포에 r-CLA 시료와 d-CLA 시료 처리 2일 후의 세포독성에는 차이가 없었다. 따라서 본 연구에서 c-CLA 시료에 함유된 색소는 10 mmHg-<TEX>$220^{\circ}C$</TEX>로 감압증류 하여 제거할 수 있었고, r-CLA 시료의 세포독성은 d-CLA 시료의 세포독성과 차이가 없었다. 이와 같은 결과는 c-CLA 시료에 함유된 색소는 세포생육에 아무런 영향을 미치지 않고 인체에 아무런 영향을 미치지 않음을 의미한다. The cytotoxicity of the colorant in conjugated linoleic acid (CLA) was investigated in human cancer cell lines and a normal human cell line. Commercially-available CLA with a brown color (designate crude CLA; c-CLA) was distilled in a vacuum (10 mmHg-<TEX>$220^{\circ}C$</TEX>, 10 mmHg-<TEX>$235^{\circ}C$</TEX>, 10 mmHg-<TEX>$240^{\circ}C$</TEX>, and 20 mmHg-<TEX>$260^{\circ}C$</TEX>) for 30 min to obtain pure CLA (distilled CLA; d-CLA) and dark brown-colored CLA (residual CLA; r-CLA) samples. No color intensity was shown in the d-CLA sample obtained under 10 mmHg-<TEX>$220^{\circ}C$</TEX> conditions of distillation when the L (brightness), a (red/blue), and b (yellow/green) parameters were analyzed, whereas the r-CLA sample showed a dark brown color. The composition of CLA isomers in both the d- and r-CLA samples, as compared to that of the c-CLA sample, was not significantly different when analyzed by gas chromatography. When the cytotoxicity of the r-CLA and d-CLA samples obtained under 10 mmHg-<TEX>$220^{\circ}C$</TEX> conditions were compared against human breast cancer cells (MCF-7), human lung cancer cells (A-549), human colon cancer cells (HT-29), human prostate cancer cells (PC-3), and human neuroblastoma cells (SK-N-SH), no significant cytotoxicity was seen in the cell lines. These results suggest that the color or colorant in the CLA samples did not have any effects on the proliferation of human cancer and normal cells and imply that the colorant in commercially available CLA samples is safe for human consumption.

Abstract 2060: NADPH oxidase NOX5 regulates the expression of the cell cycle inhibitor p27kip1 in the human melanoma UACC-257 cancer cells

Abstract NADPH oxidase (NOX) family enzymes (NOX1-5 and DUOX1-2) generate reactive oxygen species (ROS) and modulate cellular pathways that regulate proliferation. While high doses of ROS are clearly toxic, the effects of low doses of NOX-mediated ROS as signaling molecules are less clear. Recent work has proposed that NOX5 plays a role in cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma and in prostate cancer cells, yet relatively little is known of the signaling pathways mediated by NOX5. In this study we explored the functional role of NOX5 in cancer cells obtained from the NCI-60 cell line panel. Initial screening of the NCI-60 cancer cell line panel for expression of the Nox family members by real time PCR revealed several melanoma cell lines that express Nox5. Of the NOX5 expressing NCI-60 human cancer cell lines, subsequent studies were carried out in human UACC-257 melanoma cancer cells. Two molecular approaches were undertaken to determine the functional role of NOX5 in UACC-257 cells: 1) generating NOX5 over-expressing stable clones and 2) transiently silencing endogenous NOX5 and the stably over-expressed NOX5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that NOX5 is functional and generates superoxide in both UACC-257 cells and stable Nox5-over-expressing clones. NOX5 can be activated both by the protein kinase C activating phorbol ester PMA and by the Ca2+ ionophore ionomycin. Furthermore, this enzymatic activity is calcium- and flavin dehydrogenase-dependent; both the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased NOX5-mediated ROS production. Silencing NOX5 over-expression significantly inhibited both endogenous and PMA-stimulated ROS production, suggesting that the ROS production is NOX5-mediated. Furthermore, knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in increased p27Kip1 expression. Conversely, over-expression of NOX5 in UACC-257 cells resulted in decreased p27Kip1 expression both at the mRNA and protein levels. As the AKT signaling pathways are known to modulate p27Kip1 levels, we observed that knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in decreased Akt and GSK-3β phosphorylation. Studies to characterize additional mechanisms of translational and transcriptional regulation of p27Kip1 by NOX5 are ongoing. In summary, our findings suggest that NOX5 expression could contribute to cell proliferation in human cancers in part due to the generation of high local concentrations of extracellular ROS that regulate the Akt/GSK-3β/p27Kip1 signaling network. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2060. doi:1538-7445.AM2012-2060

Abstract 4205: Regulation of Hif-1α expression by NADPH oxidase 5 (Nox5) in human cancer cell lines

Abstract Reactive oxygen species (ROS) produced by NADPH oxidases (Noxs) participate in signaling cascades regulating proliferation. However, the relative contribution to tumor cell growth by the seven Nox homologues (Nox1-5 and Duox 1 and 2) remains unclear. Several studies have proposed that Nox5 plays a role in promoting cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma cells, and in prostate cancer; however, little is known about the signaling pathways mediated by Nox5. We explored the functional role of Nox5 in a variety of tumor cell lines, including the NCI-60 cell panel; real time RT-PCR revealed that several melanoma lines express Nox4 and Nox5, but no Nox1 mRNA; and that prostate cell lines express Nox5 mRNA. Of the Nox5- expressing NCI-60 human cancer cell lines, we studied UACC-257 melanoma and PC-3 prostate cancer cells. These lines were characterized for variations in their N-terminus Ca2+ binding domains; RT-PCR revealed that both cell lines expressed the Nox5α and Nox5γ forms. The functional role of Nox5 in UACC-257 and PC-3 cells was studied by generating stable Nox5-overexpressing clones, and by transiently silencing Nox5 that was expressed endogenously, as well as that in clones stably overexpressing Nox5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that Nox5 was functional and generated superoxide in UACC-257 and PC-3 clones overexpressing Nox5. Nox5 could be activated by the phorbol ester PMA and by the Ca2+ ionophore ionomycin in overexpressing clones. This enzymatic activity was Ca2+- and flavin dehydrogenase-dependent; the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased Nox5-mediated ROS production. Overexpression of Nox5 in UACC-257 cells resulted in increased normoxic Hif-1α expression that was also Ca2+-, flavin dehydrogenase- and ROS-dependent. Silencing Nox5 by RNAi significantly inhibited both endogenous and PMA-stimulated ROS production, confirming that ROS production was Nox5-mediated. Knockdown of both endogenous and overexpressed Nox5 in both UACC-257 and PC-3 cells resulted in decreased Hif-1α expression. Studies to characterize the mechanisms by which Nox5 modulates cell proliferation through the HIF-1α pathway are ongoing. Our experiments suggest that Nox5 expression may play an important role in the generation of high local concentrations of ROS that could contribute to activation of Hif-1α and promote cell proliferation in human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2011-4205

A New Pathogenomic Mechanism In FPD/AML by a Constitutional T(16;21) Involving 16p13 ATF7IP2 and 21q22 RUNX1 Loci

AbstractAbstract 4851Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant disorder characterized by platelet abnormalities and a predisposition to acute myeloid leukemia (AML). FPD/AML is caused by inherited mutations in 21q22 RUNX1. Recent publications by us and others reported on RUNX1 copy number changes as a pathogenomic mechanism next to point mutations in diagnosing FPD/AML. Given the clinical heterogeneity of FPD/AML, the syndrome could be overlooked, and has resulted in the past in stem cell transplantation (SCT) of patients from affected siblings before detection of the inherited RUNX1 mutation.Here we report on the identification of two novel missense mutations (G143R and Q235X), and a constitutional RUNX1 gene rearrangement as a result of a t(16;21)(p13;q22) as a novel pathogenomic mechanism in FPD/AML. The 16p13 breakpoint was confined to the ATF7IP2 (MCAF1) locus, a transcription factor associated with Sp1, which is involved in myeloid differentiation, and in proliferation in human cancer by epigenetic transcriptional control of Sp1-dependent maintenance of telomerase activity. This is the first report on genomic involvement of the ATF7IP2 locus in cancer. We will discuss ongoing studies to molecularly identify the breakpoints.Our data indicate that in diagnosing FPD/AML FISH has to be performed for structural abnormalities along with sequencing and copy number analysis of RUNX1. Furthermore, this is the first report on a constitutional chromosome translocation with an oncogene rearrangement as a pathogenomic mechanism in a hematological malignancy. Disclosures:No relevant conflicts of interest to declare.

Abstract 3119: Phosphorylation of epidermal growth factor receptor at Ser1046/1047 by p38 mitogen-activated protein kinase plays a critical role in its downregulation in cancer cells

Abstract The mechanism underlying downregulation of EGF receptor (EGFR) is important for the regulation of cancer cells. Although the phosphorylation of EGFR at Tyr1045 and Ser1046/1047 reportedly account for its downregulation, the exact mechanism remains unclear. We recently reported that p38 mitogen-activated protein kinase (MAPK), a stress-inducible kinase believed to negatively regulate tumorigenesis, controls EGFR downregulation via its phosphorylation at Ser1046/1047 (Cancer Lett 2009;277:108-13). To further investigate the role of phosphorylation of p38 MAPK in the action of anti-cancer drug, we examined the effects of (−)-epigallocatechin gallate (EGCG) and several HSP90 inhibitors (17-AAG, geldanamycin and 17-DMAG) on p38 MAPK in colon and pancreatic cancer cells. While EGCG and HSP90 inhibitors caused downregulation of EGFR and subsequent cell death, they induced phosphorylation of p38 MAPK, and inhibition of p38 MAPK suppressed the internalization and subsequent degradation of EGFR. Moreover, we found that they caused phosphorylation of EGFR at Ser1046/1047, not Tyr1045, and that they were also suppressed by the inhibition of p38 MAPK. Taken together, our results strongly suggest that phosphorylation of EGFR at Ser 1046/1047 via activation of p38 MAPK plays a pivotal role in anti-cancer drug-induced downregulation of EGFR. These findings may indicate new therapeutic strategies for inhibiting the proliferation of human cancers that are highly dependent on the activation of EGFR. (Adachi S et al., Carcinogenesis 2009;30:1544-52) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3119.

MiR-449a and miR-449b are direct transcriptional targets of E2F1 and negatively regulate pRb–E2F1 activity through a feedback loop by targeting CDK6 and CDC25A

The Rb-E2F pathway drives cell cycle progression and cell proliferation, and the molecular strategies safeguarding its activity are not fully understood. Here we report that E2F1 directly transactivates miR-449a/b. miR-449a/b targets and inhibits oncogenic CDK6 and CDC25A, resulting in pRb dephosphorylation and cell cycle arrest at G1 phase, revealing a negative feedback regulation of the pRb-E2F1 pathway. Moreover, miR-449a/b expression in cancer cells is epigenetically repressed through histone H3 Lys27 trimethylation, and epigenetic drug treatment targeting histone methylation results in strong induction of miR-449a/b. Our study reveals a tumor suppressor function of miR-449a/b through regulating Rb/E2F1 activity, and suggests that escape from this regulation through an aberrant epigenetic event contributes to E2F1 deregulation and unrestricted proliferation in human cancer.