PURPOSE: Glioblastoma (GB) is the most frequent and most malignant primary brain tumor in adults. Previously, it has been found that both genetic and epigenetic factors may play critical roles in its etiology and prognosis. In addition, it has been found that the epidermal growth factor receptor gene (EGFR) is frequently over-expressed and amplified in primary GBs. Here, we assessed the promoter methylation status of 10 genes relevant to GB and explored associations between these findings and the EGFR gene amplification status.
METHODS: Tumor samples were obtained from 36 patients with primary GBs. In addition, 6 control specimens were included from patients who were operated for diseases other than brain tumors. The amplification status of the EGFR gene, and its deletion mutant EGFRvIII, were evaluated using FISH and MLPA, respectively. The IDH1/2 gene mutation status was verified using Sanger sequencing. A commercial DNA methylation kit was used to assess the promoter methylation status of 10 pre-selected genes. Metabolic profiles were measured using HR-MAS NMR spectroscopy. The EGFR and ARF1 mRNA expression levels were quantified using qRT-PCR.
RESULTS: Of the 10 genes analyzed, we found that only ARF1 promoter hypermethylation was significantly associated with EGFR gene amplification. ARF1 is a GTPase that is involved in vesicle trafficking and the Golgi apparatus. Subsequent tumor metabolism measurements revealed a positive association between EGFR amplification and different membrane precursors and methyl-donor metabolites. Finally, we found that EGFR gene amplifications were associated with distinct tumor infiltration patterns, thus representing a putative novel functional association between EGFR gene amplification and ARF1 gene promoter methylation in GB.
CONCLUSIONS: The results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism(s) underlying these connections and their functional consequences remain to be established.

Cell adhesion-mediated drug resistance (CAM-DR) remains the primary obstacle in human multiple myeloma (MM) therapy. In this study, we aimed at investigating the expression and biologic function of ARF1 in MM. We determined that ARF1 expression was positively correlated with cell proliferation and knockdown of ARF1 contributed to CAM-DR. The enhancement in the adhesion of MM cells to fibronectin (FN) or the bone marrow stroma cell line HS-5 cells translated to an increased CAM-DR phenotype. Importantly, we showed that this CAM-DR phenotype was correlated with the phosphorylation of Akt and ERK in MM cells. Moreover, we sought to determine whether ARF1 could interact with p27 in RPMI8226 cells. Knockdown of ARF1 also significantly decreased pT157-p27 protein expression in RPMI8226 cells. Our research shows ARF1 may reverse CAM-DR by regulating phosphorylation of p27 at T157 in MM. Taken together, our data shed new light on the molecular mechanism of CAM-DR in MM, and targeting ARF1 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in MM.

ADP-ribosylation factor 1 (ARF1) is a small G protein that regulates many cellular processes such as reorganization of the actin cytoskeleton and is highly expressed in various tumor cells and tissues. However, the role of ARF1 in ovarian cancer progression remains unknown. In the present study, we explored the expression patterns of ARF1 in clinical ovarian cancer samples and adjacent noncancerous tissues. The results revealed that ARF1 overexpressed in EOC tissues and cell lines, compared with the adjacent non-tumorous tissues and normal ovarian cells. In addition, the immunoreactivity of ARF1 was positively correlated with EOC grade and Ki-67 expression. Knockdown of ARF1 expression notably inhibited cell proliferation and migration rate of EOC cells by the auxiliary of PI3K. Taken together, our findings provide new insights into the functional role of ARF1 on EOC cell growth and migration and it may serve as a diagnostic and therapeutic target.

ADP-ribosylation factor 1 (ARF1) is a crucial regulator in vesicle-mediated membrane trafficking and involved in the activation of signaling molecules. However, virtually nothing is known about its function in prostate cancer. Here we have demonstrated that ARF1 expression is significantly elevated in prostate cancer cells and human tissues and that the expression levels of ARF1 correlate with the activation of mitogen-activated protein kinases (MAPK) ERK1/2. Furthermore, we have shown that overexpression and knockdown of ARF1 produce opposing effects on prostate cancer cell proliferation, anchorage-independent growth and tumor growth in mouse xenograft models and that ARF1-mediated cell proliferation can be abolished by the Raf1 inhibitor GW5074 and the MEK inhibitors U0126 and PD98059. Moreover, inhibition of ARF1 activation achieved by mutating Thr48 abolishes ARF1's abilities to activate the ERK1/2 and to promote cell proliferation. These data demonstrate that the aberrant MAPK signaling in prostate cancer is, at least in part, under the control of ARF1 and that, similar to Ras, ARF1 is a critical regulator in prostate cancer progression. These data also suggest that ARF1 may represent a key molecular target for prostate cancer therapeutics and diagnosis.

Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.

To obtain further insights into the biological differences of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) and classical Hodgkin lymphoma (HL), we screened microbial culture filtrates to search for compounds that would exert a significantly greater effect on the viability of ALK+ ALCL cell lines compared to HL cell lines and identified Brefeldin A (BFA) as a suitable candidate. BFA inhibited phosphorylation of ALK and its downstream molecule, signal transducer and activator of transcription 3 (STAT3), one of the central pathways for the survival of ALK+ ALCL cells. In HL cell lines BFA did not affect CD30 expression or constitutive nuclear factor (NF)-κB activity, both of which are critical for HL cell survival. BFA induced disruption of the Golgi apparatus in ALK+ ALCL cell lines, which was accompanied by a decrease in active ADP-ribosylation factor 1 (ARF1), whereas BFA had no significant effect on these parameters in HL cell lines. These results add extra insights into the biological distinction between ALK+ ALCL and HL cells and highlight the Golgi apparatus as a target for the treatment of ALK+ ALCL.

Adhesion complex formation and disassembly is crucial for maintaining efficient cell movement. During migration, several proteins act in concert to promote remodeling of the actin cytoskeleton and we have previously shown that in highly invasive breast cancer cells, this process is regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. These are overexpressed and highly activated in these cells. Here, we report that one mechanism by which ARF1 regulates migration is by controlling assembly of focal adhesions. In cells depleted of ARF1, paxillin is no longer colocalized with actin at focal adhesion sites. In addition, we demonstrate that this occurs through the ability of ARF1 to regulate the recruitment of key proteins such as paxillin, talin and FAK to ß1-integrin. Furthermore, we show that the interactions between paxillin and talin together and with FAK are significantly impaired in ARF1 knocked down cells. Our findings also indicate that ARF1 is essential for EGF-mediated phosphorylation of FAK and Src. Finally, we report that ARF1 can be found in complex with key focal adhesion proteins such as ß1-integrin, paxillin, talin and FAK. Together our findings uncover a new mechanism by which ARF1 regulates cell migration and provide this GTPase as a target for the development of new therapeutics in triple negative breast cancer.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with reduced adipose tissue lipolysis that can be rescued by aerobic exercise. We aimed to identify differences in the gene expression of perilipins and associated targets in adipose tissue in women with PCOS before and after exercise.
DESIGN AND METHODS: We conducted a cross-sectional study in eight women with PCOS and eight women matched for BMI and age with normal cycles. Women with PCOS also completed a 16-week prospective aerobic exercise-training study. Abdominal subcutaneous adipose tissue biopsies were collected, and primary adipose-derived stromal/stem cell cultures were established from women with PCOS before 16 weeks of aerobic exercise training (n=5) and controls (n=5). Gene expression was measured using real-time PCR, in vitro lipolysis was measured using radiolabeled oleate, and perilipin 3 (PLIN3) protein content was measured by western blotting analysis.
RESULTS: The expression of PLIN1, PLIN3, and PLIN5, along with coatomers ARF1, ARFRP1, and βCOP was ∼ 80% lower in women with PCOS (all P<0.05). Following exercise training, PLIN3 was the only perilipin to increase significantly (P<0.05), along with coatomers ARF1, ARFRP1, βCOP, and SEC23A (all P<0.05). Furthermore, PLIN3 protein expression was undetectable in the cell cultures from women with PCOS vs controls. Following exercise training, in vitro adipose oleate oxidation, glycerol secretion, and PLIN3 protein expression were increased, along with reductions in triglyceride content and absence of large lipid droplet morphology.
CONCLUSIONS: These findings suggest that PLIN3 and coatomer GTPases are important regulators of lipolysis and triglyceride storage in the adipose tissue of women with PCOS.

OBJECTIVE: To evaluate 51 different housekeeping genes for their use as internal standards in myometrial and matched leiomyoma samples in proliferative and secretory phases.
METHODS: RNA from 6 myometrium and matched leiomyoma samples was obtained from pre-menopausal women who underwent hysterectomy. Reverse-transcription and real-time quantitative PCR were achieved using TaqMan high-density open-array human endogenous control panel.
RESULTS: Expression stability of 51 candidate genes was determined by GeNorm and NormFinder softwares. We identified 10 housekeeping genes, ARF1, MRPL19, FBXW2, PUM1, UBE2D2, EIF2B1, HPRT1, GUSB, ALAS1, and TRIM27, as the best set of normalization genes for comparing relative expression between leiomyoma and myometrium samples in proliferative and secretory phases.
CONCLUSIONS: Adequate reference genes for accurate normalization are essential to compare gene expression between leiomyoma and myometrial samples. Ideal housekeeping genes must have stable expression patterns regardless of the sample type and menstrual cycle phase. In this study, we propose a set of 10 candidate genes with greater expression stability than those housekeeping genes commonly used in leiomyoma and myometrium tissues. Their use will improve the sensitivity and specificity of the gene expression analysis in these tissues.

ADP-ribosylation factors (ARFs) are monomeric G proteins that regulate many cellular processes such as reorganization of the actin cytoskeleton. We have previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced migration. In this study, we propose to define the molecular mechanism by which ARF1 regulates this complex cellular response by investigating the role of this ARF GTPase on the activation process of Rac1, a Rho GTPase, associated with lamellipodia formation during cell migration. Here, we first show that inhibition of ARF1 or Rac1 expression markedly impacts the ability of MDA-MB-231 cells to migrate upon EGF stimulation. However, the effect of ARF1 depletion can be reversed by overexpression of the Rac1 active mutant, Rac1 Q(61)L. Depletion of ARF1 also impairs the ability of EGF stimulation to promote GTP-loading of Rac1. To further investigate the possible cross-talk between ARF1 and Rac1, we next examined whether they could form a complex. We observed that the two GTPases could directly interact independently of the nature of the nucleotide bound to them. EGF treatment however resulted in the association of Rac1 with its effector IRSp53, which was completely abrogated in ARF1 depleted cells. We present evidences that this ARF isoform is responsible for the plasma membrane targeting of both Rac1 and IRSp53, a step essential for lamellipodia formation. In conclusion, this study provides a new mechanism by which ARF1 regulates cell migration and identifies this GTPase as a promising pharmacological target to reduce metastasis formation in breast cancer patients.

The ADP-ribosylation factors (ARFs) 1 and 6 are small GTP-binding proteins, highly expressed and activated in several breast cancer cell lines and are associated with enhanced migration and invasiveness. In this study, we report that ARF1 has a critical role in cell proliferation. Depletion of this GTPase or expression of a dominant negative form, which both resulted in diminished ARF1 activity, led to sustained cell-growth arrest. This cellular response was associated with the induction of senescent markers in highly invasive breast cancer cells as well as in control mammary epithelial cells by a mechanism regulating retinoblastoma protein (pRB) function. When examining the role of ARF1, we found that this GTPase was highly activated in normal proliferative conditions, and that a limited amount could be found in the nucleus, associated with the chromatin of MDA-MB-231 cells. However, when cells were arrested in the G(0)/G(1) phase or transfected with a dominant negative form of ARF1, the total level of activated ARF1 was markedly reduced and the GTPase significantly enriched in the chromatin. Using biochemical approaches, we demonstrated that the GDP-bound form of ARF1 directly interacted with pRB, but not other members of this family of proteins. In addition, depletion of ARF1 or expression of ARF1T(31)N resulted in the constitutive association of pRB and E2F1, thereby stabilizing the interaction of E2F1 as well as pRB at endogenous sites of target gene promoters, preventing expression of E2F target genes, such as cyclin D1, Mcm6 and E2F1, important for cell-cycle progression. These novel findings provide direct physiological and molecular evidence for the role of ARF1 in controlling cell proliferation, dependent on its ability to regulate pRB/E2F1 activity and gene expression for enhanced proliferation and breast cancer progression.

Interaction of the Eph family of receptor protein tyrosine kinases and their ligands, ephrin family members, induces bi-directional signaling via cell-cell contacts. High expression of B-type ephrin is associated with high invasion potential of tumors, however, the mechanism by which ephrin-B promotes cancer cell invasion is poorly understood. We show that interaction of ephrin-B1 with the Eph receptor B2 (EphB2) significantly enhances processing of the extracellular domain of ephrin-B1, which is regulated by the C-terminus. Matrix metalloproteinase-8 (MMP-8) is the key protease that cleaves ephrin-B1, and the C-terminus of ephrin-B1 regulates activation of the extracellular release of MMP-8 without requirement of de novo protein synthesis. One possible mechanism by which ephrin-B1 regulates the exocytosis of MMP-8 is the activation of Arf1 GTPase, a critical regulator of membrane trafficking. In support of this hypothesis, activation of ephrin-B1 increased GTP-bound Arf1, and the secretion of MMP-8 was reduced by expression of a dominant-negative mutant of Arf1. Expression of ephrin-B1 promoted the invasion of cancer cells in vivo, which required the C-terminus of ephrin-B1. Our results suggest a novel function of the C-terminus of ephrin-B1 in activating MMP-8 secretion, which promotes the invasion of cancer cells.

Fibrolamellar carcinomas (FLC) are a rare type of primary hepatocellular carcinoma found in younger individuals. FLC are known to have relatively few consistent chromosomal alterations, although a gain of chromosome 1q has been reported. The gene expression of 4 FLC (2 primary FLC and 2 metastatic deposits) were studied using Affymetrix DNA microarray technology (Santa Clara, CA). Selected genes were confirmed by real-time polymerase chain reaction. Relatively few genes were significantly overexpressed-447 genes, case 1; 1298 genes, case 2-corresponding to approximately 0.8% and 2.3%, respectively, of the 56000 transcripts present in the arrays. Of these, 155 genes were overexpressed simultaneously by both tumors. The number of significantly overexpressed genes more than doubled in the 2 metastatic deposits (2777 and 2855 genes compared with 1298 in the primary tumor). Proteins involved in the RAS, MAPK, PIK3, and xenobiotic degradation pathways were commonly overexpressed. Because chromosome 1q is thought to contain an important oncogene, additional attention was focused on this region. Of 114 total genes found overexpressed in common among all primary and metastatic tumors, 11 of 114 genes were located on chromosome 1q: ARF1, CD46, CNIH4, ENSA, FH, NICE-3, PSMB4, RGS2, RGS5, TIMM17A, and UFC1. Primary FLC show overexpression of genes involved in the RAS, MAPK, PIK3, and xenobiotic degradation pathways. Eleven common genes were consistently overexpressed on chromosome 1q among all tumors and metastases and warrant further study as potential oncogenes.

The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.

We have investigated gene expression profiles of human ovarian carcinomas in vivo during Taxol(R) (paclitaxel) treatment and observed a difference in expression. Nude mice bearing 1A9 or 1A9PTX22 xenografts were given 60 mg/kg of paclitaxel. Therapeutic efficacy was achieved for 1A9, while 1A9PTX22 did not respond. Tumor tissues harvested 4 and 24 h after treatment were evaluated by cDNA microarray against untreated tumors. Paclitaxel caused the modulation of more genes in 1A9 than in 1A9PTX22 tumors, in accordance to their therapeutic response. Most gene expression alterations were detected 24 h after paclitaxel administration and affected genes involved in various biological functions including cell cycle regulation and cell proliferation (CDC2, CDKN1A, PLAB, and TOP2A), apoptosis (BNIP3 and PIG8), signal transduction and transcriptional regulation (ARF1, ATF2, FOS, GNA11, HDAC3, MADH2, SLUG, and SPRY4), fatty acid biosynthesis and sterol metabolism (FDPS, IDI1, LIPA, and SC5D), and IFN-mediated signaling (G1P3, IFI16, IFI27, IFITM1, and ISG15). The modulation of two representative genes, CDKN1A and TOP2A, was validated by Northern analyses on a panel of seven ovarian carcinoma xenograft models undergoing treatment with paclitaxel. We found that the changes in expression level of these genes was strictly associated with the responsiveness to paclitaxel. Our study shows the feasibility of obtaining gene expression profiles of xenografted tumor models as a result of drug exposure. This in turn might provide insights related to the drugs' action in vivo that will anticipate the response to treatment manifested by tumors and could be the basis for novel approaches to molecular pharmacodynamics.

Directional tag PCR subtractive hybridization was applied to construct a cDNA library generated from three different human osteosarcoma (OS) target cell lines (OHS, SaOS-2 and KPDXM) from which normal osteoblast (NO) sequences were subtracted. After two consecutive subtractive steps more than 98% of the common mRNAs species were depleted, leading to effective enrichment of the remaining target sequences. After differential screening of 960 clones, 81 candidates were further studied by Northern blot analysis and 73 represented separate mRNA species. Fifty-three of these showed enriched mRNA levels, of which 36 represented known and 17 not previously published cDNAs or EST sequences. The mRNAs showed a 1.4- to 504-fold enrichment compared to the mRNA levels in NO cells. The known mRNAs are: Ribosomal protein S11, KSP-37, Tethering factor SEC34, FXYD6, Alpha enolase, G-s-alpha, GPR85, DAF, RPL35A, GIF, TAPA-1, ANAPC11, DCI, hsp27, MRPS7 homolog, eIF p110 subunit, DPH2L, HMG-14, FB1 protein, chondroitin-6-sulphonase, calgizzarin, RNA polymerase II subunit, RPL13A, DHS, gp96, HHP2, acidic ribosomal phosphoprotein P2, ANT-2, ARF1, AFG3L2, SKD3, phosphoglucoisomerase, GST pi, CKI gamma 2, DNA polymerase delta small subunit and TRAP delta. Sections of human osteosarcoma biopsies and a xenograft were studied by in situ analysis. Seven cDNAs highly expressed in Northern blot analysis were tested. Their in situ expression differed between the xenograft and human sections as did that of collagen I. In the xenograft made from one of the target cell lines (OHS), a fair to strong representation of 3 cloned mRNAs was observed while collagen I mRNA was not detectable. We conclude that the molecular heterogeneity of these tumors is considerable. These results ought to have implications for future work to describe phenotypic subtypes with the aim of improving the diagnosis of human osteosarcomas.

The CDKN2A tumor-suppressor gene in chromosome band 9p21 encoding CDKN2A (also known as p16, INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutations, promoter hypermethylation, or deletions. Homozygous deletions predominate in certain cancer types (e.g., bladder cancers). To understand why deletions are unusually prevalent at this locus, deletions in bladder and renal cancer cell lines were mapped in detail and several deletion breakpoints cloned. Deletions were interstitial and encompassed 0.1 to >30 Mb. Most deletion breakpoints were located in or close to LINE-1 retrotransposon clusters. Therefore, deletions of CDKN2A may be facilitated by the presence of LINE-1 clusters that flank the locus. All cloned junctions were products of non-homologous recombination and consistently contained exact 2-bp microhomologies. Microhomologies are otherwise hallmarks of DNA double-strand break repair by non-homologous end joining, but the consistent size found at the CDKN2A deletion junctions is difficult to reconcile with the known properties of this process. Therefore, an unknown mechanism appears to be involved in the generation of CDKN2A deletions during carcinogenesis.