ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein.
ABSTRACT 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.
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ABSTRACT: Invasion of tumor cells is a key step in metastasis, which depends largely on the ability of these cells to degrade the extracellular matrix. Although we have recently shown that the GTPase ARF1 is overexpressed in highly invasive breast cancer cell lines and that EGF stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has never been addressed in the context of invasiveness. Here, we report that modulation of ARF1 expression and activity markedly impaired the ability of MDA-MB-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling MMP-9 activity. In addition, we demonstrate that this occurs through an inhibition of invadopodia maturation as well as shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn impacts MLC phosphorylation. Together, our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness, and metastasis.Molecular biology of the cell 11/2013; 25(1). DOI:10.1091/mbc.E13-06-0335 · 5.98 Impact Factor
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ABSTRACT: RNA-seq has the potential to discover genes created by complex chromosomal rearrangements. 'Fusion' genes formed by the breakage and re-joining of two different chromosomes have repeatedly been implicated in the development of cancer. However, although RNA-seq can detect these fusion events, normal cells are also characterized by read-through fusions across adjacent genes in the genome, or by transcription-induced chimeras (TICs). In this paper, we detected TICs events in normal tissue samples. The information derived from our analysis indicates the presence of a significant number of TICs in normal tissues. Some of these TICs can be associated with cancer development. In some cases TIC expression is corroborated by a large number of reads and their expression is significantly spread over different tissues. Our data highlight that TICs could be erroneously associated with cancer aberrations if RNA-seq analysis is only evaluated in tumor samples without considering the parallel analysis of normal tissue samples associated with the tumor.Complex, Intelligent and Software Intensive Systems (CISIS), 2012 Sixth International Conference on; 01/2012
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ABSTRACT: By virtual screening using a Fragment-Based Drug Design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. Using SPR, NMR and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then, the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography. This highlighted the role of stable and transient pockets at the Sec7 domain surface in the discovery and binding of interfering compounds. These results provide structural information on how small organic compounds can interfere with the Arf1-Arno Sec7 domain interaction and may guide the rational drug design of competitive inhibitors of Arno enzymatic activity.Journal of Medicinal Chemistry 10/2013; 56(21). DOI:10.1021/jm4009357 · 5.48 Impact Factor