Browsing by Subject "Small GTPase"

Now showing 1 - 4 of 4
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    ARF6 is a Novel Target for Immunotherapy in Triple Negative Breast Cancer
    (2024-07) Moulana, Fathima Ishara; Lu, Xiongbin; Pollok, Karen; Hopewell, Emily; Liu, Jing
    Triple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes with poor clinical outcomes due to lack of effective treatments owing to its hormone receptor negative status. Immune checkpoint blockade (ICB) therapy, which prevents the exhaustion of CD8+ T cells, has shown promise in treating these patients. However, only a small proportion respond, possibly due to resistance and immune evasion mechanisms by the tumor cells. A primary mechanism by which tumor cells evade immune surveillance is by reduced tumor antigen presentation, as indicated by a decreased level of antigen-MHC-I (major histocompatibility complex-I) on the surface of tumor cells. The dynamics of tumor antigens on the cell surface and how cell endocytosis contributes to antigen presentation and their recycling is little known. Here we sought to study the roles of two proteins: clathrin and ADP-Ribosylation Factor 6 (ARF6) which are essential for clathrin-mediated endocytosis and clathrin-independent endocytosis respectively, on the surface turnover of fluorophore-conjugated antigenic peptide bound to MHC-I. We employed Total Internal Reflection Fluorescence Microscopy (TIRFM) and Single Molecule Tracking (SMT) to determine the dynamics of tumor antigen endocytosis on the surface of EO771 murine TNBC cells. We found that the inhibition of ARF6 remarkably impaired the endocytosis of the antigen-MHC-I foci, leading to extended stay of the foci on the cell membrane, while inhibition of clathrin did not, suggesting that clathrin-independent endocytosis is the primary route for MHC-I-mediated antigen endocytosis. Consistent with this finding, reduced ARF6 levels promoted in vitro tumor cell killing by CD8+ T cells and suppressed tumor growth in mice when combined with ICB therapy. We further investigated the effect of pharmacological inhibition of ARF6 in murine TNBC cells and splenic CD8+ T cells using a commercially available ARF6 inhibitor NAV-2729. Treatment with NAV-2729 increased surface MHC-I levels and enhanced the secretion of T cell functional markers such as IFN-, TNF- and IL-2, suggesting the possibility of in vivo administration of ARF6 inhibitors in combination with ICB therapy. Collectively, these data suggest that ARF6 is a novel target for the combined treatment with ICB therapy to improve its efficacy in TNBC patients.
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    The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth
    (ASBMB, 2014-05-01) Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A.; Stupak, Dwayne G.; Brown, Joan Heller; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo.
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    Rnd3/RhoE Modulates HIF1α/VEGF Signaling by Stabilizing HIF1α and Regulates Responsive Cardiac Angiogenesis
    (American Heart Association, 2016-03) Yue, Xiaojing; Yang, Tingli; Lin, Xi; Yang, Xiangsheng; Yi, Xin; Jiang, Xuejun; Li, Xiaoyan; Li, Tianfa; Guo, Junli; Dai, Yuan; Shi, Jianjian; Wei, Lei; Youker, Keith A.; Torre-Amione, Guillermo; Yu, Yanhong; Andrade, Kelsey C.; Chang, Jiang; Department of Pediatrics, IU School of Medicine
    The insufficiency of compensatory angiogenesis in the heart of patients with hypertension contributes to heart failure transition. The hypoxia-inducible factor 1α-vascular endothelial growth factor (HIF1α-VEGF) signaling cascade controls responsive angiogenesis. One of the challenges in reprograming the insufficient angiogenesis is to achieve a sustainable tissue exposure to the proangiogenic factors, such as HIF1α stabilization. In this study, we identified Rnd3, a small Rho GTPase, as a proangiogenic factor participating in the regulation of the HIF1α-VEGF signaling cascade. Rnd3 physically interacted with and stabilized HIF1α, and consequently promoted VEGFA expression and endothelial cell tube formation. To demonstrate this proangiogenic role of Rnd3 in vivo, we generated Rnd3 knockout mice. Rnd3 haploinsufficient (Rnd3(+/-)) mice were viable, yet developed dilated cardiomyopathy with heart failure after transverse aortic constriction stress. The poststress Rnd3(+/-) hearts showed significantly impaired angiogenesis and decreased HIF1α and VEGFA expression. The angiogenesis defect and heart failure phenotype were partially rescued by cobalt chloride treatment, a HIF1α stabilizer, confirming a critical role of Rnd3 in stress-responsive angiogenesis. Furthermore, we generated Rnd3 transgenic mice and demonstrated that Rnd3 overexpression in heart had a cardioprotective effect through reserved cardiac function and preserved responsive angiogenesis after pressure overload. Finally, we assessed the expression levels of Rnd3 in the human heart and detected significant downregulation of Rnd3 in patients with end-stage heart failure. We concluded that Rnd3 acted as a novel proangiogenic factor involved in cardiac responsive angiogenesis through HIF1α-VEGFA signaling promotion. Rnd3 downregulation observed in patients with heart failure may explain the insufficient compensatory angiogenesis involved in the transition to heart failure.
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    Roco Proteins and the Parkinson's Disease-Associated LRRK2
    (MDPI, 2018-12-17) Liao, Jingling; Hoang, Quyen Q.; Biochemistry and Molecular Biology, School of Medicine
    Small G-proteins are structurally-conserved modules that function as molecular on-off switches. They function in many different cellular processes with differential specificity determined by the unique effector-binding surfaces, which undergo conformational changes during the switching action. These switches are typically standalone monomeric modules that form transient heterodimers with specific effector proteins in the ‘on’ state, and cycle to back to the monomeric conformation in the ‘off’ state. A new class of small G-proteins called “Roco” was discovered about a decade ago; this class is distinct from the typical G-proteins in several intriguing ways. Their switch module resides within a polypeptide chain of a large multi-domain protein, always adjacent to a unique domain called COR, and its effector kinase often resides within the same polypeptide. As such, the mechanisms of action of the Roco G-proteins are likely to differ from those of the typical G-proteins. Understanding these mechanisms is important because aberrant activity in the human Roco protein LRRK2 is associated with the pathogenesis of Parkinson’s disease. This review provides an update on the current state of our understanding of the Roco G-proteins and the prospects of targeting them for therapeutic purposes.