Rlip depletion prevents spontaneous neoplasia in TP53 null mice.
ABSTRACT: TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.
Project description:Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76(-/-) MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis-RLIP76(-/-) mice will be deficient in chemical neoplasia-was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76(-/-). PMA-induced skin carcinogenesis in RLIP76(+/+) mouse was suppressed completely by depletion of either PKC? or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76(-/-) mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76(-/-) mice. In RLIP76(-/-) mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis.
Project description:RalBP1/RLIP76 is a widely expressed multifunctional protein that binds the Ral and R-Ras small GTPases. In the mouse, RLIP76 is nonessential but its depletion or blockade promotes tumorigenesis and heightens the sensitivity of normal and tumor cells to radiation and cytotoxic drugs. However, its pathobiologic functions, which support tumorigenesis, are not well understood. Here, we show that RLIP76 is required for angiogenesis and for efficient neovascularization of primary solid tumors. Tumor growth from implanted melanoma or carcinoma cells was blunted in RLIP76(-/-) mice. An X-ray microcomputed tomography-based method to model tumor vascular structures revealed defects in both the extent and form of tumor angiogenesis in RLIP76(-/-) mice. Specifically, tumor vascular volumes were diminished and vessels were fewer in number, shorter, and narrower in RLIP76(-/-) mice than in wild-type mice. Moreover, we found that angiogenesis was blunted in mutant mice in the absence of tumor cells, with endothelial cells isolated from these animals exhibiting defects in migration, proliferation, and cord formation in vitro. Taken together, our results establish that RLIP76 is required for efficient endothelial cell function and angiogenesis in solid tumors.
Project description:R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(?1-180) or RLIP76(?180-192). Spreading and migration required RLIP76(1-180), and RLIP76(?1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(?1-180). Similarly, RLIP76(?1-180) or RLIP76(?180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.
Project description:RLIP76 (RalBP1) is a multidomain protein that is a downstream effector of the small GTP ases RalA and RalB. As well as the Ral binding domain it contains a RhoGAP domain active against Cdc42 and Rac1. RLIP76 also binds to proteins involved in endocytosis and to R-Ras. We recently solved the structure of the Ral binding domain of RLIP76 and the structure of the complex that it forms with RalB. The structure shows that, unlike the other Ral effectors characterized so far, RLIP76 forms a coiled-coil that interacts with RalB. The RLIP76 Ral binding domain binds to both the switch regions of RalB, which are the parts of the G protein that chance conformation upon nucleotide exchange. Here, we review our structure and discuss how it sheds light on the other functions of RLIP76.
Project description:RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain, and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral-binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB, and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84, and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.
Project description:Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission. Equal distribution of mitochondria to daughter cells during mitosis requires fission. Mitotic mitochondrial fission depends on both the relocalization of the large GTPase DRP1 to the outer mitochondrial membrane and phosphorylation of Ser 616 on DRP1 by the mitotic kinase cyclin B-CDK1 (ref. 2). We now report that these processes are mediated by the small Ras-like GTPase RALA and its effector RALBP1 (also known as RLIP76, RLIP1 or RIP1; refs 3, 4). Specifically, the mitotic kinase Aurora A phosphorylates Ser 194 of RALA, relocalizing it to the mitochondria, where it concentrates RALBP1 and DRP1. Furthermore, RALBP1 is associated with cyclin B-CDK1 kinase activity that leads to phosphorylation of DRP1 on Ser 616. Disrupting either RALA or RALBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B-CDK1 converge on RALA and RALBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.
Project description:2'-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNF?, and phospho-PDGFR-? were depleted in all three cell lines; MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-? inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF-Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF-PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.
Project description:Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB) of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-?B. RLIP76 (a.k.a. RALBP1), an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-?) pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-? on RLIP76, we faced the problem of choosing reference genes impervious to TNF-?. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-?, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-?, and show for the first time that RLIP76 expression is induced by TNF-? and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1), another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-? is not a general property of glutathione transporters. The reference geneset uncovered herein should aid in future gene expression studies in inflammatory conditions of the BBB.
Project description:Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope.JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep.
Project description:Breast cancer (BC) is the most common cancer in women. Estrogen, epidermal growth factor receptor 2 (ERBB2, HER2), and oxidative stress represent critical mechanistic nodes associated with BC. RLIP76 is a major mercapturic acid pathway transporter whose expression is increased in BC. In the quest of a novel molecule with chemopreventive and chemotherapeutic potential, we evaluated the effects of 2'-Hydroxyflavanone (2HF) in BC. 2HF enhanced the inhibitory effects of RLIP76 depletion and also inhibited RLIP76-mediated doxorubicin transport in BC cells. RNA-sequencing revealed that 2HF induces strong reversal of the gene expression pattern in ER+MCF7, HER2+ SKBR3 and triple-negative MDA-MB-231 BC cells with minimal effects on MCF10A normal breast epithelial cells. 2HF down regulated ER? and enhanced inhibitory effects of imatinib mesylate/Gleevec in MCF7 cells. 2HF also down regulated ER? and HER2 gene networks in MCF7 and SKBR3 cells, respectively. 2HF activated TP53 and inhibited TGF?1 canonical pathway in MCF7 and MDA-MB-231 BC cells. 2HF also regulated the expression of a number of critical prognostic genes of MammaPrint panel and their upstream targets including TP53, CDKN2A and MYC. The collective findings from this study provide a comprehensive, direct and integrated evidence for the benefits of 2HF in targeting major and clinically relevant mechanistic regulators of BC.