Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

Herlevsen, Mikael; Oxford, Gary; Owens, Charles; Conaway, Mark; Theodorescu, Dan

doi:10.1158/1535-7163.mct-06-0372

Cited by 66 publications

(65 citation statements)

References 46 publications

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“…The membrane fractions were checked for the fractionation purification by immunoblotting for the cytosolic and membrane markers ␣-tubulin (Calbiochem) and Caveolin-1 or integrin ␤1 (BD Biosciences), respectively. UMUC3 cells were transiently transfected with pFLAG-CMV4 and pFLAG-CMV4-RhoGDI2, and mass spectrometry analysis of the immunoprecipitate was carried out as described previously (27,28).…”

Section: Methodsmentioning

confidence: 99%

Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function

Moissoglu

Wang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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RhoGDI2 is a suppressor of metastasis in human bladder cancer. Although diminished RhoGDI2 expression in tumors is associated with decreased patient survival, normal expression in some metastatic tumors led us to wonder whether other mechanisms regulate RhoGDI2 function. Protein interaction analysis identified Src as a novel RhoGDI2 interaction partner. Gene expression profiling and immunohistochemistry of human tumors revealed that Src levels diminish as a function of bladder cancer stage. In addition, diminished Src levels and RhoGDI2 levels appear mutually exclusive in individual tumors, indicating that both genes are likely involved in the same signaling pathway leading to metastasis suppression. Studies confirmed that activated Src kinase binds and phosphorylates RhoGDI2 in vitro and vivo. Mutagenesis revealed that Tyr-153 and, to a lesser degree, Tyr-24 were the primary Src phosphorylation sites. Phosphorylation decreased the amount of Rac1 in RhoGDI2 complexes and increased RhoGDI2 association with cell membranes. Stable expression of phosphomimetic Tyr-153 RhoGDI2 in metastatic human bladder cancer cell lines had no effect on primary tumor growth but suppressed metastasis more potently than WT RhoGDI2. These data suggest that phosphorylation by Src enhances RhoGDI2 metastasis suppression and that loss of Src relieves metastasis suppression in tumor cells that maintain RhoGDI2 expression. Our findings also suggest caution in using Src inhibitors in the hope of delaying progression in patients with bladder cancer.bladder neoplasms ͉ guanine nucleotide dissociation inhibitors ͉ neoplasm metastasis ͉ src-family kinases B ladder cancer is common in the United States (1). Most deaths from this disease are due to metastases, commonly to lung and liver. RhoGDI2 (LyGDI/D4GDI) has been shown to be a metastasis suppressor in animal models of bladder cancer and to be lost in many metastatic human tumors (2, 3). RhoGDI2 belongs to a family of related proteins that includes RhoGDI1 and RhoGDI3 (reviewed in ref. 4). RhoGDI1 is the most widely expressed and the most intensely studied. Previously, it was thought that RhoGDI2 expression was confined to cells of hematopoietic origin, but our studies showed it is expressed in a wider range of tissues and cell types (5). Much less is known about RhoGDI3. Its expression is highest in brain, lung, and testis and, in contrast to RhoGDI1 and RhoGDI2, it is not found in the cytoplasm, but is associated with vesicular membranes. RhoGDIs are thought to inhibit the activation of small GTPases of the Rho family by sequestering the GTPases in the cytosol and blocking activation by guanine nucleotide exchange factors (GEFs) (4). Previous work identified RhoA, Rac1, and Rac2 as targets of RhoGDI2-mediated inactivation (6, 7), suggesting that RhoGDI2's function overlaps that of RhoGDI1.Although RhoGDI2 expression is reduced in many metastatic bladder cancers, Ϸ35% of patients with moderate or high levels of RhoGDI2 protein still developed metastatic disease. Although factors unrelated to...

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Section: Methodsmentioning

confidence: 99%

Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function

Moissoglu

Wang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…MVP/vaults have been associated with multidrug resistance (21)(22)(23), nuclear-cytoplasmic transport (24), innate immunity (25), autophagy (26), and signal transduction pathways (27)(28)(29)(30)(31); however, the precise function of MVP/ vault remains enigmatic. Our previous work indicating MVPinduced type I IFN production upon hepatitis C virus infection uncovers a new role of MVP in modulating innate immunity during viral infection (32).…”

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confidence: 99%

Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA– or Virus-Induced Proinflammatory Response

Peng

Shi

Xia

et al. 2016

The Journal of Immunology

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Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinflammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP-1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP−/−) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)β–liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPβ binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPβ. The interactions promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP−/− mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response.

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“…In 2007 it was shown that knockdown of the major vault protein (MVP) using siRNA disrupted lysosomal uptake of not only doxorubicin, but also the intracellular pH probe LysoSensor and the lysosomal specific antigen LAMP1. This study indicated that MVP was integral in redistributing the drug away from the nucleus to the lysosomes (Herlevsen et al, 2007). In another study, cells were exposed to various weak bases before lysosomes were isolated and their contents quantified using high performance liquid chromotography.…”

Section: Drug Sequestration Through the Endosomal Pathwaysmentioning

confidence: 90%