NFκB2 p52 stabilizes rhogdiß mRNA by inhibiting AUF1 protein degradation via a miR-145/Sp1/USP8-dependent axis.

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiß mRNA stability and expression. Positively regulation of rhogdiß mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiß mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIß axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits.

BACKGROUND: The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. METHODS: Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. RESULTS: We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled Cα- and Hα-based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. CONCLUSIONS: Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. GENERAL INTERESTS: Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques.

An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu).

Filamin A (FLNA) is an actin filament crosslinking protein with multiple intracellular binding partners. Mechanical force exposes cryptic FLNA binding sites for some of these ligands. To identify new force-dependent binding interactions, we used a fusion construct composed of two FLNA domains, one of which was previously identified as containing a force-dependent binding site as a bait in a yeast two-hybrid system and identified the Rho dissociation inhibitor 2 (RhoGDI2) as a potential interacting partner. A RhoGDI2 truncate with 81 N-terminal amino acid residues and a phosphomimetic mutant, RhoGDI(Tyr153Glu) interacted with the FLNA construct. However, neither wild-type or full-length RhoGDI2 phosphorylated at Y153 interacted with FLNA. Our interpretation of these contradictions is that truncation and/or mutation of RhoGDI2 perturbs its conformation to expose a site that adventitiously binds FLNA and is not a bona-fide interaction. Therefore, previous studies reporting that a RhoGDI(Y153E) mutant suppresses the metastasis of human bladder cancer cells must be reinvestigated in light of artificial interaction of this point mutant with FLNA.

Comparative proteomic profiling of triple-negative breast cancer reveals that up-regulation of RhoGDI-2 is associated to the inhibition of caspase 3 and caspase 9.

There are no targeted therapeutic modalities for triple-negative breast cancer (TNBC), thus it is associated with poor prognosis and worst clinical outcome. Here, our aim was to identify deregulated proteins in TNBC with potential therapeutic applications. Proteomics profiling of TNBC and normal breast tissues through two-dimensional electrophoresis and ESI-MS/MS mass spectrometry revealed the existence of 16 proteins (RhoGDI-2, HSP27, SOD1, DJ1, UBE2N, PSME1, FTL, SH3BGRL, and eIF5A-1) with increased abundance in carcinomas. We also evidenced for the first time the deregulation of COX5, MTPN and DB1 proteins in TNBC that may represent novel tumor markers. Particularly, we confirmed the overexpression of the Rho-GDP dissociation inhibitor 2 (RhoGDI-2) in distinct breast cancer subtypes, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Remarkably, targeted disruption of RhoGDI-2 by RNA interference induced mitochondrial dysfunction, and facilitated caspase-3 and -9 activation in two breast cancer cell lines. Moreover, suppression of RhoGDI-2 resulted in a robust sensitization of breast cancer cells to cisplatin therapy. In conclusion, we identified novel proteins deregulated in TNBC, and confirmed the overexpression of RhoGDI-2. We propose that RhoGDI-2 inhibition may be exploited as a potential therapeutic strategy along cisplatin-based chemotherapy in breast cancer. BIOLOGICAL SIGNIFICANCE: There are no useful biomarkers neither targeted therapeutic modalities for triple-negative breast cancer, which highly contributes to the poor prognosis of this breast cancer subtype. In this work, we used two-dimensional electrophoresis and ESI-MS/MS spectrometry to identify novel deregulated proteins in breast cancer tissues. Particularly, our results showed that RhoGDI-2, a protein that has been associated to metastasis and poor survival in human cancers, is overexpressed in different subtypes of breast tumors, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Our data also provided novel insights about the role of RhoGDI-2 in apoptosis through intrinsic pathway inhibition. Importantly, they suggested that targeted modulation of RhoGDI-2 levels might be a useful strategy for breast cancer therapy.

Centrosomal localization of RhoGDIß and its relevance to mitotic processes in cancer cells.

Rho GDP-dissociation inhibitors (RhoGDIs) are regulators of Rho family GTPases. RhoGDIß has been implicated in cancer progression, but its precise role remains unclear. We determined the subcellular localization of RhoGDIß and examined the effects of its overexpression and RNAi knockdown in cancer cells. Immunofluorescence staining showed that RhoGDIß localized to centrosomes in human cancer cells. In HeLa cells, exogenous GFP-tagged RhoGDIß localized to centrosomes and its overexpression caused prolonged mitosis and aberrant cytokinesis in which the cell shape was distorted. RNAi knockdown of RhoGDIß led to increased incidence of monopolar spindle mitosis resulting in polyploid cells. These results suggest that RhoGDIß has mitotic functions, including regulation of cytokinesis and bipolar spindle formation. The dysregulated expression of RhoGDIß may contribute to cancer progression by disrupting these processes.