F-box protein FBXO16 functions as a tumor suppressor by attenuating nuclear beta-catenin function

Aberrant activation of beta-catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/beta-catenin-signaling pathways is still poorly understood. In this study, we show that F-box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1-Cullin1-F-box protein) complex, which targets the nuclear beta-catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C-terminal domain of beta-catenin and promotes its lysine 48-linked polyubiquitination. In addition, it inhibits epithelial-to-mesenchymal transition (EMT) by attenuating the level of beta-catenin. Therefore, depletion of FBXO16 leads to increased levels of beta-catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and beta-catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCF(FBXO16) complex that targets nuclear beta-catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant beta-catenin activation

FBXW2 suppresses breast tumorigenesis by targeting AKT-Moesin-SKP2 axis

Abstract Oncogene Moesin plays critical role in initiation, progression, and metastasis of multiple cancers. It exerts oncogenic activity due to its high-level expression as well as posttranslational modification in cancer. However, factors responsible for its high-level expression remain elusive. In this study, we identified positive as well as negative regulators of Moesin. Our study reveals that Moesin is a cellular target of F-box protein FBXW2. We showed that FBXW2 suppresses breast cancer progression through directing proteasomal degradation of Moesin. In contrast, AKT kinase plays an important role in oncogenic function of Moesin by protecting it from FBXW2-mediated proteasomal degradation. Mechanistically, AKT phosphorylates Moesin at Thr-558 and thereby prevents its degradation by FBXW2 via weakening the association between FBXW2 and Moesin. Further, accumulated Moesin prevents FBXW2-mediated degradation of oncogene SKP2, showing that Moesin functions as an upstream regulator of oncogene SKP2. In turn, SKP2 stabilizes Moesin by directing its non-degradable form of polyubiquitination and therefore AKT-Moesin-SKP2 oncogenic axis plays crucial role in breast cancer progression. Collectively, our study reveals that FBXW2 functions as a tumor suppressor in breast cancer by restricting AKT-Moesin-SKP2 axis. Thus, AKT-Moesin-SKP2 axis may be explored for the development of therapeutics for cancer treatment

Protocol for detecting macrophage-mediated cancer cell phagocytosis in vitro and in vivo

Summary: Here, we present optimized approaches to identify the efficiency of cancer cell phagocytosis by macrophages in vitro and in vivo. We describe the preparation and co-culture of macrophages and cancer cells, followed by in vitro phagocytosis assay using flow cytometry and confocal microscopy, respectively. We then detail the establishment of xenograft tumor mouse model and the in vivo detecting of phagocytosis efficiency by flow cytometry and qRT-PCR. This protocol provides a convenient way to assess macrophage-mediated phagocytosis of cancer cells.For complete details on the use and execution of this protocol, please refer to Xu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Synthesis, characterization, TDDFT calculation and biological activity of tetradentate ligand based square pyramidal Cu(ii) complexes

Distorted square pyramidal complexes [Cu(L1)(H2O)]·ClO4 (1), [Cu(L2)(H2O)]·ClO4 (2) and [Cu(L3)(H2O)]·ClO4 (3) (HL1 = o-{[2-(2-aminoethylamino)ethylimino]methyl}phenol; HL2 = 2-{[2-(2-aminoethylamino)ethylimino]methyl}-6-methoxyphenol; HL3 = o-{1-[2-(2-aminoethylamino)ethylimino]ethyl}phenol) have been synthesized, and characterized by X-ray crystallography and spectroscopic analysis. All the complexes exhibit fluorescence at room temperature [λex = 267 nm, λem = 312, 329 and 357 nm, φ = 0.52 for 1; λex = 272 nm, λem = 312, 329 and 355 nm, φ = 0.46 for 2; λex = 265 nm, λem = 312, 356 and 377 nm, φ = 0.33 for 3]. The electronic structure and photophysical properties of the ligands and complexes were calculated by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods using the B3LYP, B3PW91 and MPW1PW91 functionals, with 6-31G (d-p) and LanL2DZ basis sets. The results of TD-DFT calculations are functional-dependent and among the functionals, B3LYP was able to best reproduce the experimental results. Catecholase activity of 1-3 has been investigated using 3,5-di-tert butyl catechol (3,5-DTBC) as the model substrate and found that complexes are active for catalyzing the aerobic oxidation of 3,5-DTBC to 3,5-di-tert butyl benzoquinone (3,5-DTBQ). The compound with more distorted square pyramidal geometry shows a higher rate of catalytic activity. All the complexes have been tested for their anticancer activities in human breast (MCF7) cancer cell lines. Complexes show dose dependent suppression of cell viability with IC50 values 30, 68 and >100 μM for 1, 2 and 3, respectively. Anticancer activities of 1-3 and cisplatin were compared, and found that 1-3 were relatively less active than cisplatin

Synthesis, structure, DNA/protein binding, molecular docking and in vitro anticancer activity of two Schiff base coordinated copper(II) complexes

Two Schiff bases HL and HL′, having potential tridentate O,N,N′ donor sets, have been used for the synthesis of two copper(II) complexes, namely [Cu(HL)(pdc)]2 (1) and [Cu(L′)2]2 (2′), where HL = 2-([2-(piperazin-yl)ethylimino]methyl)phenol, pdc = py-2,5-dicarboxylate and HL′ = 2-(((2-(di-isopropylamino)ethyl)imino)methyl)phenol. X-ray single crystal analysis of complex 1 shows a centro-symmetric dimer. It crystallizes with a number of lattice water molecules that form a network of H-bonds, also involving the protonated piperazinium fragment, giving rise to a 3D supramolecular architecture. Complex 2′ also crystallizes as dinuclear, formed through mutual bridging phenol oxygen atoms as [Cu(L′)2]2, whilst an ESI mass spectrometry study evidences that in solution the complex exists as mononuclear [Cu(L′)2] (2). The interaction of complexes 1 and 2 with calf thymus DNA (CT-DNA) and with bovine serum albumin (BSA) was investigated using electronic absorption and fluorescence spectroscopic techniques. In both studies the results show a higher binding affinity of complex 1 in comparison to 2. The anticancer activity of the complexes against human breast (MCF7) cancer cell lines reveals that complex 1 has moderate growth suppression activity against these cells with an IC50 value of 24 ± 6.24 μM

A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer

The transformation of a normal cell to cancer requires the derail of multiple pathways. Normal signaling in a cell is regulated at multiple stages by the presence of feedback loops, calibration of levels of proteins by their regulated turnover, and posttranscriptional regulation, to name a few. The tumor suppressor protein FBXO31 is a component of the SCF E3 ubiquitin ligase and is required to arrest cells at G1 following genotoxic stresses. Due to its growth-suppression activity, it is underexpressed in many cancers. However, the molecular mechanism underlying the translational regulation of FBXO31 remains unclear. Here we show that the oncogenic microRNAs miR-93 and miR-106a repress FBXO31, resulting in the upregulation of Slug, which is involved in epithelial-mesenchymal transition and cell invasion. FBXO31 targets and ubiquitylates Slug for proteasomal degradation. However, this mechanism is repressed in breast tumors where miR-93 and miR-106a are overexpressed. Our study further unravels an interesting mechanism whereby Slug drives the expression of miR-93 and miR-106a, thus establishing a positive feedback loop to maintain an invasive phenotype. Together, these results establish the presence of interplay between microRNAs and the ubiquitination machinery, which together regulate cancer cell invasion