Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases Their Susceptibility to Temozolomide.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor, with a very high rate of recurrence and a median survival of 15 months after diagnosis. Abundant evidence suggests that a certain sub-population of cancer cells harbors a stem-like phenotype and is likely responsible for disease recurrence, treatment resistance and potentially even for the infiltrative growth of GBM. GBM incidence has been negatively correlated with the serum levels of 25-hydroxy-vitamin D3, while the low pH within tumors has been shown to promote the expression of the vitamin D3-degrading enzyme 24-hydroxylase, encoded by the CYP24A1 gene. Therefore, we hypothesized that calcitriol can specifically target stem-like glioblastoma cells and induce their differentiation. Here, we show, using in vitro limiting dilution assays, quantitative real-time PCR, quantitative proteomics and ex vivo adult organotypic brain slice transplantation cultures, that therapeutic doses of calcitriol, the hormonally active form of vitamin D3, reduce stemness to varying extents in a panel of investigated GSC lines, and that it effectively hinders tumor growth of responding GSCs ex vivo. We further show that calcitriol synergizes with Temozolomide ex vivo to completely eliminate some GSC tumors. These findings indicate that calcitriol carries potential as an adjuvant therapy for a subgroup of GBM patients and should be analyzed in more detail in follow-up studies.

Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms.

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.

A clinoptilolite effect on cell media and the consequent effects on tumor cells in vitro.

Clinoptilolite is a nontoxic natural zeolite with properties of an ion-exchanger and adsorbent. Earlier studies showed that clinoptilolite could be an adjuvant in cancer therapy. The aim of this study was to define effects of clinoptilolite in cell media on cell viability and activity of key proteins regulating cell survival, cell division and stress response. The number of viable cells, DNA synthesis and activity of EGF-R, PKB/Akt and NF?B was reduced, while apoptosis was increased in cells that were cultured in medium supplemneted with clinoptilolite. These results might be due to adsorbtion of some serum components such as EGF to clinoptilolite. In treated medium without serum the predominant role of clinoptilolite is that of cation exchange, likely affecting calcium levels and calcium-dependent signalling pathways. These results are in line with other data that confirm enhanced apoptosis in cells incubated in treated medium. Together, data presented here demonstrate that clinoptilolite affects cellular microenvironment through mechanisms that are dependent on adsorptive and ion-exchange characteristics of this material.

SH3P2 in complex with Cbl and Src.

In this report, we describe SH3P2, an SH3-domain containing protein, as a novel Cbl-interacting molecule that is a substrate of tyrosine kinase Src. We identified a specific polyproline motif of Cbl responsible for binding of SH3P2 and Src, and observed mutual sequestration of Src and SH3P2 from monomer Cbl molecules. In adherent cells, SH3P2 associated with Cbl and fibrilar actin and was localized at focal contacts in fibroblasts as well as at the apical part of podosome rings in differentiated osteoclasts. Our data implicate that SH3P2, a novel component of adhesion sites, is involved in Cbl and Src-mediated pathways.

Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases.

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.