Background:Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear.Methods:We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR.Results:We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4me3 mark at transcriptional start sites.Conclusions:Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.

Background Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. Methods Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate microtube formation under TGF-βstimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β Results Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF- β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. Conclusion TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network.

Although histone post-translational modifications play a paramount role in controlling access to genetic information, our understanding of the precise mechanisms regulating chromatin signaling remains superficial. For instance, histone H3 trimethylated on lysine 9 (H3K9(me3)) favors the association of chromodomain proteins such as heterochromatin protein 1α (HP1α) with chromatin. However, HP1α and other such chromatin proteins are not covering all specific histone marks at all times. Thus, how are these reader-histone interactions regulated? We propose tyrosine phosphorylation within the aromatic cage of histone mark readers as a molecular switch that can either turn ON or OFF and even alter the specificity of reader-histone interactions. We have identified tyrosine phosphorylation events on the chromatin proteins HP1α and M-phase phosphoprotein 8 that regulate their association with methylated histones in vitro (synthetic peptides, calf thymus purified histones, and nucleosomes), but also in cells, thus controlling access to genetic information.

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of glioblastoma multiforme (GBM), the most aggressive type of brain cancer in adults. Here, we show that small-molecule inhibition of RHO-associated serine/threonine kinase proteins (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) 'webs', which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range Ca 2+ signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.