Kinase Regulation of HOX Transcription Factors.

The HOX genes are a group of homeodomain-containing transcription factors that play important regulatory roles in early development, including the establishment of cell and tissue identity. HOX expression is generally reduced in adult cells but is frequently re-established as an early event in tumour formation and supports an oncogenic phenotype. HOX transcription factors are also involved in cell cycle regulation and DNA repair, along with normal adult physiological process including stem cell renewal. There have been extensive studies on the mechanism by which HOX proteins regulate transcription, with particular emphasis on their interaction with cofactors such as Pre-B-cell Leukaemia Homeobox (PBX) and Myeloid Ecotropic Viral Integration Site 1 (MEIS). However, significantly less is known of how the activity of HOX proteins is regulated. There is growing evidence that phosphorylation may play an important role in this context, and in this review, we draw together a number of important studies published over the last 20 years, and discuss the relevance of phosphorylation in the regulation and function of HOX proteins in development, evolution, cell cycle regulation, and cancer.

Membrane insertion and secretion of the Engrailed-2 (EN2) transcription factor by prostate cancer cells may induce antiviral activity in the stroma.

Engrailed-2 (EN2) is a homeodomain-containing transcription factor that has roles in boundary formation and neural guidance in early development, but which is also expressed in a range of cancers. In addition to transcriptional regulation, it is secreted by cells and taken up by others through a mechanism that is yet to be fully elucidated. In this study, the distribution of EN2 protein in cells was evaluated using immunofluorescence with a set of antibodies raised against overlapping epitopes across the protein, and through the use of an EN2-GFP construct. MX2 expression in primary prostate tumors was evaluated using immunohistochemistry. We showed that EN2 protein is present in the cell membrane and within microvesicles that can be secreted from the cell and taken up by others. When taken up by normal cells from the stroma EN2 induces the expression of MX2 (MxB), a protein that has a key role in the innate immune response to viruses. Our findings indicate that EN2 secretion by tumors may be a means of preventing viral-mediated immune invasion of tissue immediately adjacent to the tumor.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7.

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids.

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As2O3) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.

Human mesenchymal stem cells exploit the immune response mediating chemokines to impact the phenotype of glioblastoma.

In contrast to the application of human mesenchymal stem cells (hMSCs) in regenerative medicine, only a limited number of studies are addressing their use in anticancer therapy. As the latter may represent a new hope to improve the survival of patients with glioblastoma multiformae (GBM), the most common and malignant form of the brain tumors, we aimed to investigate the interactions of hMSCs and GBM cells under in vitro conditions. Four hMSC clones and three different GBM cell lines were used to study their mutual paracrine interactions in cocultures compared to their monocultures, where cells were grown under the same experimental conditions. The effects on cell growth, proliferation, and invasion in Matrigel were quantified. Further, bioinformatics tools were used to relate these results to the data obtained from cytokine macroarrays and cDNA microarrays that revealed proteins and genes significantly involved in cellular cross-talk. We showed that hMSCs are responsible for the impairment of GBM cell invasion and growth, possibly via induction of their senescence. On the other hand, GBM cells inversely affected some of these characteristics in hMSCs. We found CCL2/MCP-1 to be the most significantly regulated chemokine during hMSC and U87-MG paracrine signaling in addition to several chemokines that may account for changed cocultured cells' phenotype by affecting genes associated with proliferation (Pmepa-1, NF-κB, IL-6, IL-1b), invasion (EphB2, Sod2, Pcdh18, Col7A1, Gja1, Mmp1/2), and senescence (Kiaa1199, SerpinB2). As we functionally confirmed the role of CCL2/MCP-1 in GBM cell invasion we thereby propose a novel mechanism of CCL2/MCP-1 antimigratory effects on GBM cells, distinct from its immunomodulatory role. Significant alterations of GBM phenotype in the presence of hMSCs should encourage the studies on the naive hMSC use for GBM treatment.