Chaya Brodie

Brain metastases are the most common secondary brain tumors in adults. Despite their high frequency and patient poor prognosis, very little research has been performed on lung tumor brain metastases, mainly due to the lack of appropriate experimental models. In this study, we isolated cancer stem cells (CSCs) from fresh specimens of lung tumor brain metastases. The CSCs were analyzed for sphere formation and limiting dilution analyses, stemness markers, ability to generate xenografts and for their interaction with microglia cells. We found that CSCs derived from brain metastases had a high sphere forming capacity and self-renewal ability comparable to that of glioma stem cells. The CSCs expressed the stemness markers, CD133, Sox2, Klf4, Aldh2a, CD44 and the lung tumor markers, cytokeratin 7, and CD166. Transplantation of the CSCs or organoids generated from the brain metastases formed xenografts that recapitulated the parental tumors. These xenografts were infiltrated by a large number of amoeboid microglia that expressed high levels of M2 markers. Using co-culture experiments, we further found that CSCs derived from brain metastasis induced the polarization of microglia to the M2 phenotype via secreted exosomes. Similarly, M2 micrglia cells increased the self-renewal and stemness of the CSCs. RNA seq analysis identified specific miRNAs and lncRNAs that were associated with the CSC-microglia interactions. Using specific reporters, antagomiRs and CRISPR/Cas9 we demonstrated that miR-21, miR-1246 and the lncRNA TALNEC2 played a major role in the M2 polarization of microglia cells both in vitro and in vivo. In conclusion, we generated CSCs and organoids from lung tumor-derived brain metastases and identified potential therapeutic targets for these tumors. The established CSCs can serve as valuable in vitro and in vivo models for analyzing mechanisms involved in brain metastasis, for studying the tumor-microenvironment interactions and for personalized high throughput screening of new and repurposed drugs.

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell–derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell–derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.

Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors tochemo‐ and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to γ‐radiation, has the capacity to repopulate the tumors and contribute to their malignant progression. γ‐radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by γ‐radiation in CD133+ GSCs. Irradiation of CD133+ cells induced autophagy within 24–48 hr and slightly decreased the viability of the cells. γ‐radiation induced a larger degree of autophagy in the CD133+ cells as compared with CD133− cells and the CD133+ cells expressed higher levels of the autophagy‐related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD133+ cells to γ‐radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to γ‐radiation. © 2009 UICC

In this study, we examined the role of protein kinase C (PKC)-ε in the apoptosis and survival of glioma cells using tumor necrosis factor–related apoptosis inducing ligand (TRAIL)-stimulated cells and silencing of PKCε expression. Treatment of glioma cells with TRAIL induced activation, caspase-dependent cleavage, and down-regulation of PKCε within 3 to 5 hours of treatment. Overexpression of PKCε inhibited the apoptosis induced by TRAIL, acting downstream of caspase 8 and upstream of Bid cleavage and cytochrome c release from the mitochondria. A caspase-resistant PKCε mutant (D383A) was more protective than PKCε, suggesting that both the cleavage of PKCε and its down-regulation contributed to the apoptotic effect of TRAIL. To further study the role of PKCε in glioma cell apoptosis, we employed short interfering RNAs directed against the mRNA of PKCε and found that silencing of PKCε expression induced apoptosis of various glioma cell lines and primary glioma cultures. To delineate the molecular mechanisms involved in the apoptosis induced by silencing of PKCε, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that knockdown of PKCε did not affect the expression of Bcl2 and Bax or the phosphorylation and expression of Erk1/2, c-Jun-NH₂-kinase, p38, or STAT, whereas it selectively reduced the expression of AKT. Similarly, TRAIL reduced the expression of AKT in glioma cells and this decrease was abolished in cells overexpressing PKCε. Our results suggest that the cleavage of PKCε and its down-regulation play important roles in the apoptotic effect of TRAIL. Moreover, PKCε regulates AKT expression and is essential for the survival of glioma cells.

RasGRP is a family of guanine nucleotide exchange factors that activate small GTPases and contain a C1 domain similar to the one present in protein kinase C (PKC). In this study, we examined the interaction of RasGRP3 and PKC in response to the phorbol ester PMA. In Chinese hamster ovary or LN-229 cells heterologously expressing RasGRP3, phorbol 12-myristate 13-acetate (PMA) induced translocation of RasGRP3 to the perinuclear region and a decrease in the electrophoretic mobility of RasGRP3. The mobility shift was associated with phosphorylation of RasGRP3 on serine residues and seemed to be PKCdelta-dependent because it was blocked by the PKCdelta inhibitor rottlerin as well as by a PKCdelta kinase-dead mutant. Using coimmunoprecipitation, we found that PMA induced the physical association of RasGRP3 with PKCdelta and, using in situ methods, we showed colocalization of PKCdelta and RasGRP3 in the perinuclear region. PKCdelta phosphorylated RasGRP3 in vitro. Previous studies suggest that ectopic expression of RasGRP3 increases activation of Erk1/2. We found that overexpression of either PKCdelta or RasGRP3 increased the activation of Erk1/2 by PMA. In contrast, coexpression of PKCdelta and RasGRP3 yielded a level of phosphorylation of Erk1/2 similar to that of control vector cells. Our results suggest that PKCdelta may act as an upstream kinase associating with and phosphorylating RasGRP3 in response to PMA. The interaction between RasGRP3 and PKCdelta points to the existence of complex cross-talk between various members of the phorbol ester receptors which can have important impact on major signal transduction pathways and cellular processes induced by phorbol esters or DAG

The isoforms of the PKC family are activated in response to mitogenic stimuli, to inflammatory stimuli, and to stress and play important roles in a variety of cellular functions including apoptosis....

Protein kinase Cδ (PKCδ) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCδ in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G₁/S phase of the cell cycle. Overexpression of PKCδ increased the apoptotic effect induced by etoposide, whereas the PKCδ selective inhibitor rottlerin and the PKCδ dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCδ and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCδ were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCδ in the effects of etoposide was examined using cells overexpressing a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCδ. Likewise, activation of caspase 3 and the cleavage of the PKCδ5 mutant were significantly lower in cells overexpressing PKCδ5. Using mutants of PKCδ altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCδ in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCδ as an important regulator of this effect.

Nerve growth factor (NGF) is a neurotrophic factor acting on both the peripheral and central nervous systems. In addition, it has been shown to modulate B lymphocyte function through receptorsconsisting of both p75 and TrkA proteins. The low-affinity NGFR, p75, shares structural homology with the B cell antigen, CD40, tumor necrosis factor (TNF) receptor and Fas antigen (APO-1), which play a role in cell apoptosis. We studied the effect of NGF on anti-IgM-induced apoptosis in human B lymphocytes and the role of protein kinase C (PKC) in this effect. Incubation of Ramos cells with anti-IgM (10 μg / ml) induced apoptosis which was observed after 6 h and reached plateau levels after 24 h. Addition of NGF to anti-IgM-treated cells rescued cells from apoptosis. The NGF effectwas blocked by anti-NGF antibody and by K252a, a specific inhibitor for the tyrosine kinase activity of TrkA. NGF induced translocation of PKCδ and PKCα from the cytosol to the plasma membrane and translocation of PKCζ to the nucleus. To examine the role of PKC in the inhibitory effect of NGF on anti-IgM-induced apoptosis, we used inhibitors of PKCα and PKCδ and found that these treatments did not alter the NGF effect. In contrast, treatment of the cells with oligonucleotide antisense directed against the 5′ coding sequence of PKCζ reduced the expression of PKCζ in the cells and abolished the protective effect of NGF on anti-IgM-induced apoptosis. The translocation of PKCζ and the protective effect of NGF were inhibited by the phosphatidylinositol 3 (PI3)-kinase inhibitors wortmannin and LY294002. The results of this study indicate that NGF is involved in B cell survival and that this effect is mediated by PI3-kinase-dependent activation of PKCζ.

Sindbis virus (SV) is an alphavirus used as a model for studying the pathogenesis of viral encephalitis. In this study we examined the effects and the mechanisms involved in the apoptosis induced by SV in PC-12 cells, and the role of a vFLIP in this process. Infection of PC-12 cells with a neurovirulent strain of SV, SVNI, induced cell apoptosis. Overexpression of vFLIP encoded by the HHV-8 or treatment with a caspase-8 inhibitor inhibited cell apoptosis. SVNI induced an increase in the expression of tumor necrosis factor α (TNF-α), and pre-treatment of the cells with an anti-TNF-α blocking antibody or with soluble TNF-α receptor abrogated the apoptotic effect of SVNI. Moreover, TNF-αR1 knockout mice were more resistant to the cytopathic effects of the virus as compared to control animals. Our results indicate that the apoptosis induced by SVNI is mediated by activation of caspase-8, and that TNF-α plays an important role in the apoptotic response. Cell Death and Differentiation (2001) 8, 1224–1231