TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity

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The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

Granzyme B-induced cell death involves induction of p53 tumor suppressor gene and its activation in tumor target cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.     

miR-505 acts as a tumor suppressor in gastric cancer progression through targeting HMGB1

Gastric cancer (GC) is a frequent type of malignant tumor worldwide. GC metastasis results in the majority of clinical treatment failures. MicroRNAs (miRNA) are identified to exhibit crucial roles in GC. Our current study aimed to explore the biological roles of miR-505 in GC progression. It was observed that miR-505 was robustly decreased in GC cells compared with human normal gastric epithelial GES-1 cells. Overexpression of miR-505 was able to repress GC progression in AGS and BGC-823 cells. In addition, high-mobility group box 1 (HMGB1) has been identified as a crucial oncogene in several cancer types. By carrying out bioinformatics analysis, HMGB1 was predicted as a direct target of miR-505. Meanwhile, HMGB1 was found to be significantly increased in GC cells and it was confirmed in our study that miR-505 can directly target HMGB1 in vitro. miR-505 mimics can inhibit HMGB1 messenger RNA and protein expression dramatically. Subsequently, knockdown of HMGB1 can inhibit GC cell proliferation, colony formation, and induce cell apoptosis. Furthermore, HMGB1 silence suppressed GC cell migration and invasion greatly in vitro. Finally, it was validated that miR-505 can inhibit GC progression by targeting HMGB1 in vivo. Taken these together, it was indicated that miR-505/HMGB1 axis was involved in the development of GC. miR-505 can serve as a potential prognostic indicator in GC therapy.     

Degradation of the tumor suppressor PML by Pin1 contributes to the cancer phenotype of breast cancer MDA-MB-231 cells

Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.     

Specific binding of a factor inducing differentiation to mouse myeloid leukemic M1 cells

A factor inducing differentiation of mouse myeloid leukemic M1 cells into macrophages (differentiation-inducing factor, D-factor), which was purified to homogeneity from conditioned medium of mouse Ehrlich ascites tumor cells, could be iodinated without detectable loss of biological activity. The binding of 125I-D-factor to M1 cells was specific; the binding was inhibited competitively by D-factor derived from Ehrlich cells and mouse fibroblast L929 cells, but not by other growth factors or D-factor derived from differentiated M1 cells. The latter differs from D-factor of Ehrlich cells and L929 cells in antigenicity and molecular weight. At 21 degrees C, the binding was saturated at 370 pM 125I-D-factor. M1 cells showed a high affinity for 125I-D-factor (dissociation constant, 1.0 X 10(-10) M) and expressed a small number of binding sites (170 per cell). Specific binding of 125I-D-factor was observed only to several clones derived from M1 cells, including those sensitive and resistant to induction of differentiation by D-factor.     

Rgs2 mediates pro-angiogenic function of myeloid derived suppressor cells in the tumor microenvironment via upregulation of MCP-1

Background

Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization.

Methodology/Principal Findings

In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2−/− tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1.

Conclusion

Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production.     

DNMT1 as a molecular target in a multimodality-resistant phenotype in tumor cells

We have previously shown that hydrogen peroxide-resistant permanent (OC-14) cells are resistant to the cytotoxicity of several exogenous oxidative and anticancer agents including H(2)O(2), etoposide, and cisplatin; and we refer to this process as an oxidative multimodality-resistant phenotype (MMRP). Furthermore, OC-14 cells contain increased activator protein 1 activity, and inhibition of activator protein 1 reversed the MMRP. In this study, we show that permanent Rat-1 cell lines genetically altered to overexpress c-Fos also displayed a similar MMRP to H(2)O(2), etoposide, and cisplatin as OC-14 cells. Gene expression analysis of the OC-14 cells and c-Fos-overexpressing cells showed increased DNMT1 expression. Where OC-14 and c-Fos-overexpressing cells were exposed to 5-aza-2'-deoxycytidine, which inhibits DNMT activity, a significant but incomplete reversal of the MMRP was observed. Thus, it seems logical to suggest that DNMT1 might be at least one target in the MMRP. Rat-1 cells genetically altered to overexpress DNMT1 were also shown to be resistant to the cytotoxicity of H(2)O(2), etoposide, and cisplatin. Finally, somatic HCT116 knockout cells that do not express either DNMT1 (DNMT1(-/-)) or DNMT3B (DNMT3B(-/-)) were shown to be more sensitive to the cytotoxicity of H(2)O(2), etoposide, and cisplatin compared with control HCT116 cells. This work is the first example of a role for the epigenome in tumor cell resistance to the cytotoxicity of exogenous oxidative (H(2)O(2)) or systemic (etoposide and cisplatin) agents and highlights a potential role for DNMT1 as a potential molecular target in cancer therapy.     

Hyperphosphorylation of the BARD1 tumor suppressor in mitotic cells

Although the BRCA1 tumor suppressor has been implicated in a number of cellular processes, it plays an especially important role in the DNA damage response as a regulator of cell cycle checkpoints and DNA repair pathways. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein, and many of its biological functions are mediated by the BRCA1-BARD1 complex. Here, we show that BARD1 is phosphorylated in a cell cycle-dependent manner and that the hyperphosphorylated forms of BARD1 predominate during M phase. By mobility shift analysis and mass spectrometry, we have identified seven sites of mitotic phosphorylation within BARD1. All sites exist within either an SP or TP sequence, and two sites resemble the consensus motif recognized by cyclin-dependent kinases. To examine the functional consequences of BARD1 phosphorylation, we used a gene targeting knock-in approach to generate isogenic cell lines that express either wild-type or mutant forms of the BARD1 polypeptide. Analysis of these lines in clonogenic survival assays revealed that cells bearing phosphorylation site mutations are hypersensitive to mitomycin C, a genotoxic agent that induces interstrand DNA cross-links. These results implicate BARD1 phosphorylation in the cellular response to DNA damage.     

MicroRNA-215 enhances invasion and migration by targeting retinoblastoma tumor suppressor gene 1 in high-grade glioma

Objective

To elucidate the molecular mechanism of microRNA-215 (miR-215) in the migration and invasion of high grade glioma.

Results

42 Patients were analysed for clinicopathological characteristics. qRT-PCR showed that miR-215 was up-regulated in glioma tissues compared with non-neoplastic brain tissues (P < 0.05). The up-regulated miR-215 was closely associated with high grade glioma (P < 0.01) and poor overall survival (P < 0.01). Transwell assay showed that re-expression of miR-215 enhanced migration and invasion of glioma cells. miR-215 also down-regulated retinoblastoma tumor suppressor gene 1 (RB1) expression by targeting its 3′-UTR. Reversely, re-expression of RB1 inhibited partial effect of miR-215 on migration and invasion in vitro.

Conclusions

Re-expression of miR-215 promoted cell migration and invasion of glioma by targeting RB1. miR-215 can thus be used as a biomarker for tumor progression and prognosis in human high grade glioma.

     

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint^−/−) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1^−/− mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1^−/− mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1^−/− mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1^−/− mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.     

MicroRNA-487a-3p functions as a new tumor suppressor in prostate cancer by targeting CCND1

Prostate cancer (PCa) is one of the major health problems of the aging male. The roles of dysregulated microRNAs in PCa remain unclear. In this study, we mined the public published data and found that miR-487a-3p was significantly downregulated in 38 pairs of clinical prostate tumor tissues compared with the normal tissues. We further verified this result by in situ hybridization on tissue chip and quantitative real-time polymerase chain reaction (qRT-PCR) in PCa/normal cells. miR-487a-3p targeting of cyclin D1 (CCND1) was identified using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, cell cycle, migration, and invasion were assessed by cell counting kit-8, flow cytometry analysis and transwell assay. We discovered that overexpression of miR-487a-3p suppressed PCa cell growth, migration, invasion by directly targeting CCND1. Knockdown of CCND1 in PCa cells showed similar results. Meanwhile, the expression level of CCND1 was significantly upregulated in the PCa tissues and cell lines, which presented negative correlation with the expression of miR-487a-3p. More important, we demonstrated significantly reduced growth of xenograft tumors of stable miR-487a-3p-overexpressed human PCa cells in nude mice. Taken together, for the first time, our results revealed that miR-487a-3p as a tumor suppressor of PCa could target CCND1. Our finding might reveal miR-487a-3p could be potentially contributed to the pathogenesis and a clinical biomarker or the new potential therapeutic target of PCa.     

microRNA-34a is tumor suppressive in brain tumors and glioma stem cells

We recently found that microRNA-34a (miR-34a) is downregulated in human glioma tumors as compared to normal brain, and that miR-34a levels in mutant-p53 gliomas were lower than in wildtype-p53 tumors. We showed that miR-34a expression in glioma and medulloblastoma cells inhibits cell proliferation, G1/S cell cycle progression, cell survival, cell migration and cell invasion, but that miR-34a expression in human astrocytes does not affect cell survival and cell cycle. We uncovered the oncogenes c-Met, Notch-1 and Notch-2 as direct targets of miR-34a that are inhibited by miR-34a transfection. We found that c-Met levels in human glioma specimens inversely correlate with miR-34a levels. We showed that c-Met and Notch partially mediate the inhibitory effects of miR-34a on cell proliferation and cell death. We also found that mir-34a expression inhibits in vivo glioma xenograft growth. We concluded that miR-34a is a potential tumor suppressor in brain tumors that acts by targeting multiple oncogenes. In this extra view, we briefly review and discuss the implications of these findings and present new data on the effects of miR-34a in glioma stem cells. The new data show that miR-34a expression inhibits various malignancy endpoints in glioma stem cells. Importantly, they also show for the first time that miR-34a expression induces glioma stem cell differentiation. Altogether, the data suggest that miR-34a is a tumor suppressor and a potential potent therapeutic agent that acts by targeting multiple oncogenic pathways in brain tumors and by inducing the differentiation of cancer stem cells.     

Proinflammatory-activated glioma cells induce a switch in microglial polarization and activation status,from a predominant M2b phenotype to a mixture of M1 and M2a/B polarized cells

Malignant gliomas are primary brain tumors characterized by morphological and genetic complexities, as well as diffuse infiltration into normal brain parenchyma. Within gliomas, microglia/macrophages represent the largest tumor-infiltrating cell population, contributing by at least one-third to the total tumor mass. Bi-directional interactions between glioma cells and microglia may therefore play an important role on tumor growth and biology. In the present study, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. We found that microglial cells undergo a different pattern of activation depending on the stimulus; in the presence of activated glioma-derived factors, i.e. a condition mimicking the late stage of pathology, microglia presents as a mixture of polarization phenotypes (M1 and M2a/b), with up-regulation of iNOS (inducible nitric oxide synthase), ARG (arginase) and IL (interleukine)-10. At variance, microglia exposed to basal glioma-derived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2b polarization status and up-regulation of IL-10 only. As far as viability and cell proliferation are concerned, both LI-CM [LPS (lipopolysaccharide)–IFNγ (interferon γ) conditioned media] and C-CM (control-conditioned media) induce similar effects on microglial morphology. Finally, in human glioma tissue obtained from surgical resection of patients with IV grade glioblastoma, we detected a significant amount of CD68 positive cells, which is a marker of macrophage/microglial phagocytic activity, suggesting that in vitro findings presented here might have a relevance in the human pathology as well.     

The tumor suppressor RASSF1A prevents dephosphorylation of the mammalian STE20-like kinases MST1 and MST2

The RASSF1A tumor suppressor protein interacts with the pro-apoptotic mammalian STE20-like kinases MST1 and MST2 and induces their autophosphorylation and activation, but the mechanism of how RASSF1A activates MST1/2 is unclear. Okadaic acid treatment and PP2A knockdown promoted MST1/2 phosphorylation. Data from dephosphorylation assays and reduced activation of MST1/2 seen after RASSF1A depletion suggest that dephosphorylation of MST1/2 on Thr-183 and Thr-180 by PP2A is prevented by RASSF1A, shifting the balance of MST1/2 to the activated autophosphorylated form. In addition to preventing dephosphorylation, RASSF1A also stabilized the MST2 protein. Through binding to MST1/2, RASSF1A supports maintenance of MST1/2 phosphorylation, promoting an active state of the MST kinases and favoring induction of apoptosis. This is one of the first examples of a tumor suppressor acting as an inhibitor of a specific dephosphorylation pathway.