Antibody therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule 4 (CTLA-4) and programmed cell death 1 (PD-1) has demonstrated marked tumor regression in clinical ...trials. MicroRNAs (miRNAs) can modulate multiple gene transcripts including possibly more than one immune checkpoint and could be exploited as immune therapeutics.
Using online miRNA targeting prediction algorithms, we searched for miRNAs that were predicted to target both PD-1 and CTLA-4. MiR-138 emerged as a leading candidate. The effects of miR-138 on CTLA-4 and PD-1 expression and function in T cells were determined and the therapeutic effect of intravenous administration of miR-138 was investigated in both immune-competent and -incompetent murine models of GL261 glioma.
Target binding algorithms predicted that miR-138 could bind the 3' untranslated regions of CTLA-4 and PD-1, which was confirmed with luciferase expression assays. Transfection of human CD4+ T cells with miR-138 suppressed expression of CTLA-4, PD-1, and Forkhead box protein 3 (FoxP3) in transfected human CD4+ T cells. In vivo miR-138 treatment of GL261 gliomas in immune-competent mice demonstrated marked tumor regression, a 43% increase in median survival time (P = .011), and an associated decrease in intratumoral FoxP3+ regulatory T cells, CTLA-4, and PD-1 expression. This treatment effect was lost in nude immune-incompetent mice and with depletion of CD4+ or CD8+ T cells, and miR-138 had no suppressive effect on glioma cells when treated directly at physiological in vivo doses.
MiR-138 exerts anti-glioma efficacy by targeting immune checkpoints which may have rapid translational potential as a novel immunotherapeutic agent.
Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we ...performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background & Aims MicroRNAs (miRNAs) can promote or inhibit tumor growth and are therefore being developed as targets for cancer therapies. They are diverse not only in the messenger RNAs (mRNA) they ...target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p , that can bind different mRNAs. We analyzed the expression, functions, and mechanisms of miR-28-5p and miR-28-3p in colorectal cancer (CRC) cells. Methods We measured levels of miR-28-5p and miR-28-3p expression in 108 CRC and 49 normal colorectal samples (47 paired) by reverse transcription, quantitative real-time polymerase chain reaction. The roles of miR-28 in CRC development were studied using cultured HCT116, RKO, and SW480 cells and tumor xenograft analyses in immunodeficient mice; their mRNA targets were also investigated. Results miR-28-5p and miR-28-3p were down-regulated in CRC samples compared with normal colon samples. Overexpression of miRNAs in CRC cells had different effects and the miRNAs interacted with different mRNAs: miR-28-5p altered expression of CCND1 and HOXB3 , whereas miR-28-3p bound NM23-H1 . Overexpression of miR-28-5p reduced CRC cell proliferation, migration, and invasion in vitro, whereas miR-28-3p increased CRC cell migration and invasion in vitro. CRC cells overexpressing miR-28 developed tumors more slowly in mice compared with control cells, but miR-28 promoted tumor metastasis in mice. Conclusion miR-28-5p and miR-28-3p are transcribed from the same RNA hairpin and are down-regulated in CRC cells. Overexpression of each has different effects on CRC cell proliferation and migration. Such information has a direct application for the design of miRNA gene therapy trials.
Circulating miRNAs are known to play important roles in intercellular communication. However, the effects of exosomal miRNAs on cells are not fully understood.
To investigate the role of exosomal ...miR-1246 in ovarian cancer (OC) microenvironment, we performed RPPA as well as many other in vitro functional assays in ovarian cancer cells (sensitive; HeyA8, Skov3ip1, A2780 and chemoresistant; HeyA8-MDR, Skov3-TR, A2780-CP20). Therapeutic effect of miR-1246 inhibitor treatment was tested in OC animal model. We showed the effect of OC exosomal miR-1246 uptake on macrophages by co-culture experiments.
Substantial expression of oncogenic miR-1246 OC exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFβ receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages.
Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemoresistance and tumor progression through exosomal miR-1246 in OC patients.
EGFL6, a member of the EGF-like superfamily, plays an important role during embryonic development and has been implicated in promotion of tumor angiogenesis without affecting wound healing. There is ...very little known about the function of EGFL6 in cancer cells. Here, we investigated whether EGFL6 plays a direct role in cancer cells in addition to the promotion of tumor angiogenesis. Our study showed that EGFL6 promoted epithelial-mesenchymal transition (EMT) and stemness of breast cancer cells and increased cell migration and invasion in cell culture studies. We also found that EGFL6 reduced apoptotic signaling in cancer cells and promoted tumor growth in vivo. Importantly, expression of EGFL6 in cancer cells and tumor endothelial cells not only increased tumor angiogenesis but also promoted migration of cancer cells. Such dual engagement of cancer and stromal cells suggests crosstalk mediated by EGFL6 in the tumor microenvironment. Blockade of EGFL6 using our novel anti-EGFL6 monoclonal antibody significantly reduced cancer cell migration, tumor angiogenesis, and tumor growth in mouse xenograft tumor models. Silencing EGFL6 mRNA by shRNA transfection of cancer cells also significantly reduced cancer cell migration, tumor angiogenesis, and tumor growth in mouse xenograft tumor models. Taken together, the results of this study indicate that targeting EGFL6 is a unique strategy for inhibiting both cancer cell metastasis and tumor angiogenesis.
We previously found focal adhesion kinase (FAK) inhibition sensitizes ovarian cancer to taxanes; however, the mechanisms are not well understood.
We characterized the biologic response of ...taxane-resistant and taxane-sensitive ovarian cancer models to a novel FAK inhibitor (VS-6063). We used reverse-phase protein arrays (RPPA) to identify novel downstream targets in taxane-resistant cell lines. Furthermore, we correlated clinical and pathological data with nuclear and cytoplasmic expression of FAK and YB-1 in 105 ovarian cancer samples. Statistical tests were two-sided, and P values were calculated with Student t test or Fisher exact test.
We found that VS-6063 inhibited FAK phosphorylation at the Tyr397 site in a time- and dose-dependent manner. The combination of VS-6063 and paclitaxel markedly decreased proliferation and increased apoptosis, which resulted in 92.7% to 97.9% reductions in tumor weight. RPPA data showed that VS-6063 reduced levels of AKT and YB-1 in taxane-resistant cell lines. FAK inhibition enhanced chemosensitivity in taxane-resistant cells by decreasing YB-1 phosphorylation and subsequently CD44 in an AKT-dependent manner. In human ovarian cancer samples, nuclear FAK expression was associated with increased nuclear YB-1 expression (χ²) = 37.7; P < .001). Coexpression of nuclear FAK and YB-1 was associated with statistically significantly worse median overall survival (24.9 vs 67.3 months; hazard ratio = 2.64; 95% confidence interval = 1.38 to 5.05; P = .006).
We have identified a novel pathway whereby FAK inhibition with VS-6063 overcomes YB-1-mediated paclitaxel resistance by an AKT-dependent pathway. These findings have implications for clinical trials aimed at targeting FAK.
A series of new pyrrole derivatives were designed as chemical analogs of the 1,4-dihydropyridines drugs in order to develop future new calcium channel blockers. The new tri- and tetra-substituted
...-arylpyrroles were synthesized by the one-pot reaction of 1-methyl-3-cyanomethyl benzimidazolium bromide with substituted alkynes having at least one electron-withdrawing substituent, in 1,2-epoxybutane, acting both as the solvent and reagent to generate the corresponding benzimidazolium N3-ylide. The structural characterization of the new substituted pyrroles was based on IR, NMR spectroscopy as well as on single crystal X-ray analysis. The toxicity of the new compounds was assessed on the plant cell using
L. species and on the animal cell using
Kellogg and
Straus crustaceans. The compounds showed minimal phytotoxicity on
rootlets and virtually no acute toxicity on
nauplii, while on
, it induced moderate to high toxicity, similar to nifedipine. Our research indicates that the newly synthetized pyrrole derivatives are promising molecules with biological activity and low acute toxicity.
Non-coding RNAs and especially microRNAs have been discovered to act as master regulators of cancer initiation and progression. The aim of our study was to discover and characterize the function of ...yet functionally uncharacterized microRNAs in human breast carcinogenesis.
In an unbiased approach, we utilized an established model system for breast cancer (BC) stem cell formation ("mammosphere assay") to identify whole miRNome alterations in breast carcinogenesis. Clinical samples of BC patients were used to evaluate the human relevance of the newly identified miRNA candidates. One promising candidate, miR-1287-5p, was further explored on its impact on several hallmarks of cancer. The molecular mode of action was characterized by whole transcriptome analysis, in silico prediction tools, miRNA-interaction assays, pheno-copy assays, and drug sensitivity assays.
Among several other microRNAs, miR-1287-5p was significantly downregulated in mammospheres and human BC tissue compared to normal breast tissue (p < 0.0001). Low expression levels were significantly associated with poor prognosis in BC patients. MiR-1287-5p significantly decreased cellular growth, cells in S phase of cell cycle, anchorage-independent growth, and tumor formation in vivo. In addition, we identified PIK3CB as a direct molecular interactor of miR-1287-5p and a novel prognostic factor in BC. Finally, PI3Kinase pathway chemical inhibitors combined with miR-1287-5p mimic increased the pharmacological growth inhibitory potential in triple negative BC cells.
Our data identified for the first time the involvement of miR-1287-5p in human BC and suggest a potential for therapeutic interventions in difficult to treat triple negative BC.
New pyrrolo1,2-
pyridazines were synthesized by 3 + 2 cycloaddition reaction between mesoionic oxazolo-pyridazinones and methyl/ethyl propiolate. The mesoionic compounds were generated in situ by ...action of acetic anhydride on 3(2
)pyridazinone acids obtained from corresponding esters by alkaline hydrolysis followed by acidification. The structures of the compounds were confirmed by elemental analyses and IR,
H-NMR,
C-NMR, and X-ray diffraction data. The regioselectivity of cycloaddition was evidenced by NMR spectroscopy and confirmed by X-ray analysis. The compounds were evaluated for their cytotoxicity on plant cells (
L.) and crustacean animal cells (
Kellogg and
Straus). The results indicated that the tested compounds exhibited low toxicity on the plant cell (IC
values higher than 200 µM), while on Artemia nauplii no lethality was observed. Daphnia magna assay showed that pyrrolo1,2-
pyridazines
and
could exhibit toxic effects, whereas, for the other compounds, toxicity was low to moderate. Also, the cytotoxic effects of the compounds were tested on three human adenocarcinoma-derived adherent cell lines (colon LoVo, ovary SK-OV-3, breast MCF-7). The in vitro compound-mediated cytotoxicity assays, performed by the MTS technique, demonstrated dose- and time-dependent cytotoxic activity for several compounds, the highest anti-tumor activity being observed for
,
and
, especially against colon cancer cells.
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