Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

Liquid crystalline nanogel targets skin cancer via low-frequency ultrasound treatment.

The potential of low-frequency ultrasound (LFU) combined with nanotechnology-based formulations in improving skin tumors topical treatment was investigated. The impact of solid lipid nanoparticles (SLN) and hydrophilic nanogels as coupling media on LFU-induced skin localized transport regions (LTR) and the penetration of doxorubicin (DOX) in LFU-pretreated skin was evaluated. SLN were prepared by the microemulsion technique and liquid crystalline nanogels using Poloxamer. In vitro, the skin was pretreated with LFU until skin resistivity of âˆ¼1 KΩ.cm2 using the various coupling media followed by evaluation of DOX penetration from DOX-nanogel and SLN-DOX in skin layers. Squamous cell carcinoma (SCC) induced in mice was LFU-treated using the nanogel with the LFU tip placed 5 mm or 10 mm from the tumor surface, followed by DOX-nanogel application. LFU with nanogel coupling achieved larger LTR areas than LFU with SLN coupling. In LFU-pretreated skin, DOX-nanogel significantly improved drug penetration to the viable epidermis, while SLN-DOX hindered drug transport through LTR. In vivo, LFU-nanogel pretreatment with the 10 mm tip distance induced significant tumor inhibition and reduced tumor cell numbers and necrosis. These findings suggest the importance of optimizing nanoparticle-based formulations and LFU parameters for the clinical application of LFU technology in skin tumor treatment.

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

KDM3A/Ets1/MCAM axis promotes growth and metastatic properties in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy of childhood. RMS exists as two major disease subtypes, with oncofusion-positive RMS (FP-RMS) typically carrying a worse prognosis than oncofusion-negative RMS (FN-RMS), in part due to higher propensity for metastasis. Epigenetic mechanisms have recently emerged as critical players in the pathogenesis of pediatric cancers, as well as potential new therapeutic vulnerabilities. Herein, we show that the epigenetic regulator KDM3A, a member of the Jumonji-domain histone demethylase (JHDM) family, is overexpressed, potently promotes colony formation and transendothelial invasion, and activates the expression of genes involved in cell growth, migration and metastasis, in both FN-RMS and FP-RMS. In mechanistic studies, we demonstrate that both RMS subtypes utilize a KDM3A/Ets1/MCAM disease-promoting axis recently discovered in Ewing Sarcoma, another aggressive pediatric cancer of distinct cellular and molecular origin. We further show that KDM3A depletion in FP-RMS cells inhibits both tumor growth and metastasis in vivo, and that RMS cells are highly sensitive to colony growth inhibition by the pan-JHDM inhibitor JIB-04. Together, our studies reveal an important role for the KDM3A/Ets1/MCAM axis in pediatric sarcomas of distinct cellular and molecular ontogeny, and identify new targetable vulnerabilities in RMS.

Synergic effect of OP449 and FTY720 on oral squamous cell carcinoma.

As SET protein is overexpressed and PP2A activity is reduced in oral squamous cell carcinoma (OSCC), this study aimed to assess the effects induced by OP449, a PP2A activator/SET inhibitor, on OSCC cells in vitro, and its potential either isolated or combined with FTY720, a PP2A activator/sphingosine kinase 1 antagonist, as antitumoral therapy in vivo. SET protein was analyzed in cells by immunoblotting and cancer stem cells by aldehyde dehydrogenase 1 assay (ALDH1). The cytotoxicity of OP449 was determined in five OSCC lineages by resazurin assay. Molecular actions of OP449 in SET targets were determined by immunoblotting. The coefficient of drug interaction (CDI) was used to characterize the synergism of OP449 and FTY720. The xenograft HN12 tumor model in nude mice was used to assess the antitumoral effect of OP449 and/or FTY720. HN12 (metastatic) cells showed higher SET and ALDH1 levels, and together with SCC9 cells were selected for molecular analysis. OP449 altered several SET functions/targets, such as histone H3 acetylation and NFkB. A synergism in cytotoxicity was observed when HN12 and SCC9 cells were pre-treated with 2 µM OP449 in combination with 15 µM FTY720 (CDI = 0.27 ± 0.088). Nude mice bearing xenograft HN12 tumors treated with OP449 and FTY720 showed reduced tumor mass. Moreover, NFkB was reduced in tumors after treatment. OP449 targets several SET functions, not only PP2A inhibition. Besides, OP449 plus FTY720 has a synergistic antitumoral effect on OSCC. Our results suggest new combined therapies and highlight SET and NFκB signaling as targets for OSCC therapy.

SET protein modulates H4 histone methylation status and regulates miR-137 level in oral squamous cell carcinoma.

Aim: Histone acetylation and methylation control gene expression. We investigated the impact of SET knockdown on histone methylation status and the consequences for the miRNAs levels in oral squamous cell carcinoma (OSCC). Methods: OSCC cells with and without SET knockdown were analyzed by quantitative real-time PCR to determine miRNA levels, and by immunoreactions to histone modifications. Results: The knockdown of SET increased the levels of histone H4K20me2 and miR-137. Still, SET protein binds to the miR-137 promoter region. The transfection of miR-137 mimic reduced the KI67 and Rb proteins and proliferation of OSCC cells. Conclusion: Our results show for the first time a relationship between SET and histone methylation associated with the control of miRNA expression and KI67 and Rb as targets of miR-137 in OSCC.

Anti-cancer activity of a new dihydropyridine derivative, VdiE-2N, in head and neck squamous cell carcinoma.

This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.

Relevance of CCL3/CCR5 axis in oral carcinogenesis.

The chemokine CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions. CCL3 might stimulate cancer progression by promoting leukocyte accumulation, angiogenesis and tumour growth. The expression of CCL3 and its receptors CCR1 and CCR5 was demonstrated in oral squamous cell carcinoma (OSCC), but their role was not defined. Here, the functions of CCL3 were assessed using a model of chemically induced tongue carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). Lineages of OSCC were used to analyse the effects of CCL3 in vitro. The 4NQO-induced lesions exhibited increased expression of CCL3, CCR1 and CCR5. CCL3-/- and CCR5-/- mice presented reduced incidence of tongue tumours compared to wild-type (WT) and CCR1-/- mice. Consistently, attenuated cytomorphological atypia and reduced cell proliferation were observed in lesions of CCL3-/- and CCR5-/- mice. OSCC from CCL3-/- mice exhibited lower infiltration of eosinophils and reduced expression of Egf, Fgf1, Tgf-ß1, Vegfa, Vegfb, Itga-4, Vtn, Mmp-1a, Mmp-2 and Mmp-9 than WT mice. In vitro, CCL3 induced invasion and production of CCL5, IL-6, MMP -2, -8, -9. Blockage of CCL3 in vitro using α-CCL3 or Evasin-1 (a CCL3-binding protein) impaired tumour cell invasion. In conclusion, CCL3/CCR5 axis has pro-tumourigenic effects in oral carcinogenesis. The induction of inflammatory and angiogenic pathways and eosinophils recruitment appear to be the underlying mechanism explaining these effects. These data reveal potential protective effects of CCL3 blockade in oral cancer.

ANXA1Ac2-26 peptide, a possible therapeutic approach in inflammatory ocular diseases.

The eye is immunologically privileged when inflammatory responses are suppressed. One component responsible for the suppression of inflammatory responses is the blood retinal barrier, which comprises the retinal pigment epithelium. The destruction of this barrier initiates inflammation, which can affect any part of the eye. Therefore, inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1) protein acts as a modulator of inflammation. In this study we aimed to improve the knowledge of this area by investigating how a peptide of the ANXA1 protein (ANXA1Ac2-26) modulates the morphology, proliferation, migration and expression of genes and proteins in human retinal pigment epithelium cells (ARPE-19). Determining how signaling pathways (NF-κB and UBC) are modulated by the ANXA1Ac2-26 peptide could be important for understanding the inflammatory process. ARPE-19 cells were activated by bacterial lipopolysaccharide endotoxin (LPS) and treated with ANXA1Ac2-26 peptide, in a concentration of 1.7µM and 33.8µM. We observed that the LPS activation diminished the levels of endogenous ANXA1 after 2h and 24h and ANXA1Ac2-26 peptide decreased the proliferation and re-establishes the migration of ARPE-19 cells. After using a hybridization approach, 80 differentially expressed genes were found. Five of these genes were selected (LRAT, CTGF, MAP1B, ALDH1A3 and SETD7) and all were down-regulated after treatment with the peptide. The genes CTGF and LRAT would be considered as potential molecular markers of ophthalmologic inflammation. The expression of pro-inflammatory cytokines was also decreased after the treatment, indicating the efficiency of the anti-inflammatory peptide at high concentrations, since the reduction in the levels of these mediators were observed after the treatment with ANXA1Ac2-26 peptide at 33.8µM. Our results suggest that the retinal pigment epithelial cells are a potential target of the ANXA1 protein and point to possible applications of the ANXA1Ac2-26 peptide as an innovative therapy for the treatment of ocular inflammation.

Topical Skin Cancer Therapy Using Doxorubicin-Loaded Cationic Lipid Nanoparticles and lontophoresis.

The topical administration of chemotherapeutics is a promising approach for the treatment of skin cancer; however, different pharmaceutical strategies are required to allow large amounts of drug to penetrate tumors. This work examined the potential of the anodic iontophoresis of doxorubicin-loaded cationic solid lipid nanoparticles (DOX-SLN) to increase the distribution and tumor penetration of DOX. A double-labeled cationic DOX-SLN composed of the lipids stearic acid and monoolein and a new BODIPY dye was prepared and characterized. The skin distribution and penetration of DOX were evaluated in vitro using confocal microscopy and vertical diffusion cells, respectively. The antitumor potential was evaluated in vivo through the anodic iontophoresis of DOX-SLN in squamous cell carcinoma induced in nude BALB/c mice. The encapsulation of DOX drastically altered the DOX partition coefficient and increased the distribution of DOX in the lipid matrix of the stratum corneum (SC). The association with iontophoresis created high-concentration drug reservoir zones in the follicles of the skin. Although the iontophoresis of a DOX solution increased the penetration of DOX in the viable epidermis by approximately 4-fold, the iontophoresis of cationic DOX-SLN increased the DOX penetration by approximately 50-fold. In vivo, the DOX-SLN iontophoretic treatment was effective in inhibiting tumor cell survival and tumor growth and was accompanied by an increase in keratinization and consequent cell death. These results indicate a strong and synergic effect of iontophoresis with DOX-SLN and provide a potential strategy for the treatment of skin cancer.

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis.

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.

Isolation and characterization of myofibroblast cell lines from oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) invasion is followed by several stromal events such as inflammatory and immune cell infiltration, neo-vascularization, fibroblast activation and occasionally myofibroblast emergence. Our previous studies demonstrated that myofibroblasts in the stroma of OSCC are associated with a more aggressive behavior, leading to shorter patient overall survival. Therefore, we evaluated whether OSCC-associated myofibroblasts have different characteristics compared to OSCC-associated fibroblasts. OSCC myofibroblast cell lines were isolated, cultured and characterized on the basis of the expression of specific isoform α of smooth muscle actin (α-SMA) and of the excessive production of type I collagen. To assess the proliferative potential of the cell lines, growth curves were constructed, whereas the production and activity of matrix metalloproteinases (MMP) were analyzed by ELISA and enzymography, respectively. Myofibroblast clones were positive for α-SMA and vimentin, and negative for pan-cytokeratin and CD34. In long time cultures, western blotting, flow cytometry and ELISA analysis revealed constant α-SMA expression and elevated production of type I collagen. There were no differences on proliferative potential between fibroblast and myofibroblast clones, but myofibroblast cells secreted significantly higher levels of MMP-1, -2, -9 and -13. Furthermore, MMP-2 gelatinolytic activity was significantly higher in myofibroblast clones. The results of this study suggest that myofibroblasts may contribute to OSCC invasion through elevation of MMP synthesis.

Cyclosporin A-induced gingival overgrowth is not associated with myofibroblast transdifferentiation.

Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-beta1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-beta1 expression by NG fibroblasts, it lacked to induce expression and production of isoform alpha of smooth muscle actin (alpha-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression.

Cyclosporin A-induced gingival overgrowth is not associated with myofibroblast transdifferentiation

Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-â1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-â1 expression by NG fibroblasts, it lacked to induce expression and production of isoform á of smooth muscle actin (á-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-â1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression.