Intracranial extension of adenoid cystic carcinoma: potential involvement of EphA2 expression and epithelial-mesenchymal transition in tumor metastasis: a case report

BACKGROUND: Adenoid cystic carcinoma is a malignant epithelial tumor derived from salivary glands and tends to invade the surrounding structures including nervous system. We present a case of adenoid cystic carcinoma with intracranial extension and propose a novel molecular mechanism of adenoid cystic carcinoma metastasis. CASE PRESENTATION: A 29-year-old Japanese male presented with left trigeminal nerve disturbance. Neuroimaging revealed a tumor located at the right middle cranial and infratemporal fossa. The tumor was removed via a subtemporal extradural and infratemporal fossa approach and histologically diagnosed as adenoid cystic carcinoma. Radiological and operative findings confirmed a perineural spread of the tumor along the mandibular nerve. Immunohistochemical analyses of molecular consequences in this case were performed for better understanding of the biological processes associated with adenoid cystic carcinoma metastasis. First, the neoplastic cells were not immunoreactive for E-cadherin, an epithelial marker, but for vimentin, a mesenchymal marker, suggesting changes in cell phenotype from epithelial to mesenchymal states. Correspondingly, immunoreactivity of transcriptional factors, such as Slug, Twist, matrix metalloproteinase-2 and -9, which are involved in epithelial–mesenchymal transition, were observed. Second, elevated expression of EphA2 receptor, not ephrin-A1, was notable in the neoplastic cells, suggesting morphological changes reminiscent of epithelial–mesenchymal transition and ligand-independent promotion of tumor cell migration and invasion. CONCLUSIONS: We report a case of adenoid cystic carcinoma with perineural spread and provide the first published evidence that EphA2 expression without ephrin-A1 and epithelial–mesenchymal transition might play important roles in adenoid cystic carcinoma progression

A dehydrated space-weathered skin cloaking the hydrated interior of Ryugu

Without a protective atmosphere, space-exposed surfaces of airless Solar System bodies gradually experience an alteration in composition, structure and optical properties through a collective process called space weathering. The return of samples from near-Earth asteroid (162173) Ryugu by Hayabusa2 provides the first opportunity for laboratory study of space-weathering signatures on the most abundant type of inner solar system body: a C-type asteroid, composed of materials largely unchanged since the formation of the Solar System. Weathered Ryugu grains show areas of surface amorphization and partial melting of phyllosilicates, in which reduction from Fe3+ to Fe2+ and dehydration developed. Space weathering probably contributed to dehydration by dehydroxylation of Ryugu surface phyllosilicates that had already lost interlayer water molecules and to weakening of the 2.7 µm hydroxyl (–OH) band in reflectance spectra. For C-type asteroids in general, this indicates that a weak 2.7 µm band can signify space-weathering-induced surface dehydration, rather than bulk volatile loss

Enhanced anti-tumor effect of zoledronic acid combined with temozolomide against human malignant glioma cell expressing O6-methylguanine DNA methyltransferase.

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance

A diagram of the proposed mechanism showing anti-tumor effect of TMZ in combination with ZOL against malignant glioma cells expressing MGMT.

<p>According to the results of the present study, ZOL inhibits the activity of Ras and the expression of MGMT in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. The molecular mechanism leading to the pathway indicated by the dotted arrow remains to be determined.</p

Apoptosis-mediated cell death of MGMT-expressing malignant glioma cells by co-treatment of ZOL with TMZ.

<p><b>A, Apoptotic Cell Death with TMZ and ZOL combination in human malignant glioma cell lines.</b> Cells were treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. The cytoplasmic histone-associated DNA fragments, which are indicative of ongoing apoptosis, were quantitatively measured by using the photometric enzyme-immunoassay method. Combination of TMZ and ZOL synergistically induced apoptotic cell death in T98G and LN-18. Apoptotic response of LN-229 to TMZ (100 µM) was served as a positive control. Bars, SD. *, P<0.05. <b>B, Apoptotic cell death by TMZ and ZOL combination in human malignant glioma T98G cell line.. </b><i>Left</i> Cells were treated with TMZ (100 µM) and ZOL (40 µM) for 72 hours and stained by Hoechst 33342. Apoptosis was evidenced by chromatin margination in the cell nucleus. Scale bar represents 100 µm. <i>Right</i> Cells were treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours and stained by Propidium Iodide. Cell death was more frequent in the combination treatment with Temozolomide and ZOL. Scale bar represents 100 µm. <b>C, Apoptotic cell death by TMZ and ZOL combination in human malignant glioma T98G cell line.</b> Cells were treated with TMZ (100 µM) and/or ZOL (40 µM) for 72 hours and stained by Annexin-V-FLUOS and Propidium Iodide solution. Phosphatidylserine on the outer leaflet of apoptotic cell-membranes was apparent in the combination treatment with Temozolomide and ZOL. Scale bar represents 100 µm. <b>D, Effect of TMZ and ZOL combination on cleavage of caspase-3 or PARP in human malignant glioma cell lines.. </b><i>Upper</i> Cells were treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. Expression of cleaved caspase-3 and PARP were analyzed by immunoblotting. <i>Lower</i> Relative densitometric units of the cleaved caspase-3 and PARP bands in each cell treated with or without TMZ and/or ZOL. The density of the None band is set arbitrarily at 1.0. Bars, SD. Activities of caspase-3 and PARP were prominent in the combination treatment with Temozolomide and ZOL.</p

Expression of MGMT and chemosensitivity to TMZ in human malignant glioma cell lines.

<p><b>A, Expression of <i>MGMT</i> gene in human malignant glioma cell lines.</b> RNA was isolated from ten malignant glioma cell lines. RT-PCR was done to assess <i>MGMT</i> mRNA expression. Expression of <i>MGMT</i> mRNA was detected in T98G, YH-13 and LN-18. <b>B, Expression of MGMT protein in human malignant glioma cell lines.</b> Protein extracts were prepared from ten malignant glioma cell lines. MGMT expression was analyzed by immunoblotting. β-actin was used as a loading control. Expression of MGMT protein was detected in T98G, YH-13 and LN-18. <b>C, Expression and localization of MGMT protein in human malignant glioma cell lines.</b> Cultured cells were fixed and incubated with anti-MGMT antibody. Reactants were processed with the standard streptavidin-biotin immunoperoxidase method. Diaminobenzidine was used as the final chromogen. Expression and intranuclear localization of MGMT protein was observed in T98G, YH-13and LN-18. <b>D, Growth-inhibitory effect of TMZ in human malignant glioma cell lines.</b> One thousand cells per well were placed onto a 96-well tissue culture plate and incubated overnight. TMZ was added at concentrations of 0–800 µM and incubated for 0–7 days before an MTS assay was performed. T98G and LN18 did not exhibit growth inhibition by TMZ even at a longer exposure.</p

<i>In vitro</i> growth-inhibitory effect of co-treatment of ZOL with TMZ on MGMT-expressing malignant glioma cells.

<p><b>A, Growth-inhibitory effect of ZOL in human malignant glioma T98G cell line.</b> One thousand cells per well were placed onto a 96-well tissue culture plate and incubated overnight. ZOL was added at concentrations of 0–80 µM and incubated for 0–5 days before an MTS assay was performed. T98G did not exhibit substantial growth inhibition by ZOL (40 µM) at 120 hours. <b>B, Growth-inhibitory effect of TMZ and ZOL combination in human malignant glioma cell lines. </b><i>Upper</i> One thousand cells per well were placed onto a 96-well tissue culture plate and incubated overnight. TMZ (100 µM) and/or ZOL (40 µM) was added and incubated for 0–5 days before an MTS assay was performed. This figure is representative of three independent experiments. Bars, SD. *, P<0.05. <i>Lower</i> In the same experiment as <i>Upper</i>, an MTS assay after 120 hours incubation was highlighted. T98G and LN-18 exhibited significant growth inhibition by 100 µM TMZ plus 40 µM ZOL despite modest inhibition by each drug. <b>C, Isobologram analysis of growth-inhibitory effect of TMZ and ZOL combination in human malignant glioma T98G cell line.</b> Cells were treated with TMZ and/or ZOL as a single drug or in combination. Combination effect was analyzed at the 50% growth-inhibition ratio endpoints. The isobologram was based upon the dose-response curves, as determined by MTS assay. The X- and Y-axis indicate the ZOL and TMZ concentrations (µM), respectively. The isobologram plotted at the 50% endpoint was curved downward, suggesting synergistic interaction between TMZ and ZOL in T98G.</p

Effect of co-treatment of ZOL with TMZ on MGMT expression in MGMT-expressing malignant glioma cells.

<p><b>A, Expression of <i>MGMT</i> gene in human malignant glioma cell lines.</b> RNA was isolated from cells treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. <i>MGMT</i> mRNA expression was assessed by RT-PCR. <i>GAPDH</i> was used as a loading control. ZOL down-regulated <i>MGMT</i> mRNA expression in T98G and LN-18. <b>B, Expression of MGMT protein in human malignant glioma cell lines. </b><i>Upper</i> Protein extracts were prepared from cells treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. MGMT expression was analyzed by immunoblotting. β-actin was used as a loading control. <i>Lower</i> Relative densitometric units of the Ras-GTP bands in each cell treated with or without TMZ and/or ZOL. The density of the None band is set arbitrarily at 1.0. Bars, SD. ZOL down-regulated MGMT protein expression in T98G and LN-18.</p

<i>In vivo</i> growth-inhibitory effect of co-treatment of ZOL with TMZ on MGMT-expressing malignant glioma xenografts.

<p><b>A, Anti-tumor effect of TMZ and ZOL combination in human malignant glioma LN-18 cell line.</b> BALB/cA nude mice (female, 5–6 weeks old) bearing LN-18 tumor were separated into four treatment groups; None, TMZ, ZOL and TMZ + ZOL. TMZ (10 mg/kg) was administered i.p. on day 1, 2, 3, 4, 5. ZOL (5 mg/kg) was injected i.p. on day 1, 3, 5 of each week for 2 weeks. At day 35, combination of TMZ and ZOL apparently inhibited tumor growth of LN-18. <b>B, Anti-tumor effect of TMZ and ZOL combination in human malignant glioma LN-18 cell line. </b><i>Left</i> BALB/cA nude mice bearing LN-18 tumor were treated with or without TMZ and/or ZOL. Tumor length and width were measured in situ with digital calipers once a week for 5 weeks (35 days). Tumor volume was calculated once a week for 5 weeks (35 days) according to the following equation: tumor volume (mm³)  = π/6×length× (width)². Points represent mean values ± SE. Combination of TMZ and ZOL significantly inhibited tumor growth of LN-18. <i>Right</i> In the same experiment as <i>Left</i>, body weight was measured once a week for 5 weeks (35 days). Points represent mean values ± SE. Approximate 10% body weight loss occurred in treated mice, whereas body weight was recovered during the observation period.</p

miR-101, miR-548b, miR-554, and miR-1202 are reliable prognosis predictors of the miRNAs associated with cancer immunity in primary central nervous system lymphoma.

MicroRNAs (miRNAs) inhibit protein function by silencing the translation of target mRNAs. However, in primary central nervous system lymphoma (PCNSL), the expression and functions of miRNAs are inadequately known. Here, we examined the expression of 847 miRNAs in 40 PCNSL patients with a microarray and investigated for the miRNA predictors associated with cancer immunity-related genes such as T helper cell type 1/2 (Th-1/Th-2) and regulatory T cell (T-reg) status, and stimulatory and inhibitory checkpoint genes, for prognosis prediction in PCNSL. The aim of this study is to find promising prognosis markers based on the miRNA expression in PCNSL. We detected 334 miRNAs related to 66 cancer immunity-related genes in the microarray profiling. Variable importance measured by the random survival forest analysis and Cox proportional hazards regression model elucidated that 11 miRNAs successfully constitute the survival formulae dividing the Kaplan-Meier curve of the respective PCNSL subgroups. On the other hand, univariate analysis shortlisted 23 miRNAs for overall survival times, with four miRNAs clearly dividing the survival curves-miR-101/548b/554/1202. These miRNAs regulated Th-1/Th-2 status, T-reg cell status, and immune checkpoints. The miRNAs were also associated with gene ontology terms as Ras/MAP-kinase, ubiquitin ligase, PRC2 and acetylation, CDK, and phosphorylation, and several diseases including acquired immunodeficiency syndrome, glioma, and those related to blood and hippocampus with statistical significance. In conclusion, the results demonstrated that the four miRNAs comprising miR-101/548b/554/1202 associated with cancer immunity can be a useful prognostic marker in PCNSL and would help us understand target pathways for PCNSL treatments