Treatment of nasopharyngeal carcinoma cells with the histone-deacetylase inhibitor abexinostat: cooperative effects with cis-platin and radiotherapy on patient-derived xenografts.
ABSTRACT: EBV-related nasopharyngeal carcinomas (NPCs) still raise serious therapeutic problems. The therapeutic potential of the histone-deacetylase (HDAC) inhibitor Abexinostat was investigated using 5 preclinical NPC models including 2 patient-derived xenografts (C15 and C17). The cytotoxicity of Abexinostat used either alone or in combination with cis-platin or irradiation was assessed in vitro by MTT and clonogenic assays using 2 EBV-negative (CNE1 and HONE1) and 3 EBV-positive NPC models (C15, C17 and C666-1). Subsequently, the 3 EBV-positive models were used under the form of xenografts to assess the impact of systemic treatments by Abexinostat or combinations of Abexinostat with cis-platin or irradiation. Several cell proteins known to be affected by HDAC inhibitors and the small viral non-coding RNA EBER1 were investigated in the treated tumors. Synergistic cytotoxic effects of Abexinostat combined with cis-platin or irradiation were demonstrated in vitro for each NPC model. When using xenografts, Abexinostat by itself (12.5 mg/kg, BID, 4 days a week for 3 weeks) had significant anti-tumor effects against C17. Cooperative effects with cis-platin (2 mg/kg, IP, at days 3, 10 and 17) and irradiation (1 Gy) were observed for the C15 and C17 xenografts. Simultaneously two types of biological alterations were induced in the tumor tissue, especially in the C17 model: a depletion of the DNA-repair protein RAD51 and a stronger in situ detection of the small viral RNA EBER1. Overall, these results support implementation of phase I/II clinical trials of Abexinostat for the treatment of NPC. A depletion of RAD51 is likely to contribute to the cooperation of Abexinostat with DNA damaging agents. Reduction of RAD51 combined to enhanced detection of EBER 1 might be helpful for early assessment of tumor response.
Project description:The aim of this experiment was to investigate the gene expression profile of two xenografted nasopharyngeal carcinoma tumors - C15 and C17 which are international reference tools for biological investigations of this type of disease. C15 and C17 transcriptome was profiled by comparison with human untransformed diploid fibroblasts, MRC5. These fibroblasts are very sensitive to contact inhibition and were collected after 3 days of confluence, therefore in a quiescent state. We acknowledge that malignant nasopharyngeal carcinoma cells and MRC5 cells do not belong to the same lineage (epithelial cells versus fibroblasts). However this combination was expected to highlight genes involved in proliferation of NPC cells. Competitive hybridisation of cDNA for each pair C15 and MRC5, C17 and MRC5 was performed on Agilent microarrays containing 22 000 60mer oligonucleotides related to 16 000 human transcripts. The analysis was focused mainly on genes overexpressed in both C15 and C17 by comparison with MRC5
Project description:Despite prominent role of radiotherapy in lung cancer management, there is an urgent need for strategies increasing therapeutic efficacy. Reversible epigenetic changes are promising targets for combination strategies using HDAC inhibitors (HDACi). Here we evaluated on two NSCLC cell lines, the antitumor effect of abexinostat, a novel pan HDACi combined with irradiation in vitro in normoxia and hypoxia, by clonogenic assays, demonstrating that abexinostat enhances radiosensitivity in a time dependent way with mean SER10 between 1.6 and 2.5 for A549 and H460. We found, by immunofluorescence staining, flow cytometry assays and western blotting, in abexinostat treated cells, increasing radio-induced caspase dependent apoptosis and persistent DNA double-strand breaks associated with decreased DNA damage signalling and repair. Interestingly, we demonstrated on nude mice xenografts that abexinostat potentiates tumor growth delay in combined modality treatments associating not only abexinostat and irradiation but also when adding cisplatin. Altogether, our data demonstrate in vitro and in vivo anti-tumor effect potentiation by abexinostat combined with irradiation in NSCLC. Moreover, our work suggests for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.
Project description:To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC), methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5'-end of the tetratricopeptide repeat domain 40 (TTC40) gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5'-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5'-TTC40 gene hypermethylation. In conclusion, we found that the 5'-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5'-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.
Project description:Epstein-Barr virus (EBV) was the first human virus found to encode microRNAs (miRNAs), but the function of these miRNAs has been obscure. Nasopharyngeal carcinoma (NPC) is associated with EBV infection, and the EBV-encoded LMP1 is believed to be a key factor in NPC development. However, detection of LMP1 protein in NPC is variable. Here, we report that EBV-encoded BART miRNAs target the 3' UTR of the LMP1 gene and negatively regulate LMP1 protein expression. These miRNAs also modulate LMP1-induced NF-kappaB signaling and alleviate the cisplatin sensitivity of LMP1-expressing NPC cells. Consistent with a previous study on the NPC C666-1 cell line and C15 xenograft, we found abundant expression of BART miRNAs in NPC tissues. Furthermore, DNA sequencing revealed that the 3' UTR of LMP1 is highly conserved in NPC-derived EBV isolates. The data provide insight into the discrepancy between LMP1 transcript and protein detection in NPC and highlight the role of the EBV miRNAs in regulating LMP1 downstream signaling to promote cancer development.
Project description:Undifferentiated nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr virus (EBV) as oncogenic driver. NPC is often diagnosed late due to initial vague complaints and obscured location. Prior studies suggest that measurement of EBV DNA load and RNA transcripts in nasopharyngeal (NP) brushings is useful for minimally invasive NPC diagnosis. However, whether these EBV markers relate to local virus replication or reflect tumor origin remains to be demonstrated. To resolve this, we analysed EBV-DNA characteristics and quantified latent and lytic viral RNA transcripts in NP brushings and matching frozen NP-biopsy specimens from patients suspected of having NPC. We observed non-fragmented and Cp-promotor methylated EBV-DNA in both NP brushings and biopsies suggestive of tumor origin. Using quantitative RT-PCR we determined expression levels of 7 critical latent (EBER1, Qp-EBNA1, EBNA2, BART, LMP1, LMP2, BARF1) and 5 lytic (Zta, Rta, TK, PK and VCA-p18) RNA transcripts. Although latent and early lytic RNA transcripts were frequently detected in conjunction with high EBV viral load, in both brushings and biopsies the latent transcripts prevailed and reflected a typical NPC-associated latency-II transcription profile without EBNA2. Late lytic RNA transcripts were rare and detected at low levels mainly in NP brushings, suggestive of abortive viral reactivation rather than complete virus replication. EBV-IgA serology (EBNA1, VCA, Zta) did not correlate to the level of viral reactivation in situ. Overall, viral RNA profiling, DNA fragmentation and methylation analysis in NP brushings and parallel biopsies indicate that NP brush sampling provides a true and robust indicator of NPC tumor presence.
Project description:Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1-/- mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1-/- only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.
Project description:Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV-modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or infiltrating dendritic cells may play a role in the inflammation-to-oncogenesis transition of HPV-associated cervical cancer through modulation of innate immune signals.
Project description:Poison ivy (Toxicodendron radicans (L.) Kuntze) shows accession-level differentiation in a variety of morphometric traits, suggesting local adaptation. To investigate whether the presumed defense compound urushiol also demonstrates accession-level accumulation differences, in vitro nascent germinated poison ivy seedlings from geographically isolated populations were germinated in vitro and then assayed for known urushiol congener accumulation levels. Significant accession-level differences in the accumulation levels of total C15- and C17-, total C15-, total C17-, specific C15 congeners, and specific C17 congeners of urushiol were identified. In addition, hereto novel C15- and C17-urushiol isomers were identified as well. Cardanols are assumed to be the penultimate metabolites giving rise to urushiols, but this assumption was not previously empirically validated. C15-cardanol congeners and isomers corresponding to expected substrates needed to produce the observed C15-urushiol congeners and isomers were identified in the same poison ivy seedling extracts. Total C15-cardanol and C15-cardanol congeners also showed significant accession-level differences. Based on the observed C15-cardanol congeners in poison ivy, the penultimate step in urushiol biosynthesis was proposed to be a cardanol-specific hydroxylase activity.
Project description:The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.
Project description:Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system.