Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. .

We performed whole transcriptome analysis of osteosarcoma bone samples. Initially, we sequenced total RNA from 36 fresh-frozen samples (18 tumoral bone samples and 18 non-tumoral paired samples) matching in pairs for each osteosarcoma patient. We also performed independent gene expression analysis of formalin-fixed paraffin-embedded samples to verify the RNAseq results. Formalin-fixed paraffin-embedded samples allowed us to analyze the effect of chemotherapy. Data were analyzed with DESeq2, edgeR and Reactome packages of R. We found 5365 genes expressed differentially between the normal bone and osteosarcoma tissues with an FDR below 0.05, of which 3399 genes were upregulated and 1966 were downregulated. Among those genes, BTNL9, MMP14, ABCA10, ACACB, COL11A1, and PKM2 were expressed differentially with the highest significance between tumor and normal bone. Functional annotation with the reactome identified significant changes in the pathways related to the extracellular matrix degradation and collagen biosynthesis. It was suggested that chemotherapy may induce the modification of ECM with important collagen biosynthesis. Taken together, our results indicate that changes in the degradation of extracellular matrix seem to be an important mechanism of osteosarcoma and efficient chemotherapy induces the genes related to bone formation. Impact statement Osteosarcoma is a rare disease but it is of interest to many scientists all over the world because the current standard treatment still has poor results. We sequenced total RNA from 36 fresh-frozen paired samples (18 tumoral bone samples and 18 non-tumoral paired samples) from osteosarcoma patients. We found that differences in the gene expressions between the normal and affected bones reflected the changes in the regulation of the degradation of collagen and extracellular matrix. We believe that these findings contribute to the understanding of OS and suggest ideas for further studies.

Surrounded by speakers of Indo-European, Dravidian and Tibeto-Burman languages, around 11 million Munda (a branch of Austroasiatic language family) speakers live in the densely populated and genetically diverse South Asia. Their genetic makeup holds components characteristic of South Asians as well as Southeast Asians. The admixture time between these components has been previously estimated on the basis of archaeology, linguistics and uniparental markers. Using genome-wide genotype data of 102 Munda speakers and contextual data from South and Southeast Asia, we retrieved admixture dates between 2000–3800 years ago for different populations of Munda. The best modern proxies for the source populations for the admixture with proportions 0.29/0.71 are Lao people from Laos and Dravidian speakers from Kerala in India. The South Asian population(s), with whom the incoming Southeast Asians intermixed, had a smaller proportion of West Eurasian genetic component than contemporary proxies. Somewhat surprisingly Malaysian Peninsular tribes rather than the geographically closer Austroasiatic languages speakers like Vietnamese and Cambodians show highest sharing of IBD segments with the Munda. In addition, we affirmed that the grouping of the Munda speakers into North and South Munda based on linguistics is in concordance with genome-wide data.

Osteosarcoma (OS) is a rare malignant bone tumor. It affects mostly young persons and has poor outcome with the present treatment. No improvement was observed since the introduction of chemotherapy. The better understanding of osteosarcoma development could indicate better management strategy. Repetitive DNA elements were found to play a role in cancer mechanism especially in epithelial tumors but not yet analyzed in osteosarcoma. We conducted the study to analyse the expression profile of repetitive elements (RE) in osteosarcoma.Methods: Fresh bone paired (tumor and normal bone) samples were obtained from excised parts of tumors of 18 patients with osteosarcoma. We performed sequencing of RNA extracted from 36 samples (18 tumor tissues and 18 normal bone for controls), mapped raw reads to the human genome and identified the REs. EdgeR package was used to analyse the difference in expression of REs between osteosarcoma and normal bone.Results: 82 REs were found differentially expressed (FDR < 0.05) between osteosarcoma and normal bone. Out of all significantly changed REs, 35 were upregulated and 47 were downregulated. HERVs (THE1C-int, LTR5, MER57F and MER87B) and satellite elements (HSATII, ALR-alpha) were the most significantly differential expressed elements between osteosarcoma and normal tissues. These results suggest significant impact of REs in the osteosarcoma. The role of REs should be further studied to understand the mechanism they have in the genesis of osteosarcoma.

The human genome encodes thousands of natural antisense long noncoding RNAs (lncRNAs); they play the essential role in regulation of gene expression at multiple levels, including replication, transcription and translation. Dysregulation of antisense lncRNAs plays indispensable roles in numerous biological progress, such as tumour progression, metastasis and resistance to therapeutic agents. To date, there have been several studies analysing antisense lncRNAs expression profiles in cancer, but not enough to highlight the complexity of the disease. In this study, we investigated the expression patterns of antisense lncRNAs from osteosarcoma and healthy bone samples (24 tumour-16 bone samples) using RNA sequencing. We identified 15 antisense lncRNAs (RUSC1-AS1, TBX2-AS1, PTOV1-AS1, UBE2D3-AS1, ERCC8-AS1, ZMIZ1-AS1, RNF144A-AS1, RDH10-AS1, TRG-AS1, GSN-AS1, HMGA2-AS1, ZNF528-AS1, OTUD6B-AS1, COX10-AS1 and SLC16A1-AS1) that were upregulated in tumour samples compared to bone sample controls. Further, we performed real-time polymerase chain reaction (RT-qPCR) to validate the expressions of the antisense lncRNAs in 8 different osteosarcoma cell lines (SaOS-2, G-292, HOS, U2-OS, 143B, SJSA-1, MG-63, and MNNG/HOS) compared to hFOB (human osteoblast cell line). These differentially expressed IncRNAs can be considered biomarkers and potential therapeutic targets for osteosarcoma.

BackgroundOsteosarcoma (OS) is a prevalent primary malignant bone tumour with unknown etiology. These highly metastasizing tumours are among the most frequent causes of cancer-related deaths. Thus, there is an urgent need for different markers, and with our study, we were aiming towards finding novel biomarkers for OS.MethodsFor that, we analysed the whole exome of the tumorous and non-tumour bone tissue from the same patient with OS applying next-generation sequencing. For data analysis, we used several softwares and combined the exome data with RNA-seq data from our previous study.ResultsIn the tumour exome, we found wide genomic rearrangements, which should qualify as chromotripsis—we detected almost 3,000 somatic single nucleotide variants (SNVs) and small indels and more than 2,000 copy number variants (CNVs) in different chromosomes. Furthermore, the somatic changes seem to be associated to bone tumours, whereas germline mutations to cancer in general. We confirmed the previous findings that the most significant pathway involved in OS pathogenesis is probably the WNT/β-catenin signalling pathway. Also, the IGF1/IGF2 and IGF1R homodimer signalling and TP53 (including downstream tumour suppressor gene EI24) pathways may have a role. Additionally, the mucin family genes, especially MUC4 and cell cycle controlling gene CDC27 may be considered as potential biomarkers for OS.ConclusionsThe genes, in which the mutations were detected, may be considered as targets for finding biomarkers for OS. As the study is based on a single case and only DNA and RNA analysis, further confirmative studies are required.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-014-0020-0) contains supplementary material, which is available to authorized users.

Alternative splicing of RNA is an essential mechanism that increases proteomic diversity in eukaryotic cells. Aberrant alternative splicing is often associated with various human diseases, including cancer. We conducted whole-transcriptome analysis of 18 osteosarcoma bone samples (paired normal—tumor biopsies). Using RNA-seq, we identified statistically significant (FDR <0.05) 26 differentially expressed transcript variants of leptin receptor overlapping transcript ( LEPROT) gene. Some of the transcripts were overexpressed in normal cells, whereas others were overexpressed in tumor cells. The function of LEPROT is not completely understood. Herein, we highlight a possible association between OS and aberrant alternative splicing events and its interaction with the expression of LEPROT. We also discuss the role of LEPROT in regulating growth hormone and its receptor, and the relationship with initiation and progression of OS. This research study may help to understand the association of alternative splicing mechanism in OS and in tumorigenesis more generally. Further, LEPROT gene can also be considered as a potential biomarker of osteosarcoma. Impact statement Osteosarcoma (OS, also known as osteogenic sarcoma) is the most common primary malignancy of bone in children and adolescents. The molecular mechanisms of OS are extremely complicated and its molecular mediators remain to be elucidated. We sequenced total RNA from 18 OS bone samples (paired normal—tumor biopsies). We found statistically significant (FDR <0.05) 26 differentially expressed transcript variants of LEPROT gene with different expressions in normal and tumor samples. These findings contribute to the understanding of molecular mechanisms of OS development and provide encouragement to pursue further research.

Nicotine dependence is an addiction to tobacco products and a global public health concern. Association between the SLC6A4 polymorphisms and nicotine dependence is controversial. Two variable number tandem repeat (VNTR) domains, termed HTTLPR and STin2, in the SLC6A4 gene are well characterized transcriptional regulatory elements. Their polymorphism in the copy number of the repeat correlates with the particular personality and psychiatric traits. We analyzed nicotine dependence in 1,804 participants from Central Vietnam. The Fagerström Test (FTND) was used to evaluate the nicotine dependence and PCR was used to determine the SLC6A4 HTTLPR and STin2 VNTRs. The HTTLPR VNTR was associated with difficulties to refrain from smoking in a prohibiting environment. The STIn2 10/10 genotype was associated with (1) years of smoking, (2) difficulties to quit the first cigarette, and (3) higher number of cigarettes smoked per day (CPD). Stratification analysis was used to find the genetic interaction between these two VNTRs in nicotine dependence as they may synergistically regulate the SLC6A4 expression. Smokers with the S/S HTTLPR genotypes showed a much stronger association between STin2 10/10 variant and CPD. This finding is consistent with the molecular evidence for the functional interaction between HTTLPR and STin2 in cell line models, where STin2 has described as a stronger expressional regulator. Similarly, we found that STin2 is a much stronger modifier of smoking with 10/10 genotype related to higher nicotine dependence. The present study supports genetic interaction between HTTLPR and STin2 VNTRs in the regulation of nicotine dependence with the dominance of the STin2 effects. This finding could be explained by their differential effect on the SLC6A4 expression.