Quantitative analysis reveals crosstalk mechanisms of heat shock-induced attenuation of NF-κB signaling at the single cell level.
PLoS computational biology
Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular "all-or-nothing" encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were "analogue". In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited "all-or-nothing" encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKβ subunits in whole cell lysates. However, a combination of "all-or-nothing" crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.
10.1371/journal.pcbi.1006130
Nanoparticle enhanced combination therapy for stem-like progenitors defined by single-cell transcriptomics in chemotherapy-resistant osteosarcoma.
Wang Li,Huang Xiaojia,You Xinru,Yi Tianqi,Lu Bing,Liu Jiali,Lu Guohao,Ma Minglin,Zou Changye,Wu Jun,Zhao Wei
Signal transduction and targeted therapy
The adaptation of osteosarcoma cells to therapeutic pressure impedes the efficacy of chemotherapy for osteosarcoma. However, the characteristics and cellular organization of therapy-resistant cells in osteosarcoma tumors remain elusive. Here, we utilized single-cell transcriptomics to systematically map the cell-type-specific gene expression in a chemotherapy-resistant osteosarcoma tumor. Our data demonstrated the VEGFR2-JMJD3-abundant subsets as quiescent stem-like cells, thereby establishing the hierarchy of therapy-resistant actively cycling progenitor pools (JMJD3-abundant) in osteosarcoma. VEGFR2 inhibitor and JMJD3 inhibitor synergistically impeded osteosarcoma cell propagation and tumor growth. Although osteosarcoma cells are predisposed to apoptosis induced by the synergistic therapy through activation of the CHOP pro-apoptotic factor via the endoplasmic reticulum (ER) stress, the stem-like/progenitor cells exhibit an adaptive response, leading to their survival. Reduction in cellular glutathione levels in stem-like/progenitor cells caused by the treatment with a glutathione synthesis inhibitor increases ER stress-induced apoptosis. Importantly, the marked therapeutic improvement of synergistic therapy against stem-like/progenitor cells was achieved by using glutathione-scavenging nanoparticles, which can load and release the drug pair effectively. Overall, our study provides a framework for understanding glutathione signaling as one of the therapeutic vulnerabilities of stem-like/progenitor cells. Broadly, these findings revealed a promising arsenal by encapsulating glutathione-scavenging nanoparticles with co-targeting VEGFR2 and JMJD3 to eradicate chemotherapy-resistant osteosarcoma.
10.1038/s41392-020-00248-x
A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma.
Mirabello Lisa,Koster Roelof,Moriarity Branden S,Spector Logan G,Meltzer Paul S,Gary Joy,Machiela Mitchell J,Pankratz Nathan,Panagiotou Orestis A,Largaespada David,Wang Zhaoming,Gastier-Foster Julie M,Gorlick Richard,Khanna Chand,de Toledo Silvia Regina Caminada,Petrilli Antonio S,Patiño-Garcia Ana,Sierrasesúmaga Luis,Lecanda Fernando,Andrulis Irene L,Wunder Jay S,Gokgoz Nalan,Serra Massimo,Hattinger Claudia,Picci Piero,Scotlandi Katia,Flanagan Adrienne M,Tirabosco Roberto,Amary Maria Fernanda,Halai Dina,Ballinger Mandy L,Thomas David M,Davis Sean,Barkauskas Donald A,Marina Neyssa,Helman Lee,Otto George M,Becklin Kelsie L,Wolf Natalie K,Weg Madison T,Tucker Margaret,Wacholder Sholom,Fraumeni Joseph F,Caporaso Neil E,Boland Joseph F,Hicks Belynda D,Vogt Aurelie,Burdett Laurie,Yeager Meredith,Hoover Robert N,Chanock Stephen J,Savage Sharon A
Cancer discovery
UNLABELLED:Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10(-9); OR, 2.43; 95% confidence interval, 1.83-3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene. SIGNIFICANCE:Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma.
10.1158/2159-8290.CD-15-0125
Exome sequencing of osteosarcoma reveals mutation signatures reminiscent of BRCA deficiency.
Nature communications
Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.
10.1038/ncomms9940
A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.
Nature medicine
Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.
10.1038/s41591-020-0844-1
Infiltrating Myeloid Cells Drive Osteosarcoma Progression via GRM4 Regulation of IL23.
Kansara Maya,Thomson Kristian,Pang Puiyi,Dutour Aurelie,Mirabello Lisa,Acher Francine,Pin Jean-Philippe,Demicco Elizabeth G,Yan Juming,Teng Michele W L,Smyth Mark J,Thomas David M
Cancer discovery
The glutamate metabotropic receptor 4 () locus is linked to susceptibility to human osteosarcoma, through unknown mechanisms. We show that gene-targeted mice demonstrate accelerated radiation-induced tumor development to an extent comparable with mice. GRM4 is expressed in myeloid cells, selectively regulating expression of IL23 and the related cytokine IL12. Osteosarcoma-conditioned media induce myeloid cell expression in a GRM4-dependent fashion, while suppressing the related cytokine . Both human and mouse osteosarcomas express an increased IL23:IL12 ratio, whereas higher IL23 expression is associated with worse survival in humans. Consistent with an oncogenic role, mice are strikingly resistant to osteosarcoma development. Agonists of GRM4 or a neutralizing antibody to IL23 suppressed osteosarcoma growth in mice. These findings identify a novel, druggable myeloid suppressor pathway linking GRM4 to the proinflammatory IL23/IL12 axis. SIGNIFICANCE: Few novel systemic therapies targeting osteosarcoma have emerged in the last four decades. Using insights gained from a genome-wide association study and mouse modeling, we show that GRM4 plays a role in driving osteosarcoma via a non-cell-autonomous mechanism regulating IL23, opening new avenues for therapeutic intervention...
10.1158/2159-8290.CD-19-0154
A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion.
Pierron Gaëlle,Tirode Franck,Lucchesi Carlo,Reynaud Stéphanie,Ballet Stelly,Cohen-Gogo Sarah,Perrin Virginie,Coindre Jean-Michel,Delattre Olivier
Nature genetics
The identification of subtype-specific translocations has revolutionized the diagnostics of sarcoma and has provided new insight into oncogenesis. We used RNA-seq to investigate samples from individuals diagnosed with small round cell tumors of bone, possibly Ewing sarcoma, but which lacked the canonical EWSR1-ETS translocation. A new fusion was observed between BCOR (encoding the BCL6 co-repressor) and CCNB3 (encoding the testis-specific cyclin B3) on the X chromosome. RNA-seq results were confirmed by RT-PCR and through cloning of the tumor-specific genomic translocation breakpoints. In total, 24 BCOR-CCNB3-positive tumors were identified among a series of 594 sarcoma cases. Gene profiling experiments indicated that BCOR-CCNB3-positive cases are biologically distinct from other sarcomas, particularly Ewing sarcoma. Finally, we show that CCNB3 immunohistochemistry is a powerful diagnostic marker for this subgroup of sarcoma and that overexpression of BCOR-CCNB3 or of truncated CCNB3 activates S phase in NIH3T3 cells. Thus, the intrachromosomal X-chromosome fusion described here represents a new subtype of bone sarcoma caused by a newly identified gene fusion mechanism.
10.1038/ng.1107
Genome-wide association study identifies two susceptibility loci for osteosarcoma.
Savage Sharon A,Mirabello Lisa,Wang Zhaoming,Gastier-Foster Julie M,Gorlick Richard,Khanna Chand,Flanagan Adrienne M,Tirabosco Roberto,Andrulis Irene L,Wunder Jay S,Gokgoz Nalan,Patiño-Garcia Ana,Sierrasesúmaga Luis,Lecanda Fernando,Kurucu Nilgün,Ilhan Inci Ergurhan,Sari Neriman,Serra Massimo,Hattinger Claudia,Picci Piero,Spector Logan G,Barkauskas Donald A,Marina Neyssa,de Toledo Silvia Regina Caminada,Petrilli Antonio S,Amary Maria Fernanda,Halai Dina,Thomas David M,Douglass Chester,Meltzer Paul S,Jacobs Kevin,Chung Charles C,Berndt Sonja I,Purdue Mark P,Caporaso Neil E,Tucker Margaret,Rothman Nathaniel,Landi Maria Teresa,Silverman Debra T,Kraft Peter,Hunter David J,Malats Nuria,Kogevinas Manolis,Wacholder Sholom,Troisi Rebecca,Helman Lee,Fraumeni Joseph F,Yeager Meredith,Hoover Robert N,Chanock Stephen J
Nature genetics
Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. To better understand the genetic etiology of osteosarcoma, we performed a multistage genome-wide association study consisting of 941 individuals with osteosarcoma (cases) and 3,291 cancer-free adult controls of European ancestry. Two loci achieved genome-wide significance: a locus in the GRM4 gene at 6p21.3 (encoding glutamate receptor metabotropic 4; rs1906953; P = 8.1 × 10⁻⁹) and a locus in the gene desert at 2p25.2 (rs7591996 and rs10208273; P = 1.0 × 10⁻⁸ and 2.9 × 10⁻⁷, respectively). These two loci warrant further exploration to uncover the biological mechanisms underlying susceptibility to osteosarcoma.
10.1038/ng.2645
Immuno-genomic landscape of osteosarcoma.
Wu Chia-Chin,Beird Hannah C,Andrew Livingston J,Advani Shailesh,Mitra Akash,Cao Shaolong,Reuben Alexandre,Ingram Davis,Wang Wei-Lien,Ju Zhenlin,Hong Leung Cheuk,Lin Heather,Zheng Youyun,Roszik Jason,Wang Wenyi,Patel Shreyaskumar,Benjamin Robert S,Somaiah Neeta,Conley Anthony P,Mills Gordon B,Hwu Patrick,Gorlick Richard,Lazar Alexander,Daw Najat C,Lewis Valerae,Futreal P Andrew
Nature communications
Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.
10.1038/s41467-020-14646-w
Identification of the Novel Target Genes for Osteosarcoma Therapy Based on Comprehensive Bioinformatic Analysis.
Zhou Xi,Fan Yu,Ye Weiliang,Jia Binghan,Yang Yuemei,Liu Yong
DNA and cell biology
Osteosarcoma is one of the most common primary malignant tumors of the bone and tends to develop in teenage years. Although multitreatments for the diagnosis and therapy of osteosarcoma have been developed, there are still needs of new methods to prevent and treat the osteosarcoma. Here, we performed bioinformatic analysis to screen for the key genes, molecules, and pathways involved in osteosarcoma survival. Four microarray data sets (GSE99671, GSE87624, GSE65071, and GSE28423), which include data from human bone and osteosarcoma samples, were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed mRNAs and miRNAs were identified. Kyoto Encyclopedia of Genes and Genomes enriched pathways, miRNA-mRNA target, gene/disease relationship, and overall survival was elucidated using related websites and software according to bioinformatic analysis protocols. We found three critical genes miR-29c, blood vessel epicardial substance (BVES), and proteasome 20S subunit beta 2 (PSMB2) through the GEO database and predicting miRNA-mRNA target. Among these genes, BVES and PSMB2 presented a high expression level in osteosarcoma based on GSE99671 and GSE87624 data sets, while miR-29c showed a low expression level in osteosarcoma based on GSE65071 and GSE28423 data sets. Furthermore, we found that the high expression level of miR-29c and BVES associated with better prognosis, while highly expressed PSMB2 associated with poor prognosis. The abnormally expressed mRNAs and miRNAs, which were identified by integrated bioinformatic analysis, provided insights into the molecular mechanisms of osteosarcoma. Notably, we found three critical genes that could be used as novel therapeutic targets for preventing or diagnosing osteosarcoma. Finally, PSMB2 may be the target of miR-29c.
10.1089/dna.2020.5377