Osteosarcoma Lung Metastasis Research Articles

Melittin inhibits lung metastasis of human osteosarcoma: Evidence of wnt/β-catenin signaling pathway participation

Melittin is a major active peptide component of bee venom that has been demonstrated to show anti-tumor effects. Osteosarcoma is a type of bone tumor with a high degree of malignancy, and metastasis is the main challenge of osteosarcoma therapy. This study aimed to investigate the role of melittin in the lung metastasis of osteosarcoma. 143 B cells were treated with different concentrations of melittin in vitro. Wound-healing and transwell assays were performed to determine the cell migration and invasion potential. Quantitative real-time PCR and Western blot experiments were performed to evaluate the expression levels of Wnt/β-catenin signaling pathway-related factors after treatment with melittin. The orthotopic implantation model and hematoxylin-eosin staining were used to investigate the effect of melittin treatment on tumor formation and lung metastasis. Immunohistochemical staining and Western blot experiments were performed to indicate the melittin-mediated expression changes in Wnt/β-catenin signaling pathway-related factors. The cell migration and invasion potential were observed to be inhibited in a dose-dependent manner upon treatment with melittin. Treatment with medium and high concentrations of melittin attenuated the mRNA and protein expression of LRP5, β-catenin, MMP-2, cyclin D, c-Myc, survivin, MMP-9, and VEGF genes in vitro. Melittin significantly inhibited the growth of tibia xenografts in nude mice and decreased the number of lung metastatic nodules. Consistent with the results observed in vitro, treatment with melittin at medium and high concentrations attenuated the expression of Wnt/β-catenin signaling pathway-related factors in vivo. In vitro, Wnt/β-catenin signaling pathway was involved in Melittin-mediated -migration and invasion potential of 143 B cells. Similarly, as observed in the in vivo experiments, Wnt/β-catenin signaling pathway was also associated with the role of melittin on lung metastasis of osteosarcomas.

RGS12 is a novel tumor suppressor in osteosarcoma that inhibits YAP-TEAD1-Ezrin signaling.

Osteosarcoma (OS) is the most common primary malignancy of the bone that predominantly affects children and adolescents. Hippo pathway is a crucial regulator of organ size and tumorigenesis. However, how Hippo pathway regulates the occurrence of osteosarcoma is largely unknown. Here, we reported the regulator of G protein signaling protein 12 (RGS12) is a novel Hippo pathway regulator and tumor suppressor of osteosarcoma. Depletion of Rgs12 promotes osteosarcoma progression and lung metastasis in an orthotopic xenograft mouse model. Our data showed that knockdown of RGS12 upregulates Ezrin expression through promoting the GNA12/13-RhoA-YAP pathway. Moreover, RGS12 negatively regulates the transcriptional activity of YAP/TEAD1 complex through its PDZ domain function to inhibit the expression and function of the osteosarcoma marker Ezrin. PDZ domain peptides of RGS12 can inhibit the development of intratibial tumor and lung metastases. Collectively, this study identifies the RGS12 is a novel tumor suppressor in osteosarcoma through inhibiting YAP-TEAD1-Ezrin signaling pathway and provides a proof of principle that targeting RGS12 may be a therapeutic strategy for osteosarcoma.

KDM4A-mediated histone demethylation of SLC7A11 inhibits cell ferroptosis in osteosarcoma

Osteosarcoma (OS) is the most common type of bone tumor that seriously affects limb function and induces great pain in patients. Lung metastasis and chemotherapy resistance are two key issues leading to the poor prognosis of OS patients, therefore new treatment targets and strategies are urgently needed. In our study, we uncovered the role of histone demethylase KDM4A in regulating OS cell ferroptosis and tumor progression. KDM4A was significantly upregulated in OS specimens and high KDM4A expression was associated with poorer prognosis in OS patients. Our data indicated that targeting KDM4A significantly increased OS cell death, enhanced cisplatin response, and attenuated migration ability in vitro. KDM4A depletion dramatically inhibited tumor progression and lung metastasis of OS in vivo Further experiments confirmed that KDM4A knockdown promoted OS cell ferroptosis, a special non-apoptotic form of cell death. KDM4A regulates SLC7A11 transcription and OS cell ferroptosis by controlling H3K9me3 demethylation in the promoter region of SLC7A11. Our findings deepened the recognition of epigenetic regulatory mechanism in OS tumorigenesis, chemoresistance, and metastasis, suggesting that KDM4A activity may be a potential therapeutic target for future OS treatment.

MALDI-TOF Mass Spectrometry Profiling of Plasma Exosomes Evaluates Osteosarcoma Metastasis

Osteosarcoma is the most common primary sarcoma of bone among adolescents, often characterized by early lung metastasis resulting in high mortality. Recently, exosomes have been used in liquid biopsy for monitoring tumors. Herein, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to profile human plasma exosomes for the evaluation of osteosarcoma lung metastasis. Forty osteosarcoma patients with (n = 20) or without (n = 20) lung metastasis as well as 12 heathy controls were recruited. Exosomes were isolated from human plasma for MALDI-TOF MS analysis. Multivariate statistical analyses were performed on the MALDI-TOF mass spectra. It was demonstrated that the strategy can differentiate osteosarcomas from healthy controls and further discriminate osteosarcoma lung metastasis from non-lung metastasis. Seven exosomal potential protein biomarkers of osteosarcoma lung metastasis were identified. The proposed method has a great clinical application prospect in the diagnosis of osteosarcoma and monitoring of osteosarcoma lung metastasis.

Rab22a-NeoF1 fusion protein promotes osteosarcoma lung metastasis through its secretion into exosomes

It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN142). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

The lncRNA NEAT1 promotes the epithelial-mesenchymal transition and metastasis of osteosarcoma cells by sponging miR-483 to upregulate STAT3 expression

BackgroundOsteosarcoma is one of the most prevalent primary bone tumours in adolescents. Accumulating evidence shows that aberrant expression of the long non-coding RNA (lncRNA) NEAT1 and microRNA-483 (miR-483) contribute to the epithelial-mesenchymal transition (EMT), invasion and metastasis of tumour cells. However, the potential regulatory effects of NEAT1 and miR-483 on the EMT of osteosarcoma remain elusive.MethodsThe expression of the NEAT1, miR-483, signal transducer and activator of transcription-1 (STAT1), STAT3, and EMT-associated markers was measured using qRT-PCR or western blotting. NEAT1 overexpression or knockdown was induced by lentivirus-mediated transfection. A luciferase reporter assay was employed to confirm the association between NEAT1/miR-483 and miR-483/STAT3. RNA immunoprecipitation (RIP) was also performed to verify the NEAT1 and miR-483 interaction. Wound healing and transwell assays were implemented to assess the migration and invasion of U2OS cells. Unilateral subcutaneous injection of U2OS into nude mice was performed to investigate tumour metastasis in vivo.ResultsThe expression of miR-483 was downregulated in both osteosarcoma cell lines and osteosarcoma tissues. The overexpression of miR-483 suppressed the migration, invasion, and expression of EMT-associated proteins in U2OS cells, while simultaneous overexpression of STAT3 partially relieved this suppression. Mechanistically, miR-483 specifically targeted the 3′ untranslated region (3′UTR) of STAT3 and repressed its expression. However, NEAT1 sponged miR-438, increased STAT3 expression, and repressed STAT1 expression, subsequently increasing the EMT of osteosarcoma cells. The knockdown of NEAT1 in transplanted U2OS cells impaired the liver and lung metastases of osteosarcoma in nude mice. Moreover, NEAT1 silencing inhibited the mesenchymal- epithelial transition (MET) of osteosarcoma at metastasis sites.ConclusionsThe lncRNA NEAT1/miR-483/STAT3 axis plays a crucial role in regulating the metastasis of osteosarcoma and potentially represents one appealing therapeutic target for osteosarcoma treatment in the future.

A Novel Orthotopic Mouse Model of Lung Metastasis Using Fluorescent Patient-derived Osteosarcoma Cells.

A mouse model of metastatic osteosarcoma is imperative to identify effective agents for metastatic osteosarcoma, which is a recalcitrant disease. In the present study, we established osteosarcoma patient-derived cells (OS-PDCs) and transfected them with green fluorescent protein (GFP). The OS-PDCs were transfected with GFP-lentivirus. GFP-expressing OS-PDCs (2.0×105) were then injected into the tibia of nude mice to establish the patient-derived orthotopic cell (PDOC) model (n=3). Six weeks after injection, the primary tumor and each organ were resected and imaged. Primary orthotopic tumors were established in two out of three mice. The GFP-expressing OS-PDCs in the PDOC model were visualized. Multiple GFP-expressing lung metastases were detected in one of the two mice with primary tumor. The present study proves the concept that a GFP-expressing PDOC model can mimic clinical lung-metastatic osteosarcoma. This model can serve as a paradigm to screen for effective drugs for osteosarcoma lung metastasis.

Magnetic transfection with superparamagnetic chitosan-loaded IGFBP5 nanoparticles and their in vitro biosafety.

We prepared the superparamagnetic chitosan nanoparticles (SPCIONPs) to study the application of them as gene vectors using a magnetic transfection system for the targeted treatment of lung metastasis of osteosarcoma. The SPCIONPs were characterized by transmission electron microscopy, Fourier transform infrared spectrometry, superconducting quantum interference device and atomic force microscopy. Their biosafety was determined by cell counting kit-8 (CCK8) and live–dead staining assays. The transfection in vitro was detected by laser confocal microscopy. SPCIONPs, which can bind closely to plasmids and protect them from DNA enzyme degradation, were prepared with an average particle size of approximately 22 nm and zeta potential of 11.3 mV. The results of the CCK8 and live–dead staining assays showed that superparamagnetic chitosan nanoparticles loaded with insulin-like growth factor-binding protein 5 (SPCIONPs/pIGFBP5) induced no significant cytotoxicity compared to the control group. The result of transfection in vitro suggested that pIGFBP5 emitted a greater amount of red fluorescence in the SPCIONPs/pIGFBP5 group than that in the chitosan-loaded IGFBP5 (CS/pIGFBP5) group. In conclusion, the prepared SPCIONPs had good biosafety and could be effectively used to transfer pIGFBP5 into 143B cells, and they thus have good application prospects for the treatment of lung metastasis of osteosarcoma.

KDM6B-mediated histone demethylation of LDHA promotes lung metastasis of osteosarcoma.

Rationale: Osteosarcoma (OS), the most common type of bone tumor, which seriously affects the patients' limb function and life quality. OS has a strong tendency of lung metastasis, and the five-year survival rate of patients with metastatic osteosarcoma is less than 20%. Thus, new treatment targets and strategies are urgently needed.Methods: The expression of the histone demethylase KDM6B and H3K27me3 levels in OS specimens were analyzed using quantitative PCR and immunohistochemical assays. The biological functions of KDM6B were determined using in vitro transwell, wound healing assays, and an in vivo orthotopic injection-induced lung metastasis model. Subsequently, chromatin immunoprecipitation sequencing (ChIP-seq) combined with transcriptomic RNA sequencing (RNA-seq), and subsequent ChIP-qPCR, western blot, and aerobic glycolysis assays were used to explore the mechanism of KDM6B function and validate the candidate target gene of KDM6B.Results: KDM6B expression was significantly upregulated in OS patients, and high KDM6B expression was associated with poorer prognosis in OS patients. Targeting KDM6B significantly inhibited OS cell migration in vitro and lung metastasis in vivo. RNA-seq and ChIP-seq analysis revealed that KDM6B increases lactate dehydrogenase LDHA expression in OS cells by directly mediating H3K27me3 demethylation. The phenotypes of inhibited cell metastasis in KDM6B-knockdown OS cells was reversed upon overexpression of LDHA. Finally, a small molecule inhibitor targeting KDM6B significantly inhibited OS cell migration in vitro and lung metastasis in vivo.Conclusions: Collectively, we elucidated that upregulated KDM6B facilitates tumor metastasis in OS via modulating LDHA expression. Our findings deepen the recognition of OS metastasis mechanism and suggest that KDM6B might be a new potential therapeutic target for the treatment of OS (especially highly metastatic OS).

H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts.

Background: Collagen type VI alpha 1 (COL6A1) has been found to be dysregulated in several human malignancies. However, the role of COL6A1 in osteosarcoma (OS) progression remains largely unclear. Here, we aimed to explore the clinical significance and biological involvement of COL6A1 in the OS cell migration and invasion.Material and Methods: We used immunohistochemistry, qRT-PCR and western blot to detect the expression of COL6A1 in 181 OS patient samples. Chromatin immunoprecipitation (ChIP) and PCR were carried out to verify the regulatory interaction of p300, c-Jun and COL6A1 promoter. The invasion and migration function of COL6A1 in OS was detected in vitro and in vivo. RNA sequence was performed to detect the downstream pathway of COL6A1, and then co-immunoprecipitation (co-IP), ubiquitination assays and rescue experiments were performed to determine the regulatory effect of COL6A1 and signal transducers and activators of transcription (STAT1). Exosomes derived from OS cell lines were assessed for the ability to promote cancer progression by co-cultured assay and exosomes tracing.Results: COL6A1 was commonly upregulated in OS tissues, especially in lung metastasis tissues, which was associated with a poor prognosis. c-Jun bound p300 increased the enrichment of H3K27ac at the promoter region of the COL6A1 gene, which resulted in the upregulation of COL6A1 in OS. Overexpression of COL6A1 promoted OS cell migration and invasion via interacting with SOCS5 to suppress STAT1 expression and activation in an ubiquitination and proteasomal degradation manner. Most interestingly, we found that exosomal COL6A1 derived from OS cells convert normal fibroblasts to cancer-associated fibroblasts (CAFs) by secreting pro-inflammatory cytokines, including IL-6 and IL-8. The activated CAFs could promote OS cell invasion and migration by mediating TGF-β/COL6A1 signaling pathway.Conclusion: Our data demonstrated that upregulation of COL6A1 activated by H3K27 acetylation promoted the cell migration and invasion by suppressing STAT1 pathway in OS cells. Moreover, COL6A1 can be packaged into OS cell-derived exosomes and activate CAFs to promote OS metastasis.

Diallyl trisulfide regulates cell apoptosis and invasion in human osteosarcoma U2OS cells through regulating PI3K/AKT/GSK3β signaling pathway.

To investigate the effects and the mechanisms of action of Diallyl trisulfide (DATS) on the proliferation and metastasis of human osteosarcoma (OS) U2OS. U2OS cells were treated by different concentrations of DATS at different time points. Cell proliferations were measured by MTT assay. DATS induced cell cycle distribution and apoptosis were evaluated by flow cytometry (FCM) with Annexin-V. Cell migration and invasion were detected by wound healing assay and transwell assay. The effects of DATS in U2OS cell growth and metastasis were also detected in a mouse OS xenograft model. A time- and concentration-dependent cytotoxic effect of DATS was observed in U2OS cells. FCM with PI staining and Annexin-V -FITC indicated that DATS induces apoptosis and a G0/G1 cell cycle arrest of U2OS cells at all concentrations from 25 μmol/l to 100 μmol/l. DATS also inhibits the migration and invasion of U2OS cells. Western blot showed that the expression levels of p-AKT, p-GSK3β, Bcl-2, Vimentin and β-catenin were decreased, while the expression levels of Bad, Bax and E-cadherin were significantly increased in DATS treated U2OS cells. Analysis using a mouse xenograft model indicated that xenografts of DATS treatment group had a significant decrease in tumor volume and weight compared to the control group. Lung metastasis models in mice demonstrated that treatment of DATS inhibits lung metastasis of OS in vivo. These data suggested that DATS inhibits OS development and progression through the regulation of PI3K/AKT/GSK3β signaling pathways, accompanied by downregulation of Bcl-2, Vimentin and β-catenin, as well as upregulation of Bad, Bax and E-cadherin. Therefore, our data demonstrated that DATS exerted its anticancer effects by inhibiting cell proliferation, migration and invasion in vitro and in vivo. These results provide evidence for the use of the natural product DATS either alone or in combination with standard therapy for OS.

Long non-coding RNA EWSAT1 promoted metastasis and actin cytoskeleton changes via miR-24-3p sponging in osteosarcoma.

Non‐coding RNAs are closely associated with tumorigenesis in multiple malignant tumours, including osteosarcoma (OS). Long non‐coding RNA Ewing sarcoma‐associated transcript 1 (EWSAT1) plays a role in metastasis, and actin cytoskeletal changes in OS remain unclear. In the current study, we showed that EWSAT1 expression was up‐regulated in OS and that an elevation in the EWSAT1 expression level was correlated with poor prognosis in patients with OS. Functionally, we showed that knockdown of EWSAT1 suppressed migration and induced actin stress fibre degradation in MNNG/HOS and 143B cells. Moreover, we found that ROCK1 was a key downstream effector in EWSAT1‐mediated cell migration and actin stress fibre changes. Furthermore, we demonstrated that ROCK1 and EWSAT1 shared a similar microRNA response element of microRNA‐24‐3p (miR‐24‐3p). Moreover, we verified that miR‐24‐3p suppressed ROCK1 and its mediated migration and actin stress fibres change by direct targeting. EWSAT1 promoted ROCK1‐mediated migration and actin stress fibre formation through miR‐24‐3p sponging. Lastly, through an in vivo study, we demonstrated that EWSAT1 promoted lung metastasis in OS. According to the above‐mentioned results, we suggest that EWSAT1 acts as an oncogene and that EWSAT1/miR‐24‐3p/ROCK1 axial could be a new target in the treatment of OS.

Tumor cell MT1-MMP is dispensable for osteosarcoma tumor growth, bone degradation and lung metastasis

The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.

Abstract A03: Autophagy and HSP27: A potential link to define autophagy fate in osteosarcoma

Abstract Survival in osteosarcoma (OS) has not improved in over a decade, thus the need for new therapeutic strategies. Autophagy, a catabolic process used by cells to survive under stress, has been implicated in resistance to chemotherapy. We previously demonstrated aerosol gemcitabine (GCB) efficacy against OS lung metastasis and showed GCB to induce autophagy through the Akt/mTOR signaling pathway. Whether autophagy contributes to tumor response or resistance to GCB has been the focus of our studies. What determines whether chemotherapy-induced autophagy will lead to survival or death is unknown. We found phosphorylated heat shock protein 27 (pHSP27) to potentially define the outcome of chemotherapy-induced autophagy in OS. Small heat shock proteins, specifically HSP27, are upregulated in many cancers, including OS, and associated with treatment resistance. The purpose of this study is to investigate if induction of pHSP27 predicts the role of chemotherapy-induced autophagy in OS lung metastasis. We hypothesize that when pHSP27 is induced, blocking autophagy will lead to increased sensitivity to GCB. Furthermore, inhibition of pHSP27 may revert the effect of GCB-induced autophagy in OS cells by decreasing sensitivity to GCB. We showed in the LM7 and CCH-OS-D human metastatic OS cells in vitro, that GCB induces autophagy as determined by the conversion of microtubule-associated light chain 3 one to two (LC3I/LC3II), increase in Beclin 1, and decrease in p62 protein expression. Sensitivity of LM7 cells to GCB was enhanced after autophagy inhibition by hydroxychloroquine (HCQ), a pharmacologic inhibitor and shRNA targeting Beclin 1, suggesting autophagy as a prosurvival mechanism whereas in CCH-OS-D cells inhibition of autophagy decreased cell sensitivity to GCB, concluding here that induction of autophagy leads to cell death. We demonstrated that pHSP27 is significantly higher in GCB-treated LM7 cells as compared to CCH-OS-D cells. We also showed that inhibition of HSP27 by shRNA targeting HSP27 has no effect on GCB-induced autophagy in LM7 cells. Treatment of shHSP27LM7 with GCB resulted in an increase in LC3I/LC3II conversion and a decrease in p62. We further revealed this in vivo by conducting a pilot study in which shHSP27LM7 cells were injected i.v. into nude mice. 6 weeks later, lung metastases were confirmed. Animals were divided into two groups: untreated and aerosol GCB treated (1mg/mL three times a week). Mice were sacrificed after 2 weeks of therapy. Transmitted electron microscopy demonstrated an increase in the number of autophagosomes in lung metastasis from mice treated with GCB, confirming induction of autophagy in the absence of HSP27. Lastly, in order to demonstrate that inhibition of HSP27 will change GCB-induced autophagy response in LM7 cells, we treated shHSP27LM7 cells with the combination GCB+HCQ and showed increased viability compared to the LM7 GipZ control cells, confirming preliminarily our hypothesis that induction of pHSP27 will determine chemotherapy-induced autophagy response in OS. Citation Format: Grace Nehme, Kumar Felix, Andrew Wahba, Diana M. Fandino, Nancy Gordon. Autophagy and HSP27: A potential link to define autophagy fate in osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A03.

VEGFR2 Promotes Metastasis and PD-L2 Expression of Human Osteosarcoma Cells by Activating the STAT3 and RhoA-ROCK-LIMK2 Pathways.

The survival rate of osteosarcoma, the most prevalent primary bone tumor, has not been effectively improved in the last 30 years. Hence, new treatments and drugs are urgently needed. Antiangiogenic therapy and immunotherapy have good antitumor effects in many kinds of tumors. It is hypothesized that there may be a synergistic effect between immune checkpoint inhibitors and antiangiogenic therapy. Nevertheless, its potential mechanism is still unclear. Vascular endothelial growth factor receptor-2 (VEGFR2) expression was detected by immunohistochemistry in 18 paired osteosarcoma tissues. Moreover, we investigated the effects of apatinib treatment and VEGFR2 knockdown on osteosarcoma as well as the relevant underlying mechanism. Immunohistochemistry assays showed that, compared with that in primary osteosarcoma, VEGFR2 expression was higher in lung metastases. VEGFR2 was positively correlated with PD-L2 expression in osteosarcoma lung metastasis. Transwell assays indicated that VEGFR2 inhibition reduced osteosarcoma cell metastatic abilities in vitro. We also demonstrated that VEGFR2 inhibition downregulated the STAT3 and RhoA-ROCK-LIMK2 pathways, thereby attenuating migration and invasion. Additionally, VEGFR2 inhibition targeted STAT3, through which it reduced PD-L2 expression in osteosarcoma cells. VEGFR2 inhibition markedly attenuated osteosarcoma lung metastatic ability in vivo. In this study, we presented the pro-metastatic functional mechanism of VEGFR2 in osteosarcoma. VEGFR2 inhibition exhibits antitumor effects through antiangiogenic effects and inhibition of immune escape, which possibly provides potential clinical treatment for metastatic osteosarcoma.

Identification and Functional Analysis of EPOR+ Tumor-Associated Macrophages in Human Osteosarcoma Lung Metastasis.

Background Tissue-resident macrophages can be educated to tumor-associated macrophages (TAMs) by the tumor microenvironment and many types of macrophages express erythropoietic receptor (EPOR); However, little is known about the expression of EPOR on TAMs and the identity of EPOR+ TAMs in osteosarcoma lung metastasis has thus far remained elusive. Methods EPOR-eGFPcre mice were used to determine the expression of EPOR on lung tissue-resident macrophages. Flow cytometry, RT-PCR, and Western blot were examined to define the identity of EPOR+ TAMs in 106 osteosarcoma lung metastasis specimens. Moreover, the clinicopathologic factors and prognosis of patients with CD163+EPOR+ macrophages were compared. Results We found that a subpopulation of mouse lung tissue-resident macrophages express EPOR and EPO enhances the proliferation of EPOR+ macrophages in mouse lung. A subpopulation of CD163+ macrophages expresses EPOR in human osteosarcoma lung metastasis specimens. CD163+EPOR+TAMs increase 2.5 times in human osteosarcoma lung metastasis tissues; CD206, CD163, and PD1, which are known to have a significant role in TAM function had high expression in CD163+EPOR+ TAMs compared with CD163+EPOR− TAMs. Furthermore, CD163+EPOR+ TAMs had higher M2 marker and cytokine expression in osteosarcoma tissues compared with para-osteosarcoma tissues. EPO enhanced the expression of M2 cytokines in primary CD163+EPOR+ TAMs. Importantly, the percentage of CD163+EPOR+ TAMs had a positive linear association with malignant phenotypes as well as poor disease-free survival and overall survival time. Conclusions We have characterized TAMs expressing EPOR and CD163+EPOR+ macrophages as TAMs in osteosarcoma lung metastasis patients, which are highly associated with tumor aggressiveness.

Abstract A07: Role of YAP/TEAD and YAP/Smad signaling pathways in osteosarcoma tumor growth and lung metastasis dissemination

Abstract Osteosarcoma (OS) is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastases are present at diagnosis. Because Hippo/YAP cascade plays a crucial role in cancer progression, we investigated its implication in OS development. Using proximity ligation assay and immunoprecipitation approaches, and promoter/reporter assays, we demonstrated a functional interaction between YAP and the transcriptional factor TEAD-1 in OS cell lines. To understand the specific role of this YAP/TEAD-1 interaction to regulate OS tumor growth, we generated OS cells that overexpressed a YAP protein able (YAP127A) or not able (YAP94A) to interact with TEAD-1. Using in vitro experiments and a preclinical model of OS, we demonstrated that YAP overexpression stimulates respectively OS cell proliferation and tumor growth only when YAP is able to interact with TEAD-1. Furthermore, the overexpression of YAPS94A reduces specific TEAD-1 transcriptional response and in vivo tumor growth. In contrast, the ability of YAP to stimulate OS cell migration does not depend on its ability to interact with TEAD-1. In addition, the effect of YAP overexpression (YAPS94A and YAPS127A) on OS cell migration is associated with cytoskeleton reorganization. Since we previously demonstrated the crucial role of the TGF-β/Smad3 signaling pathway in OS migration and metastatic development, we study the interaction between YAP and Smad3 in OS cells. Using proximity ligation assay and immunoprecipitation approaches, and promoter/reporter assays, we demonstrated a functional interaction between YAP and the transcriptional factor Smad3 in OS cell lines regardless of YAP’s ability to interact with TEAD-1. Finally, we investigated the effect of verteporfin, identified as an inhibitor of the Hippo signaling pathway, on OS progression. In vitro and in vivo experiments showed that verteporfin inhibits YAP/TEAD-1 cascade, OS cell proliferation, and reduces primary tumor growth. In addition, verteporfin reduces in vivo lung metastases development. In this context, in vitro experiments demonstrated that verteprofin inhibits the TGF-β/Smad3 cascade and the response of TGF-β key targets involved in the metastasis dissemination process. Together these results demonstrate that YAP/TEAD-1 interactions are crucial in OS tumor growth and suggest that YAP/SMAD3 interactions are involved in OS lung metastasis dissemination. Furthermore, we show that verteporfin may be a promising therapeutic strategy against tumor progression of OS, specifically against lung metastasis dissemination. Citation Format: Sarah Morice, Mathilde Mullard, Sarah Renault, Jérôme Amiaud, Régis Brion, Isabelle Corre, Anne Gomez-Brouchet, Françoise Redini, Franck Verrecchia. Role of YAP/TEAD and YAP/Smad signaling pathways in osteosarcoma tumor growth and lung metastasis dissemination [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A07.

LncRNA MALAT1 facilitates lung metastasis of osteosarcomas through miR-202 sponging

Lungs are the primary metastatic sites for osteosarcomas responsible for associated mortality. Recent data has documented role of long non-coding RNAs (lncRNAs) in proliferation and growth of osteosarcoma cells. We evaluated a role of lncRNAs in the lung metastasis of osteosarcoma with the goal of identifying a unique signature. Comparison of different lncRNAs in tumor samples from osteosarcoma with and without lung metastasis led to identification of MALAT1 as the most differentially upregulated lncRNA in the osteosarcoma patients with lung metastasis. MALAT1 was also high in osteosarcoma cells KRIB and MALAT1’s targeted downregulation in these cells led to decreased invasive potential and identification of miR-202 as the miRNA that is sponged by MALAT1. In the lung metastasis in vivo model, parental KRIB cells metastasized to lungs and such metastasis was significantly inhibited in KRIB cells with downregulated MALAT1. Ectopic miR-202 expression attenuated KRIB downregulation-mediated effects on lung metastasis. In yet another in vivo model involving parental SAOS-2 and lung-metastatic derivatives SAOS-2-LM, MALAT1 expression was found to be elevated in lung metastatic cells, which also correlated with reduced miR-202. In conclusion, MALAT1-miR-202 represents a potential lncRNA-miRNA signature that affects lung metastasis of osteosarcomas and could potentially be targeted for therapy.

Abstract PR10: Disruption of IL6-mediated paracrine signaling to prevent pulmonary metastasis

Abstract Outcomes for patients diagnosed with osteosarcoma (OS) hinge primarily on whether or not they develop pulmonary metastasis. Looking to identify novel opportunities for therapeutic intervention, we have sought mechanisms that facilitate colonization of lung tissues by osteosarcoma cells. We have shown that metastatic colonization of lung by OS cells requires their IL6 and CXCL8 production, which is elicited upon the interaction of OS cells with lung tissues. The lung-derived signals that trigger this behavior and the effector mechanisms by which IL6 and CXCL8 facilitate metastatic colonization remain unknown. Here, we hypothesized that the interaction of OS cells lung epithelium triggers production of cytokines that establish paracrine signal amplification loops. We postulated that OS cell-derived IL6 and CXCL8 stimulate lung-resident cells to produce secondary signaling molecules that are, in turn, capable of stimulating OS cells. If our paracrine loop hypothesis is correct, we would expect that these lung cell-derived factors elicit further production of IL6 and CXCL8, resulting in a feed-forward paracrine signaling. Our lab has shown that soluble factors produced by lung epithelium profoundly increase IL6 and CXCL8 production by OS cells. In order to identify the specific factors responsible for this effect, we used RNA-seq to identify soluble ligands produced by lung epithelial cells that could activate cognate receptors produced by OS cells. Epithelial cell production of a subset of these soluble ligands increased when cells were exposed to supernatants from OS cells. We then performed a similar analysis using single-cell RNA-seq in mouse lungs bearing metastatic OS tumors, which identified many of the same ligands and receptors. Eleven candidate epithelial-to-tumor ligand-receptor interactions were identified in both the in vitro and the in vivo analysis. Interpreting these results in the context of prior known biology, one of these factors, IL-1, stood out as a prime candidate for a lung-to-tumor signal that would elicit IL6 and CXCL8 production in disseminated OS cells. We have since shown that IL-1 is upregulated in lung cells upon interaction with OS cells, that the cognate receptor IL1R1 is increased on OS cells following exposure to lung cell conditioned media, and that stimulation of OS cells with IL-1β drastically increases production of both IL6 and CXCL8. We have also verified production of IL-1α/β in lung epithelial cells and are working to confirm these findings in additional human-derived lung cell models. Our results suggest that this lung cell-tumor cell paracrine loop may play a mechanistic role in the colonization of lung tissue by OS. If true, the therapeutic effects of inhibiting this imminently targetable pathway on OS lung metastasis should be explored. Such studies are currently under way. This abstract is also being presented as Poster A61. Citation Format: John Hinckley, Amy Gross, Ryan Roberts. Disruption of IL6-mediated paracrine signaling to prevent pulmonary metastasis [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR10.

Chromosomal translocation-derived aberrant Rab22a drives metastasis of osteosarcoma.

Osteosarcoma is a type of aggressive malignant bone tumour that frequently metastasizes to lungs, resulting in poor prognosis. However, the molecular mechanisms of lung metastasis of osteosarcoma remain poorly understood. Here we identify exon-intron fusion genes in osteosarcoma cell lines and tissues. These fusion genes are derived from chromosomal translocations that juxtapose the coding region for amino acids 1-38 of Rab22a (Rab22a1-38) with multiple inverted introns and untranslated regions of chromosome 20. The resulting translation products, designated Rab22a-NeoFs, acquire the ability to drive lung metastasis of osteosarcoma. The Rab22a1-38 moiety governs the function of Rab22a-NeoFs by binding to SmgGDS-607, a GTP-GDP exchange factor of RhoA. This association facilitates the release of GTP-bound RhoA from SmgGDS-607, which induces increased activity of RhoA and promotes metastasis. Disrupting the interaction between Rab22a-NeoF1 and SmgGDS-607 with a synthetic peptide prevents lung metastasis in an orthotopic model of osteosarcoma. Our findings may provide a promising strategy for a subset of osteosarcoma patients with lung metastases.