Therapeutic Strategy For Osteosarcoma Research Articles

Deciphering the Effect of a Cu(II)-hydrazone Complex on Intracellular Cell Signalling Pathways in a Human Osteosarcoma 2D and 3D Models.

New therapeutic strategies for osteosarcoma (OS) have demonstrated the potential efficacy of copper compounds as anticancer drugs and as a substitute for the often used platinum compounds. OS is a type of bone cancer, primarily affecting young adults and children.The main objective of this work is to discover the molecular targets and cellular pathways related to the antitumor properties of a Cu(II)-hydrazone toward human OS 2D and 3D systems. Cell viability study using MG-63 cells was evaluated in OS monolayer and spheroids. CuHL significantly reduced cell viability in OS models (IC50 2D: 2.6±0.3 μM; IC50 3D: 9.9±1.4 μM) (p<0.001). Also, CuHL inhibits cell proliferation and it induces cells to apoptosis. The main mechanism of action found for CuHL are the interaction with DNA, genotoxicity, the ROS generation and the proteasome activity inhibition. Besides, 67 differentially expressed proteins were found using proteomic approaches. Of those 67 proteins, 40 were found overexpressed and 27 underexpressed. The response to stress and to unfolded protein, as well as ATP synthesis were the most affected biological process among upregulated proteins, whilst proteins related to DNA replication and redox homeostasis were downregulated.

Menadione and protocatechuic acid: A drug combination with antitumor effects in murine osteosarcoma cells

Osteosarcoma (OS) is a primary malignant bone tumor that has an abnormal expression of oncogenesis and tumor suppressors and causes dysregulation of various signaling pathways. Thus, novel therapeutic strategies for OS are needed to overcome the resistance of traditional treatments. This study evaluated the cytotoxic and anticancer effects of the association between menadione (MEN) and protocatechuic acid (PCA) in murine OS cells (UMR-106). The concentrations were 3.12 μM of isolated MEN, 500 μM of isolated PCA, and their associations. We performed cell viability assays, morphology modification analysis, cell migration by the wound-healing method, apoptosis by flow cytometry, reactive oxygen species (ROS) production, gene expression of NOX by RT-qPCR, and degradation of MMP-2 and 9 by zymography. Our results showed that the association of MEN+PCA was more effective in OS cells than the compounds alone. The association decreased cell viability, delayed cell migration, and decreased the expression of NOX-2 and ROS. In addition, the MEN+PCA association induced a slight increase in the apoptotic process. In summary, the association can enhance the compound's antitumor effects and establish a higher selectivity for tumor cells, possibly caused by significant mitochondrial damage and antioxidant properties.

Autophagy Modulation as a Potential Therapeutic Strategy in Osteosarcoma: Current Insights and Future Perspectives.

Autophagy, the process that enables the recycling and degradation of cellular components, is essential for homeostasis, which occurs in response to various types of stress. Autophagy plays an important role in the genesis and evolution of osteosarcoma (OS). The conventional treatment of OS has limitations and is not always effective at controlling the disease. Therefore, numerous researchers have analyzed how controlling autophagy could be used as a treatment or strategy to reverse resistance to therapy in OS. They highlight how the inhibition of autophagy improves the efficacy of chemotherapeutic treatments and how the promotion of autophagy could prove positive in OS therapy. The modulation of autophagy can also be directed against OS stem cells, improving treatment efficacy and preventing cancer recurrence. Despite promising findings, future studies are needed to elucidate the molecular mechanisms of autophagy and its relationship to OS, as well as the mechanisms underlying the functioning of autophagic modulators. Careful evaluation is required as autophagy modulation may have adverse effects on normal cells, and the optimization of autophagic modulators for use as drugs in OS is imperative.

Abstract 5010: Inhibition of discoidin domain receptor 1 (DDR1) as a new therapeutic strategy for osteosarcoma

Abstract Osteosarcoma is the most common type of bone cancer. Current management for osteosarcoma includes neoadjuvant chemotherapy and surgery. Unfortunately, some patients eventually develop recurrent or metastatic diseases and treatment options are extremely limited. Discoidin domain receptor 1 (DDR1) is a unique collagen-activated tyrosine kinase that participates in various human diseases, including cancer. DDR1 promotes the adhesion, proliferation, differentiation, migration, and metastasis of cancer cells. However, the expression and function of DDR1 remain unknown in osteosarcoma. The purpose of this study is to assess the expression, clinical prognostic relationship, and functional roles of DDR1 in osteosarcoma. The correlation between DDR1 expression in tumor tissues and clinicopathological features and prognosis was assessed via immunohistochemical staining of a unique tissue microarray (TMA) constructed from osteosarcoma specimens. Furthermore, DDR1 expression in osteosarcoma cell lines was determined by Western blot. DDR1-specific siRNA and a highly selective DDR1 inhibitor, 7rh, were applied to determine the impact of DDR1 expression on osteosarcoma cell growth and proliferation. In addition, the effect of DDR1 inhibition on clonogenicity was evaluated using a clonogenic assay, and a 3D cell culture model was used to mimic DDR1 effects in an in vivo environment. The results demonstrate that higher DDR1 expression significantly correlates with recurrence, metastasis, and shorter overall survival in osteosarcoma patients. The expression of DDR1 is also inversely correlated to the response to neoadjuvant chemotherapy. Therapeutically, DDR1 knockdown with siRNA or selective inhibition with 7rh decreases the proliferation and growth of osteosarcoma cells. In conclusion, our study supports DDR1 expression as an independent predictor of poor prognosis and a promising therapeutic target for osteosarcoma. Citation Format: Jinglu Wang, Robert Walker, Francis Hornicek, Zhenfeng Duan. Inhibition of discoidin domain receptor 1 (DDR1) as a new therapeutic strategy for osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5010.

Macrophage Repolarization as a Therapeutic Strategy for Osteosarcoma.

Macrophages are versatile immune cells and can adapt to both external stimuli and their surrounding environment. Macrophages are categorized into two major categories; M1 macrophages release pro-inflammatory cytokines and produce protective responses that lead to antimicrobial or antitumor activity. M2 or tumor-associated macrophages (TAM) release anti-inflammatory cytokines that support tumor growth, invasion capacity, and metastatic potential. Since macrophages can be re-polarized from an M2 to an M1 phenotype with a variety of strategies, this has emerged as an innovative anti-cancer approach. Osteosarcoma (OS) is a kind of bone cancer and consists of a complex niche, and immunotherapy is not very effective. Therefore, immediate attention to new strategies is required. We incorporated the recent studies that have used M2-M1 repolarization strategies in the aspect of treating OS cancer.

ZCCHC4 Promotes Osteosarcoma Progression by Upregulating ITGB1.

Zinc finger CCHC-type containing 4 (ZCCHC4), RNA binding protein, has been reported to mediate rRNA methylation and affect tumor cell proliferation. However, the role of ZCCHC4 in the regulation of osteosarcoma (OS) remains unknown. ZCCHC4 was highly expressed in OS tissues and cell lines. Overexpression or silencing of ZCCHC4 promoted or inhibited cell proliferation, epithelial-mesenchymal transition (EMT), and motility. Additionally, we proved that ZCCHC4 facilitates OS progression through upregulating integrin β1 (ITGB1). In the animal model, ZCCHC4 knockdown reduced OS tumor growth and metastases in vivo. Our findings showed that ZCCHC4 promoted the progression of OS through upregulating ITGB1 and suggested that inhibition of ZCCHC4 could be a novel therapeutic strategy for OS.

Injectable agarose hydrogels and doxorubicin-encapsulated iron-gallic acid nanoparticles for chemodynamic-photothermal synergistic therapy against osteosarcoma.

Osteosarcoma is a malignant bone cancer that usually occurs in children and adolescents. Although chemotherapy, radiotherapy and other methods have been used to treat osteosarcoma, these therapeutic regimens fail to cure this disease completely. Herein, doxorubicin-encapsulated iron-gallic acid (FeGA-DOX) nanoparticles (NPs) were fused with agarose hydrogels (AG) for synergistic therapy of osteosarcoma. Under near-infrared laser irradiation, the local temperature of FeGA-DOX NPs was increased. Therefore, tumour cells were killed using photothermal therapy, and AG dissolved to release FeGA-DOX into the cells. Doxorubicin generates hydrogen peroxide, which is then converted to reactive oxygen species (ROS) via FeGA-DOX by the Fenton reaction, inducing tumour cell apoptosis. ROS induced by chemodynamic therapy compensates for the incomplete cure of osteosarcoma cells. The AG-encapsulated NPs could mediate synergistic chemodynamic and photothermal therapy with self-sufficient H2O2, providing a novel therapeutic strategy for osteosarcoma.

Compound Library Screening for Synergistic Drug Combinations: mTOR Inhibitor and Proteasome Inhibitor Effective Against Osteosarcoma Cells.

The development of new drugs is urgently needed for new treatment strategies that can improve the prognosis of osteosarcoma (OS). In this study, we attempted to identify combinations of new molecular-targeted agents for OS. A library containing 324 compounds was used. For the first screening, MG-63 OS cells were treated with each of the compounds and cell viability was measured. After the best candidate compound was decided, the compound was included in a second screening. The combination of most effective compounds was decided. The antiproliferative effect of the combination was examined and the cell signaling mechanism was evaluated by western blot analysis. 143B OS-bearing mice were used for in vivo antitumor testing. In the first screening, bortezomib was chosen as the effective drug. In the second screening with bortezomib, everolimus was chosen. This combination showed a synergistic inhibitory effect on cell proliferation when compared to monotherapy with each of these drugs alone. Compared to monotherapy, the combination therapy enhanced the levels of cleaved poly (ADP-ribose) polymerase, caspase-3, caspase-8 and caspase-9, phospho-c-Jun N-terminal kinase, and P38. In contrast, the levels of c-MYC proto-oncogene bHLH transcription factor, survivin, and phospho-cyclin D1 were reduced. The combination effectively induced apoptosis and interfered with cell cycle progression. In the in vivo analysis, the combination therapy significantly inhibited tumor growth. The combination of everolimus and bortezomib demonstrated a synergistic effect against OS both in vitro and in vivo. These results indicate that this combination may be useful as a novel therapeutic strategy for OS.

The prognostic value and immune landscapes of m1A/m5C/m6A-associated lncRNA signature in osteosarcoma.

RNA methylation modifications, mainly including N1-methyladenosine (m1A), 5-methylcytosine (m5C), and N6-methyladenosine (m6A), are widely existed in osteosarcoma and involved in the biological processes of cancers. However, there is still no study regarding the relationship between osteosarcoma and m1A/m5C/m6A-associated long non-coding RNAs (lncRNAs). Here, expression data of osteosarcoma from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database were retrieved to identify ER-related lncRNAs associated with the overall survival (OS) of osteosarcoma patients. Then, Lasso penalized Cox regression analysis was applied to construct a lncRNAs risk signature. Meanwhile, patients were stratified into two clusters based on the identified m1A/m5C/m6A-associated lncRNAs. The prognostic value and immune landscape of the identified signature and clusters were further evaluated. Two m1A/m5C/m6A-associated lncRNAs were incorporated into our risk signature. The functional analyses indicated that the prognostic model was correlated with patient survival, and cancer metastasis and growth. Meanwhile, the signature model was significantly associated with the infiltration of immune cells, immune microenvironment, as well as several immune checkpoint genes. Similar results were detected for the lncRNAs clusters, which were significantly correlated with immune infiltration, cancer microenvironment, and immune-associated genes, and contributed to predicting the prognosis of patients. Moreover, our risk signature and clusters might help guide the application of immunotherapeutic drugs for osteosarcoma patients. Finally, a nomogram based on the risk score was established. Overall, a risk signature based on two m1A/m5C/m6A-associated lncRNAs was generated and presented predictive value for the prognosis and immune landscapes of osteosarcoma patients. This signature can be further used in the development of novel therapeutic strategies for osteosarcoma.

Abstract 1061: Exportin 1 (XPO1) inhibition alone or in combination as a novel therapeutic strategy in osteosarcoma

Abstract Background: Osteosarcoma is an aggressive bone cancer in which therapeutic advancements have been limited over the last 30 years, in part due to genomic heterogeneity. The combination of high-throughput drug screening platforms that efficiently pinpoint drug sensitivities with patient-derived cross-species models is an innovative approach to address the critical need to identify novel treatment strategies for osteosarcoma patients. Methods: We performed high-throughput drug screens on patient-derived osteosarcoma cell lines D418 (canine) and 17-3x (human), followed by validation of the top compounds, to identify drug sensitivities and novel therapeutic combinations. Results: High-throughput drug screens using 2100 bioactive compounds show that osteosarcoma cell lines D418 and 17-3x exhibited sensitivity to standard-of-care chemotherapy drugs, inhibitors of XPO1 nuclear export, and proteasome inhibitors. The XPO1 inhibitor, verdinexor (VER), and the proteasome inhibitor, bortezomib (BORT), induced dose-dependent cytotoxicity in multiple osteosarcoma cell lines (D418, IC50VER: 3187 nM, IC50BORT: 2.8 nM; 17-3x IC50VER: 679 nM, IC50BORT: 10.9 nM). In addition, dual XPO1 and proteasome inhibition synergistically reduced cell proliferation in D418 (synergy score=12.89) and 17-3x (synergy score=17.87) cell lines (p &amp;lt;0.05). Selinexor (SEL), an FDA approved XPO1 inhibitor used in combination with bortezomib to treat multiple myeloma, also demonstrated dose-dependent single-agent activity in patient-derived osteosarcoma cell lines (D418, IC50SEL: 370 nM; 17-3x IC50SEL: 101 nM). With drug screening of 119 oncology compounds in combination with selinexor in 17-3x cells, XPO1 inhibition again shows synergistic activity with proteasome inhibition in osteosarcoma. Conclusions: Inhibition of XPO1-mediated nuclear export is a promising therapeutic strategy in osteosarcoma. These effects may be further potentiated when used in combination with other agents, such as proteasome inhibitors. Additional drug screening and validation assays are underway to identify novel synergistic agents for use in combination with XPO1 inhibitors in osteosarcoma. Citation Format: Laurie Graves, Gabrielle Rupprecht, Erdem Altunel, Etienne M. Flamant, Sneha Rao, Dharshan Sivara, Alexander L. Lazarides, Sarah M. Hoskinson, Maya U. Sheth, Serene Cheng, So Young Kim, Kathryn E. Ware, Anika Agarwal, Mark M. Cullen, Casey Syal, Laura E. Selmic, Jeffrey I. Everitt, Shannon J. McCall, Cindy Eward, Trinayan Kashyap, Marie Maloof, Christopher J. Walker, Yosef Landesman, Lars Wagner, William C. Eward, David S. Hsu, Jason A. Somarelli. Exportin 1 (XPO1) inhibition alone or in combination as a novel therapeutic strategy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1061.

Selected Articles from This Issue

Central nervous system (CNS) metastases are a prominent cause of morbidity and mortality in patients with ALK-positive (ALK+) non-small cell lung cancer (NSCLC). ASCEND-7 was one of the first prospective studies designed to use homogenous eligibility criteria, and assessments to evaluate intra/extracranial responses and activity of an ALK targeted agent, in the presence or absence of prior treatment, in patients with ALK+ NSCLC with active brain metastases and leptomeningeal metastasis. The phase II ASCEND-7 (NCT02336451) study was designed to assess the efficacy and safety of ALK inhibitor (ALKi) ceritinib in patients with ALK+ NSCLC metastatic to the brain and/or leptomeninges. Chow and colleagues report that ceritinib showed antitumor activity in patients with ALK+ NSCLC with active CNS disease and could be considered in the management of intracranial metastasis.Results from cohort 1 of the phase II, open-label KEYNOTE-224 study demonstrated that pembrolizumab monotherapy was efficacious and tolerable in patients with advanced hepatocellular carcinoma previously treated with sorafenib. In this article, Verset and colleagues report on cohort 2 of the KEYNOTE-224 study that investigated pembrolizumab monotherapy as a therapeutic approach for patients with advanced hepatocellular carcinoma, which had not been previously treated with systemic therapy. The results showed that pembrolizumab monotherapy in this population had promising efficacy, including objective response rate with durable antitumor activity and no new safety signals. These findings support further evaluation of pembrolizumab alone or in combination with other agents for the treatment of hepatocellular carcinoma.Podoplanin (PDPN) is highly expressed in several types of tumors and is known to promote tumor growth and metastasis through binding with a C-type lectin-like receptor 2 (CLEC-2) on platelets. In osteosarcoma, PDPN-CLEC-2 binding induced PDGFs release from platelets and resulted in PDGFRs mediated growth promotion of osteosarcoma. In this study, Takemoto and colleagues developed a humanized anti-PDPN neutralizing antibody, named AP201, aiming for the clinical application for PDPN highly expressing cancer such as osteosarcoma. AP201 potently neutralized PDPN-CLEC-2 binding and inhibited tumor growth and metastasis in osteosarcoma xenograft models. Thus, targeting PDPN with a neutralizing antibody could be a novel therapeutic strategy for osteosarcoma treatment.Kaposi sarcoma (KS) remains among the most common malignancies occurring with HIV infection (HIV-KS). Oral anti-inflammatory, anti-angiogenic, and immunomodulatory agent, lenalidomide, is a potentially attractive alternative to standard chemotherapy for KS. Here, Reid and colleagues present results of a phase I/II trial to determine the maximum tolerated dose (MTD) and response rates for lenalidomide in HIV-KS. The MTD was not reached, but the authors recommended 25 mg as the phase II dose (RP2D). Reported adverse events were consistent with the established safety profile of lenalidomide and 60% of participants receiving RP2D obtained a partial response. Correlative analysis performed in a subset of participants demonstrated a significant increase in proportions of blood T cells with a regulatory phenotype, and plasma cytokines trended toward a less inflammatory pattern. Clinical response was associated with loss of KSHV transcription. Following on this study, multicenter domestic and international AIDS Malignancy Consortium trials are underway to confirm the tolerability and activity of immunomodulatory therapy in KS.

Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway

BackgroundTetraspanins are members of the 4-transmembrane protein superfamily (TM4SF) that function by recruiting many cell surface receptors and signaling proteins into tetraspanin-enriched microdomains (TEMs) that play vital roles in the regulation of key cellular processes including adhesion, motility, and proliferation. Tetraspanin7 (Tspan7) is a member of this superfamily that plays documented roles in hippocampal neurogenesis, synaptic transmission, and malignant transformation in certain tumor types. How Tspan7 influences the onset or progression of osteosarcoma (OS), however, remains to be defined. Herein, this study aimed to explore the relationship between Tspan7 and the malignant progression of OS, and its underlying mechanism of action.MethodsIn this study, the levels of Tspan7 expression in human OS cell lines were evaluated via qRT-PCR and western blotting. The effect of Tspan7 on proliferation was examined using CCK-8 and colony formation assays, while metastatic role of Tspan7 was assessed by functional assays both in vitro and in vivo. In addition, mass spectrometry and co-immunoprecipitation were performed to verify the interaction between Tspan7 and β1 integrin, and western blotting was used to explore the mechanisms of Tspan7 in OS progresses.ResultsWe found that Tspan7 is highly expressed in primary OS tumors and OS cell lines. Downregulation of Tspan7 significantly suppressed OS growth, metastasis, and attenuated epithelial-mesenchymal transition (EMT), while its overexpression had the opposite effects in vitro. Furthermore, it exhibited reduced OS pulmonary metastases in Tspan7-deleted mice comparing control mice in vivo. Additionally, we proved that Tspan7 interacted with β1 integrin to facilitate OS metastasis through the activation of integrin-mediated downstream FAK-Src-Ras-ERK1/2 signaling pathway.ConclusionIn summary, this study demonstrates for the first time that Tspan7 promotes OS metastasis via interacting with β1 integrin and activating the FAK-Src-Ras-ERK1/2 pathway, which could provide rationale for a new therapeutic strategy for OS.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12935-022-02591-1.

Ailanthone Inhibits Proliferation, Migration and Invasion of Osteosarcoma Cells by Downregulating the Serine Biosynthetic Pathway

Osteosarcoma (OS) is the most common primary bone sarcoma, chemoresistance becomes an obstacle to its treatment. Metabolic reprogramming is a hallmark of malignancy, targeting the metabolic pathways might provide a reasonable therapeutic strategy for OS. Here we demonstrated that Ailanthone (AIL), a major component of the Chinese medicine Ailanthus altissima, significantly suppressed OS cell growth in vitro and in vivo. Furthermore, AIL dose-dependently inhibited cell migration and invasion, induced cell cycle arrest and apoptosis in OS cells. Combined transcriptomics, proteomics and metabolomics analyses revealed that AIL induced widespread changes in metabolic programs in OS cells, while the serine biosynthetic pathway (SSP) was the most significantly altered pathway. qRT-PCR and Western blot assay confirmed that the transcript and protein levels of the SSP genes (PHGDH, PSAT1 and PSPH) were downregulated dose-dependently by AIL. In addition, we found out that many downstream pathways of the SSP including the one-carbon pool by folate, purine metabolism, pyrimidine metabolism, DNA replication and sphingolipid metabolism were downregulated after AIL treatment. In the revere test, PHGDH overexpression but not exogenous serine supplementation clearly attenuated the effects of AIL on OS cells. Taken together, AIL exerts antitumor effects on OS through mediating metabolic reprogramming, at least in part, by suppressing the SSP. Our findings suggest that AIL could emerge as a potential therapeutic strategy in OS.

MiR-1270 enhances the proliferation, migration, and invasion of osteosarcoma via targeting cingulin.

Osteosarcoma (OS), characterized by high morbidity and mortality, is the most common bone malignancy worldwide. MicroRNAs (miRNAs) play a crucial role in the initiation and development of OS. The purpose of this study was to investigate the roles of miR-1270 in OS. RT-qPCR and Western blot were applied to detect the mRNA and protein level, respectively. CCK-8, colony formation, and TUNEL assays were conducted to determine the cell viability, proliferation, and apoptosis of OS cells. Wound healing and transwell assay were performed to detect the migration and invasion ability of OS cells. Bioinformatics analysis and dual-luciferase reporter assay were used to predict the target genes of miR-1270. Tumor xenograft in vivo assay was carried out to determine miR-1270 effect on the tumor size, volume, and weight. In this study, miR-1270 was overexpressed in OS tissues and cells. However, miR-1270 knockdown inhibited the proliferation, migration and invasion, and promoted the OS cells’ apoptosis. Mechanistically, cingulin (CGN) was predicted and proved to be a target of miR-1270 and partially alleviated the effects of miR-1270 on the proliferation, migration and invasion ability of OS cells. Taken together, knockdown of miR-1270 may inhibit the development of OS via targeting CGN. This finding may provide a novel therapeutic strategy for OS.

LncRNA MATN1-AS1 for Prediction of Prognosis in Osteosarcoma Patients and Its Cellular Function.

Long non-coding RNAs show essential roles in various cancer processes. This study aimed at the expression features, prognosis significance, and biological effect of lnc MATN1-AS1 in osteosarcoma (OS). Five kinds of cell lines and 117 pairs of tissues were analyzed by qRT-PCR for quantification of lnc MATN1-AS1 and miR-1299 level. Clinical data were analyzed using Chi-Square Tests to show the association with lnc MATN1-AS1 level. Kaplan-Meier analysis and Cox regression were used to judge the prognostic value. Cell counting kit-8 and Transwell assay were conducted, respectively, to analyze the effect of lnc MATN1-AS1 on cell proliferation and metastasis. The target miRNA was predicted. lnc MATN1-AS1 level was significantly elevated in OS cells and tissues and related to Enneking staging, lung metastasis, and histologic type. Patients with high lnc MATN1-AS1 level showed a shorter overall survival and recurrence-free survival. Lnc MATN1-AS1 knockdown inhibited OS cell proliferation, migration, and invasion by sponging miR-1299. Lnc MATN1-AS1 has oncogenic features and prognostic significance in OS and is a novel therapeutic strategy for OS.

Iron Chelator Induces Apoptosis in Osteosarcoma Cells by Disrupting Intracellular Iron Homeostasis and Activating the MAPK Pathway.

Osteosarcoma is a common malignant bone tumor in clinical orthopedics. Iron chelators have inhibitory effects on many cancers, but their effects and mechanisms in osteosarcoma are still uncertain. Our in vitro results show that deferoxamine (DFO) and deferasirox (DFX), two iron chelators, significantly inhibited the proliferation of osteosarcoma cells (MG-63, MNNG/HOS and K7M2). The viability of osteosarcoma cells was decreased by DFO and DFX in a concentration-dependent manner. DFO and DFX generated reactive oxygen species (ROS), altered iron metabolism and triggered apoptosis in osteosarcoma cells. Iron chelator-induced apoptosis was due to the activation of the MAPK signaling pathway, with increased phosphorylation levels of JNK, p38 and ERK, and ROS generation; in this process, the expression of C-caspase-3 and C-PARP increased. In an orthotopic osteosarcoma transplantation model, iron chelators (20 mg/kg every day, Ip, for 14 days) significantly inhibited the growth of the tumor. Immunohistochemical analysis showed that iron metabolism was altered, apoptosis was promoted, and malignant proliferation was reduced with iron chelators in the tumor tissues. In conclusion, we observed that iron chelators induced apoptosis in osteosarcoma by activating the ROS-related MAPK signaling pathway. Because iron is crucial for cell proliferation, iron chelators may provide a novel therapeutic strategy for osteosarcoma.

Deregulation of CLTC interacts with TFG, facilitating osteosarcoma via the TGF-beta and AKT/mTOR signaling pathways.

Although the treatment of osteosarcoma has improved, the overall survival rate of this common type of osseous malignancies has not changed for four decades. Thus, new targets for better therapeutic regimens are urgently needed. In this study, we found that high expression of clathrin heavy chain (CLTC) was an independent prognostic factor for tumor‐free survival (HzR, 3.049; 95% CI, 1.476–6.301) and overall survival (HzR, 2.469; 95% CI, 1.005–6.067) of patients with osteosarcoma. Down‐regulation of CLTC resulted in tumor‐suppressive effects in vitro and in vivo. Moreover, we found that CLTC was transcriptionally regulated by a transcription factor—specificity protein 1 (SP1), which binds to the CLTC promoter at the −320 to −314‐nt and +167 to +173‐nt loci. Mechanistic investigations further revealed that CLTC elicited its pro‐tumor effects by directly binding to and stabilizing trafficking from the endoplasmic reticulum to the Golgi regulator (TFG). Importantly, overexpression of TFG rescued both the tumor‐suppressive effect and inhibition of the TGF‐β and AKT/mTOR pathways caused by CLTC down‐regulation, which indicated that the activity of CLTC was TFG‐dependent. Immunohistochemistry analysis confirmed that CLTC expression was positively correlated with TFG expression. These findings collectively highlight CLTC as a new prognostic biomarker for patients with osteosarcoma, and the interruption of the SP1/CLTC/TFG axis may serve as a novel therapeutic strategy for osteosarcoma.

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.

Long Noncoding RNA HAGLROS Promotes Cell Invasion and Metastasis by Sponging miR-152 and Upregulating ROCK1 Expression in Osteosarcoma.

Background Long noncoding RNAs (lncRNAs) played a crucial role in a number of biological processes. lncRNA HAGLROS was demonstrated to facilitate cell proliferation and migration in various cancers. However, the functions and molecular mechanisms of HAGLROS in osteosarcoma remained to be elucidated. Methods qRT-PCR assay was used to detect the relative expression of HAGLROS in osteosarcoma tissue samples and cells. CCK-8 and Transwell assays were performed to assess the effects of HAGLROS on OS cells proliferation and invasion. Luciferase reporter assay verified the interaction between ROCK1 and miR-152. Results In our study, we found that the expression of HAGLROS increased osteosarcoma samples and cell lines compared with normal tissues and cells. HAGLROS knockdown inhibited certain functions of U2OS and SW1353 cells in vitro. Moreover, HAGLROS depletion inhibited tumor growth and metastasis in vivo. Mechanically, we found that HAGLROS sponged miR-152 to promote ROCK1 expression in U2OS and SW1353 cells. Conclusion In summary, our study indicated that HAGLROS could promote osteosarcoma progression by sponging miR-152 to promote ROCK1 expression. The results showed HAGLROS/miR-152/ROCK1 axis might act as a novel therapeutic strategy for osteosarcoma.

Positive feedback loop SP1/MIR17HG/miR-130a-3p promotes osteosarcoma proliferation and cisplatin resistance

Long noncoding RNAs (lncRNAs) have been identified to be critical regulator in the osteosarcoma (OS) tumorigenesis. However, the role of lncRNA MIR17HG in the OS proliferation and chemotherapy resistance is still unclear. Here, this research aims to investigate the function of lncRNA MIR17HG in the OS proliferation and cisplatin resistance. Clinically, results revealed that higher MIR17HG expression was associated with shorter overall survival. Functional investigations indicated that MIR17HG promoted the proliferation, invasion and cisplatin resistance of OS cells in vitro, and the MIR17HG knockdown inhibited the growth in vivo. Mechanistically, MIR17HG targeted the miR-130a-3p/SP1 axis, moreover, transcription factor SP1 bind with the MIR17HG promoter region to promote its expression. Taken together, MIR17HG displays the tumor-promotive role in the progression of OS through SP1/MIR17HG/miR-130a-3p/SP1 feedback loop. Our findings might help us to offer novel therapeutic strategies for OS.