Osteosarcoma Chemoresistance Research Articles

ZBTB7A Enhances Osteosarcoma Chemoresistance by Transcriptionally Repressing lncRNALINC00473-IL24 Activity

Chemoresistance remains a major drawback to osteosarcoma treatment. ZBTB7A, a member of the POK transcription repressor family, was shown to play an important role in tumorigenesis. However, the effect of ZBTB7A on osteosarcoma chemoresistance is completely unknown. In this study, we found that ZBTB7A is increased in cisplatin-resistant osteosarcoma cells and that elevated ZBTB7A inhibits cisplatin-induced apoptosis by repressing LINC00473 expression. Further mechanistic studies revealed that ZBTB7A directly binds to the promoter and suppresses the transcription of LINC00473. Additionally, our data indicate that LINC00473 interacts with the transcript factor C/EBPβ, facilitating its binding to the promoter of IL24, leading to decrease chemoresistance. Thus, these findings indicate that the ZBTB7A-mediated LINC00473-C/EBPβ-IL24 pathway is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

1678P - A new chemotherapy-based combination to prevent osteosarcoma progression

Background: Despite the intensification of chemotherapy regimen, 5 years survival rates for patients with metastatic or relapsed osteosarcoma (OS) remains of 20%. The secreted factor netrin 1 (Nt1) is overexpressed in many human cancers to block apoptosis. Recent studies showed that blocking Nt1 interaction with its receptors potentiates chemotherapy efficacy suggesting that combining chemotherapies with Nt1 interference could be a promising approach for chemoresistant tumors like OS. Methods: Analyses of the ATGSarc database (http://atg-sarc.sarcomabcb.org/), indicate that Sarcoma with complex genomic (SCG) with a higher expression of Nt1 have a poorer outcome (p < 0.002). In addition, q PCR performed on human sarcomas samples showed that Nt1 is higher expressed in OS compared to other SCG (7.75 fold increase - p < 0.02). These data indicated that Nt1 could be a potential target for OS treatment. Thus, we evaluated the antitumoral effects of anti Nt1 monoclonal antibody (aNt1) combined to doxorubicin (Dox) in a rat syngeneic and metastatic OS model. In this model, treatments were administered either on progressive OS or post operatively to prevent OS relapse. At the end of the experiments tumors and lung were collected for IHC analyses. Results: As pre operative treatment, Dox/aNt1 combination caused a marked delay in OS progression (median end point reached at day 17 and day 22 respectively in Dox and Dox/aNt1 group, (p < 0.02) and dramatically slowed down metastatic spreading: lung metastases (d > 5mm) were found respectively in 75% and 17% of Dox and Dox/aNt1 treated rats As post operative treatment, Dox/aNt1combination significantly increased animals survival (median end point reached at day 15 and day 21 respectively in Dox and in Dox/aNt1 group; (p < 0.02). Moreover, 19 days after tumor resection, 10% of the Dox treated tumors hadn’t relapsed versus 40% in the Dox/aNt1 treated group. A variation in tumor vascular density caused by the treatment was found in the Anti Nt1 treated groups as shown by CD146 staining. Conclusions: Our study reporting the antiproliferative and antimetastatic effects and of Dox/aNt1 Combination in OS indicate that this combined treatment could be a way to overcome OS chemoresistance. Legal entity responsible for the study: Dutour Aurélie Funding: None Disclosure: All authors have declared no conflicts of interest.

Pleiotrophin promotes chemoresistance to doxorubicin in osteosarcoma by upregulating P-glycoprotein.

Chemoresistance is a major hindrance to successful treatment of osteosarcoma (OS). Pleiotrophin (PTN), a neurotrophic growth factor, has been linked to the malignant characteristics of various cancer types. We retrospectively examined the correlation between PTN expression and chemoresistance in OS in a cohort of 133 OS patients. Immunohistochemistry revealed that PTN expression correlated with the necrosis rate and local OS recurrence. In a prognostic analysis, high PTN expression was associated with poor overall and disease-free survival, and was an independent adverse prognostic factor for disease-free survival. In doxorubicin-treated OS cells, PTN knockdown enhanced cellular chemosensitivity, increased the apoptosis rate and inhibited clone formation, while PTN overexpression had the opposite effects. In a xenograft model, PTN knockdown and overexpression respectively enhanced and reduced cellular sensitivity to doxorubicin. PTN upregulated anaplastic lymphoma kinase (ALK), p-Glycogen Synthase Kinase (GSK)3β, β-catenin and multidrug resistance protein 1/P-glycoprotein (MDR1/P-gp). In rescue assays with the β-catenin inhibitor XAV939 and the MDR1/P-gp inhibitor verapamil, PTN promoted chemoresistance to doxorubicin in OS cells by activating ALK/GSK3β/β-catenin signaling, thereby upregulating MDR1/P-gp. Therefore, PTN could be used as a biomarker predicting chemotherapeutic responses, and downregulating PTN could be a promising therapeutic strategy to prevent chemoresistance in OS patients.

Abstract 4505: Activating transcription factor-6α dependent regulation of Rheb-mTOR and Notch signaling contributes to chemoresistance in osteosarcoma

Abstract The standard treatment for patients with newly diagnosed osteosarcoma (OS) consists of surgery in combination with multi-agent chemotherapy. However, the optimal treatment strategy for patients with refractory and/or metastatic disease is yet to be defined and continues to be a persistent challenge in osteosarcoma treatment. The high degree of genetic aberrations and tumor heterogeneity has impeded the identification and testing of new and effective therapeutic targets. Therefore, finding and characterizing cellular mechanisms, which contribute to OS chemoresistance could be one promising strategy for designing therapies that can change the outlook for patients with this disease. Due to the well-known role of unfolded protein response (UPR) in promoting chemoresistance in solid tumors our objective here is to examine how this promotes chemoresistance in patients with OS. Using in silico pathway analysis of gene expression datasets from patients with OS we found that protein processing in the ER was one of the pathways that was significantly enriched in OS tumors compared to normal tissue. We then examined whether the UPR sensors ATF6α, PERK and IRE-1α were also activated in OS cells in vitro. Using western blotting, qPCR and immunofluorescence we confirmed that all three UPR pathways were activated in human OS cell lines. However we found that only ATF6α activation significantly enhanced chemoresistance to cisplatin, irinotecan and combinatorial treatment with cisplatin and the mTOR inhibitor rapamycin. This occurred via inhibition of Bax activation, suppression of RHEB-mTOR and NOTCH signaling. Our findings highlight a novel mechanism of chemo-resistance in OS. Furthermore, retrospective analysis of banked OS patient samples for ATF6α expression followed by a preliminary multivariate analysis using Cox regression model showed that high levels of nuclear ATF6α, was an independent prognostic indicator while also taking into account the presence of metastases and tumor site. Moreover, we found that while 50% patients with high nuclear ATF6α levels had a poor histologic response to treatment only 32% patients with low nuclear ATF6α expression suggesting that ATF6α activation could be associated with resistance to chemotherapy and poor outcome for OS patients. Our findings emphasize a role for ATF6α as the nexus of a versatile signaling network that regulates pathways that are crucial for the pathogenesis and therapy resistance of osteosarcoma. Hence therapeutic targeting of the ATF6α pathway holds promise as an innovative and effective treatment strategy for OS. Citation Format: Suma Yarapureddy, Pooja Hingorani, Saravana Kumar, Omar Asad, Jazmine Abril, Veena Sharath, Paul Dickman, Aparna C. Sertil. Activating transcription factor-6α dependent regulation of Rheb-mTOR and Notch signaling contributes to chemoresistance in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2017-4505

MiR-491 Inhibits Osteosarcoma Lung Metastasis and Chemoresistance by Targeting αB-crystallin

Dysregulated microRNAs (miRNAs) play an important role in osteosarcoma (OS) progression. In the present study, we investigate the clinical significance of serum miR-491 level and the potential role of miR-491 in OS lung metastasis and chemoresistance. Clinical data show that the level of miR-491 was decreased in serum from OS patients compared with healthy control subjects, and that a decreased serum miR-491 level is correlated with increased metastasis, poor chemoresponse, and lower survival rate in OS patients. Invitro and invivo experiments show that overexpression of miR-491 suppresses OS cell lung metastasis, whereas it enhances cisplatin (CDDP)-induced tumor growth inhibition and apoptosis. In contrast, inhibition of miR-491 stimulates OS cell lung metastasis and suppresses CDDP-induced tumor growth inhibition and apoptosis. Furthermore, we demonstrate that miR-491 exerts its role by directly targeting αB-crystallin (CRYAB) in OS. Our findings suggest that serum level of miR-491 has potential as a biomarker for predicting OS progression and prognosis of OS patients. Additionally, restoration of miR-491 may be a novel strategy for inhibiting OS lung metastasis and overcoming OS cell resistance to chemotherapy.

LncRNA FENDRR sensitizes doxorubicin-resistance of osteosarcoma cells through down-regulating ABCB1 and ABCC1.

Long noncoding RNAs (LncRNAs) act as crucial regulators in various cancers including osteosarcoma (OS), yet their potential roles and molecular mechanisms in OS chemoresistance remain unclear. In the present study, we investigated the role and potential regulatory mechanism of the most down-regulated expressed lncRNA, FENDRR screened by our previous lncRNA microarray analysis between the paired doxorubicin-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63). FENDRR expression was down-regulated in the doxorubicin-resistant OS cell lines and tissues and negatively correlated to the poor prognosis of OS patients. Overexpression of FENDRR suppressed doxorubicin-resistance, G2/M phase of cell cycle, and promoted cell apoptosis of osteosarcoma cells in vitro and tumor growth in vivo whereas FENDRR knockdown had the opposite effects. In addition, we found that FENDRR was mainly located in the cytoplasm and could regulate the drug resistance of osteosarcoma cells by negatively affecting posttranscriptional expression of ABCB1 and ABCC1. Together, our study demonstrated that lncRNA FENDRR may act as an inhibitory molecule of doxorubicin-resistance through down-regulating the expression of ABCB1 and ABCC1 genes in osteosarcoma cells. These findings may extend the function of FENDRR in tumor progression and provide a novel target for reversing OS chemoresistance.

Knockdown of STMN1 enhances osteosarcoma cell chemosensitivity through inhibition of autophagy.

Chemoresistance is a major cause for the poor prognosis of osteosarcoma (OS) patients. However, our understanding of mechanisms underlying chemoresistance in OS are limited. The present study aimed to investigate the effect of stathmin 1 (STMN1) on paclitaxel-induced chemoresistance, as well as the underlying mechanism. Western blot analysis data revealed that the expression level of STMN1 was dramatically increased in OS cell lines (HOS, Saos-2, U-2OS and MG-63), when compared to normal osteoblast hFOB1.19 cells. Furthermore, treatment with paclitaxel led to upregulation of STMN1 in U-2OS cells, accompanied by activation of autophagy, which may attenuate the cytotoxicity of paclitaxel in OS cells. Following knockdown of STMN1 expression, paclitaxel-induced autophagy was significantly reduced, accompanied by increased cytotoxicity of paclitaxel to U-2OS cells. In addition, blockade of mammalian target of rapamycin signaling attenuated the inhibitory effect of STMN1 knockdown on autophagy in OS cells. In conclusion, the present study demonstrated that knockdown of STMN1 enhances osteosarcoma cell chemosensitivity to paclitaxel through inhibition of autophagy. Therefore, STMN1 may be a potential target for the treatment of chemoresistant OS.

MicroRNA-140-5p regulates osteosarcoma chemoresistance by targeting HMGN5 and autophagy

Chemotherapy is an important treatment modality for osteosarcoma. However, it often fails because of chemoresistance, especially multidrug resistance. Previously, we found several genes were involved in chemoresistance development. In this report, we used high-throughput microRNA (miRNA) expression analysis to reveal that expression of miR-140-5p was associated with chemosensitivity in osteosarcoma. The exact roles of miR-140-5p in the chemoresistance of osteosarcoma were then investigated, we found that knockdown of miR-140-5p enhanced osteosarcoma cells resistance to multiple chemotherapeutics while overexpression of miR-140-5p sensitized tumors to chemotherapy in vitro. Moreover, in vivo, knockdown of miR-140-5p also increased the osteosarcoma cells resistance to chemotherapy. Luciferase assay and Western blot analysis showed that HMGN5 was the direct target of miR-140-5p which could positively regulated autophagy. Silencing these target genes by siRNA or inhibition of autophagy sensitized osteosarcoma cells to chemotherapy. These findings suggest that a miR-140-5p/HMGN5/autophagy regulatory loop plays a critical role in chemoresistance in osteosarcoma. In conclusion, our data elucidated that miR-140-5p promoted autophagy mediated by HMGN5 and sensitized osteosarcoma cells to chemotherapy. These results suggest a potential application of miR-140-5p in overall survival, chemoresistance prognosis and treatment.

Enhanced Stim1 expression is associated with acquired chemo-resistance of cisplatin in osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor. Although cisplatin is the primary chemotherapy used in osteosarcoma treatment, the cisplatin resistance remains a big challenge for improving overall survival. The store-operated calcium (Ca2+) entry (SOCE) and its major mediator Stim1 have been shown to be implicated in a number of pathological processes typical for cancer. In this study, we showed that Stim1 expression was significantly increased in chemo-resistant osteosarcoma tissues compared with chemo-sensitivity tissues. Patients with Sitm1 expression exhibited poorer overall survival than Stim1-negative patients. Moreover, un-regulation of Stim1 expression and SOCE were also observed in cisplatin-resistant MG63/CDDP cells compared with their parental cells. Cisplatin treatment obviously reduced Stim1 expression and SOCE in cisplatin-sensitivity MG63 cells, but had no effects on MG63/CDDP cells. In addition, cisplatin resulted in a more pronounced increase of endoplasmic reticulum (ER) stress in MG63 cells than in their resistant variants, which was evidenced by the activation of molecular markers of ER stress, GRP78, CHOP and ATF4. Knockdown of Stim1 using siRNA remarkably enhanced cisplatin-induced apoptosis and ER stress in MG63/CDDP cells, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of Stim1 markedly reversed apoptosis and ER stress following cisplatin treatment. Taken together, these results demonstrate that Stim1 as well as Ca2+ entry contributes cisplatin resistance via inhibition of ER stress-mediated apoptosis, and provide important clues to the mechanisms involved in cisplatin resistance for osteosarcoma treatment. Stim1 represents as a target of cisplatin and blockade of Stim1-mediated Ca2+ entry may be a useful strategy to improve the efficacy of cisplatin to treat osteosarcoma.

Long non-coding RNA CTA sensitizes osteosarcoma cells to doxorubicin through inhibition of autophagy.

Recently, several long non-coding RNAs (lncRNAs) have been implicated in osteosarcoma (OS). However, the regulatory roles of lncRNAs in chemotherapy resistance of OS still remain unclear. This study aimed to screen a novel lncRNA that contributes to chemotherapeutic resistance of OS, and to explore the underlying mechanisms. Our data showed that lncRNA CTA was markedly downregulated in OS tissues compared to their matched non-tumor tissues, and low expression of lncRNA CTA was significantly associated with the advanced clinical stage and tumor size. In addition, OS patients with low lncRNA CTA levels showed a worse prognosis when compared with those with high expression of lncRNA CTA. Furthermore, we report that lncRNA CTA has an inverse relationship with miR-210 expression in OS tissues. LncRNA CTA could be activated by doxorubicin (DOX), and could promote OS cell apoptosis by competitively binding miR-210, while inhibit cell autophagy. On the other hand, lncRNA CTA was downregulated in DOX-resistant OS cells. Overexpression of lncRNA CTA reduced autophagy and subsequently overcame DOX resistance of OS in vitro and in vivo. Therefore, we demonstrate that lncRNA CTA is an essential regulator in DOX-induced OS cell apoptosis, and the lncRNA CTA-miR-210 axis plays an important role in reducing OS chemoresistance.

MiR-34a-5p promotes multi-chemoresistance of osteosarcoma through down-regulation of the DLL1 gene

MiR-34a-5p has been implicated in the tumorigenesis and progression of several types of cancer. However, the role of miR-34a-5p in osteosarcoma (OS) remains largely unknown. This study was performed in two multi-chemosensitive (G-292 and MG63.2) and two resistant (SJSA-1 and MNNG/HOS) OS cell lines. MiR-34a-5p promotes OS multi-chemoresistance via its repression of the Delta-like ligand 1 (DLL1) gene, the ligand of the Notch pathway, and thus negatively correlates with OS chemoresistance. The siRNA-mediated repression of the DLL1 gene suppressed cell apoptosis and de-sensitized G-292 and MG63.2 cells, while overexpression of DLL1 sensitized SJSA-1 and MNNG/HOS cells to drug-induced cell death. In agreement with the changes in the drug-induced cell death, the activity of the ATF2/ATF3/ATF4 signaling pathway was significantly altered by a forced reversal of miR-34a-5p or DLL1 levels in OS cells. DLL1 is a target of miR-34a-5p and negatively regulates the multi-chemoresistance of OS. This study suggested that miR-34a-5p, DLL1 and the ATF2/ATF3/ATF4 signaling pathway-associated genes are the potential diagnostic and/or therapeutic targets for an effective chemotherapy of OS. Our results also provide novel insights into the effective chemotherapy for OS patients.

The miR-34a-5p promotes the multi-chemoresistance of osteosarcoma via repression of the AGTR1 gene

BackgroundChemoresistance hinders the curative cancer chemotherapy. MicroRNAs (miRNAs) are key players in diverse biological processes including the chemoresistance of cancers.MethodsA RNA-seq-based miR-omic analysis of osteosarcoma (OS) cells was performed to detect the levels of miR-34a-5p. Bioinformatics analysis revealed that AGTR1 is one of the target genes of miR-34a-5p. The mRNA and protein levels of AGTR1 were detected in both the miR-34a-5p-mimic transfected G-292 and miR-34a-5p-antagomiR transfected SJSA-1 cells. The involvement of AGTR1 with OS chemoresistance was validated by the experiments with siRNA-mediated repression or overexpression of the AGTR1 gene.ResultsWe showed that miR-34a-5p promotes the multi- chemoresistance of OS. The angiotensin II type 1 receptor (AGTR1) gene, is one of the targets of miR-34a-5p in OS and thus negatively correlates with OS chemoresistance by systematic investigations of a multi-drug sensitive (G-292) and resistant (SJSA-1) OS cell lines. Down-regulation of the AGTR1 expression by siRNA passivates G-292 cells and suppresses cell apoptosis, while over-expression of AGTR1 sensitizes SJSA-1 cells and thus promotes the drug-triggered cell death.ConclusionsThe miR-34a-5p and its target gene AGTR1 are the potential targets for an effective chemotherapy of OS. Our results also provide novel insights into the effective chemotherapy for OS patients.

SKA1: A therapeutic target for chemo-resistance in human osteosarcoma

SKA1: A therapeutic target for chemo-resistance in human osteosarcoma

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

ABSTRACTRecent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Progress in microRNA in osteosarcoma

Osteosarcoma is a primary malignant bone tumor with high invasiveness and metastasis that occurs mainly in children and adolescents. The pathogenesis of osteosarcoma remains unclear. MicroRNAs (miRNAs), a class of nucleotides non-coding RNAs, can regulate gene expression at the post-transcriptional level through binding to target mRNAs. Besides, MiRNAs takes control of many important pathophysiological processes in molecular level, including the initiation and invasiveness of osteosarcoma.This review summarizes the roles of miRNAs in initiation,development,transformation and chemoresistance of osteosarcoma to bring new ideas for both pathogenesis exploration and targeted therapy of osteosarcoma. Key words: Osteosarcoma; MicroRNA; Pathogenesis

Long non-coding RNA LINC00161 sensitises osteosarcoma cells to cisplatin-induced apoptosis by regulating the miR-645-IFIT2 axis

Chemotherapeutic insensitivity remains a major obstacle to osteosarcoma treatment. Recently, increasing evidence has suggested that long non-coding RNAs (lncRNAs) play an essential role in tumourigenesis. However, the potential biological roles and regulatory mechanisms of novel lncRNAs in response to cisplatin treatment are poorly understood. Here, we found that lncRNA LINC00161 was induced by cisplatin in osteosarcoma cells. Elevated LINC00161 increased cisplatin-induced apoptosis and reversed the cisplatin-resistant phenotype of osteosarcoma cells by upregulating IFIT2. Further mechanistic studies revealed that LINC00161 could sponge endogenous miR-645 and inhibit its activity leading to IFIT2 increase. In addition, we identified that LINC00161 enhanced cisplatin-induced apoptosis through regulation of the miR-645-IFIT2 pathway. Thus, these findings demonstrate that LINC00161 is an essential regulator in cisplatin-induced apoptosis, and the LINC00161-miR-645-IFIT2 signalling axis plays an important role in reducing osteosarcoma chemoresistance.

MiR-20a-5p represses multi-drug resistance in osteosarcoma by targeting the KIF26B gene.

BackgroundChemoresistance hinders curative cancer chemotherapy in osteosarcoma (OS), resulting in only an approximately 20 % survival rate in patients with metastatic disease at diagnosis. Identifying the mechanisms responsible for regulating chemotherapy resistance is crucial for improving OS treatment.MethodsThis study was performed in two human OS cell lines (the multi-chemosensitive OS cell line G-292 and the multi-chemoresistant OS cell line SJSA-1). The levels of miR-20a-5p and KIF26B mRNA expression were determined by quantitative real-time PCR. KIF26B protein levels were determined by western blot analysis. Cell viability was assessed by MTT assay. Apoptosis was evaluated by flow cytometry.ResultsWe found that miR-20a-5p was more highly expressed in G-292 cells than in SJSA-1 cells. Forced expression of miR-20a-5p counteracted OS cell chemoresistance in both cell culture and tumor xenografts in nude mice. One of miR-20a-5p’s targets, kinesin family member 26B (KIF26B), was found to mediate the miR-20a-5p-induced reduction in OS chemoresistance by modulating the activities of the MAPK/ERK and cAMP/PKA signaling pathways.ConclusionsIn addition to providing mechanistic insights, our study revealed that miR-20a-5p and KIF26B contribute to OS chemoresistance and determined the roles of these genes in this process, which may be critical for characterizing drug responsiveness and overcoming chemoresistance in OS patients.

Combining GRP78 suppression and MK2206-induced Akt inhibition decreases doxorubicin-induced P-glycoprotein expression and mitigates chemoresistance in human osteosarcoma.

P-glycoprotein (P-gp) overexpression is associated with poor prognosis and drug-resistance in osteosarcoma (OS), but the underlying mechanisms remain incompletely understood. Here, we examined the regulation of P-gp, GRP78, and phospho-Akt in doxorubicin (DOX)-treated OS cells. DOX induced P-gp expression, which was associated with increased GRP78 levels and Akt activation in vitro and in vivo. Functional analysis showed that Akt induces P-gp and GRP78 expression, which contributes to the DOX-induced Akt activation. Examination of the relationship between Akt and GRP78 demonstrated that GRP78 suppression attenuates the Akt activity in OS parental sensitive and resistant cells, indicating that GRP78 is required for full Akt activity. Inhibition of Akt activity using MK2206 decreased GRP78 expression in OS cells, which enhanced the inhibitory effect of MK2206 on P-gp expression. GRP78 knockdown combined with MK2206 suppressed the development of DOX resistance in OS cells and inhibited the in vivo tumor growth in the presence of DOX. These results support the development of novel therapeutic strategies that target GRP78 and Akt to sensitize OS cells for chemotherapy.

Mesenchymal stem cells promote osteosarcoma cell survival and drug resistance through activation of STAT3.

Increasing evidence suggests that the tumor microenvironment plays a key role in the development of drug resistant tumor cells. In this study, we tried to determine whether the mesenchymal stem cells (MSCs) in the tumor microenvironment contribute to the increased chemoresistance of osteosarcoma. We found that exposure of Saos-2 and U2-OS cells to MSCs conditioned medium (CM) increased the viable cells in the presence of therapeutic concentrations of doxorubicin or cisplatin. Meanwhile, the MSC CM-associated pro-proliferative effects were accompanied by reduced caspase 3/7 activity and Annexin V binding. We confirmed that STAT3 activation by IL-6 regulates MSCs-induced chemoresistance. Blockade of this signal re-sensitized drug-resistant Saos-2 cells to drug treatment. Using a osteosarcoma mouse model with co-injection of MSCs with Saos-2cells, we found that inhibition of STAT3 prolonged the survival time of tumor bearing mice by suppressing tumor growth and increasing the sensitivity of tumor cells to doxorubicin. Finally, we demonstrated that increased expression of p-STAT3, multidrug resistance protein (MRP) and P-glycoprotein (MDR-1) was associated with high chemotherapy resistance in clinical osteosarcoma samples. Collectively, our findings suggest that MSCs within the tumor microenvironment may represent a new target to enhance chemotherapeutic efficacy in osteosarcoma patients.

Hippo/YAP signaling pathway is involved in osteosarcoma chemoresistance.

BackgroundOsteosarcoma is the most common bone malignancy in children and adolescents, and 20%–30% of the patients suffer from poor prognosis because of individual chemoresistance. The Hippo/yes-associated protein (YAP) signaling pathway has been shown to play a role in tumor chemoresistance, but no previous report has focused on its involvement in osteosarcoma chemoresistance. This study aimed to investigate the role of the Hippo/YAP signaling pathway in osteosarcoma chemoresistance and to determine potential treatment targets.MethodsUsing the Cell Titer-Glo Luminescent cell viability assay and flow cytometry analysis, we determined the proliferation and chemosensitivity of YAP-overexpressing and YAP-knockdown osteosarcoma cells. In addition, using western blotting and the real-time polymerase chain reaction technique, we investigated the alteration of the Hippo/YAP signaling pathway in osteosarcoma cells treated with chemotherapeutic agents.ResultsMammalian sterile 20-like kinase 1 (MST1) degradation was increased, and large tumor suppressor kinase 1/2 (LATS1/2) total protein levels were decreased by methotrexate and doxorubicin, which increased activation and nuclear translocation of YAP. Moreover, YAP increased the proliferation and chemoresistance of MG63 cells.ConclusionsThe Hippo/YAP signaling pathway plays a role in osteosarcoma chemoresistance, and YAP is a potential target for reducing chemoresistance.