LM7 Cells Research Articles

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Riluzole-induced apoptosis in osteosarcoma is mediated through Yes-associated protein upon phosphorylation by c-Abl Kinase

Our lab has previously demonstrated Riluzole to be an effective drug in inhibiting proliferation and inducing apoptosis in both human and mouse osteosarcoma. Yes-associated protein is a transcription co-activator, known to be involved in cell proliferation or apoptosis depending on its protein partner. In the present study we investigated the role of YAP in apoptosis in osteosarcoma, we hypothesized that YAP may be activated by Riluzole to induce apoptosis in osteosarcoma. By knocking down the expression of YAP, we have demonstrated that Riluzole failed to induce apoptosis in YAP deficient osteosarcoma cells. Riluzole caused translocation of YAP from the cytoplasm to the nucleus, indicating YAP’s role in apoptosis. Both Riluzole-induced phosphorylation of YAP at tyrosine 357 and Riluzole-induced apoptosis were blocked by inhibitors of c-Abl kinase. In addition, knockdown of c-Abl kinase prevented Riluzole-induced apoptosis in LM7 cells. We further demonstrated that Riluzole promoted interaction between YAP and p73, while c-Abl kinase inhibitors abolished the interaction. Subsequently, we demonstrated that Riluzole enhanced activity of the Bax promoter in a luciferase reporter assay and enhanced YAP/p73 binding on endogenous Bax promoter in a ChIP assay. Our data supports a novel mechanism in which Riluzole activates c-Abl kinase to regulate pro-apoptotic activity of YAP in osteosarcoma.

A Novel Orthotopic Implantation Technique for Osteosarcoma Produces Spontaneous Metastases and Illustrates Dose-Dependent Efficacy of B7-H3-CAR T Cells

The outcome for metastatic pediatric osteosarcoma (OS) remains poor. Thus, there is an urgent need to develop novel therapies, and immunotherapy with CAR T cells has the potential to meet this challenge. However, there is a lack of preclinical models that mimic salient features of human disease including reliable development of metastatic disease post orthotopic OS cell injection. To overcome this roadblock, and also enable real-time imaging of metastatic disease, we took advantage of LM7 OS cells expressing firefly luciferase (LM7.ffLuc). LM7.ffLuc were implanted in a collagen mesh into the tibia of mice, and mice reliably developed orthotopic tumors and lung metastases as judged by bioluminescence imaging and histopathological analysis. Intratibial implantation also enabled surgical removal by lower leg amputation and monitoring for metastases development post-surgery. We then used this model to evaluate the antitumor activity of CAR T cells targeting B7-H3, an antigen that is expressed in a broad range of solid tumors including OS. B7-H3-CAR T cells had potent antitumor activity in a dose-dependent manner and inhibited the development of pulmonary metastases resulting in a significant survival advantage. In contrast T cells expressing an inactive B7-H3-CAR had no antitumor activity. Using unmodified LM7 cells also enabled us to demonstrate that B7-H3-CAR T cells traffic to orthotopic tumor sites. Hence, we have developed an orthotopic, spontaneously metastasizing OS model. This model may improve our ability not only to predict the safety and efficacy of current and next generation CAR T cell therapies but also other treatment modalities for metastatic OS.

Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features.

Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas-FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas-FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.

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.

Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness

Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.

Abstract 3186: Tegavivint suppresses progression and metastasis of osteosarcoma via blockade of Wnt signaling/ALDH1 axis: Preclinical study of a novel Wnt/β-catenin pathway inhibitor

Abstract Osteosarcoma (OS) is the most common bone cancer in children and adolescents, and patients with metastatic disease still have extremely poor prognosis and no effective targeted therapy. It has been reported that the activation of Wnt/β-catenin pathway is closely associated with OS development and metastatic progression. Tegavivint (BC2059), a novel small molecule inhibitor of the Wnt/β-catenin pathway, has been reported to be active against multiple types of cancer cells in vitro, and anti-tumor efficacy has been published in animal models of acute myeloid leukemia and multiple myeloma. In this study, we investigated the antitumor activity of Tegavivint against metastatic OS both in vitro and in vivo using established human OS cell lines (143B, SaOS-2, LM7) and patient-derived xenograft (PDX) models. In vitro, Tegavivint effectively inhibited tumor cell survival in a dose-dependent manner in all established OS cell lines and OS PDX-derived cells. Subsequent in vivo studies using an orthotopic model of LM7 cells engrafted in the tibia of NSG mice demonstrated complete regression of the primary tumors in all treated mice as well as a significant reduction in lung metastasis by the treatment with Tegavivint. We further examined the activity of Tegavivint using a pair of PDX models derived from an inherently chemo-resistant OS patient at different disease stages: PDX63 was derived from the pre-treatment biopsy of the primary tumor, and PDX84 was derived from the relapsed metastatic lung lesion of the same patient after chemotherapy. The growth of subcutaneously engrafted PDX63 tumors was significantly suppressed by the treatment with Tegavivint alone and Tegavivint enhanced the antitumor activity of doxorubicin. Analysis of mRNA expression by qPCR in the tumor tissue demonstrated significant downregulation of critical genes by Tegavivint, including c-Myc and ALDH1, which is a potential marker for cancer stem cells. Furthermore, in an innovative lung metastasis model using PDX84 tumor-dissociated cells intravenously (IV) injected in NSG mice, we demonstrated that Tegavivint treatment markedly reduced the number of lung metastases. Finally, PDX84-derived cells were sorted into ALDH1-high and ALDH1-low populations. ALDH1-high cells showed higher expression of β-catenin and higher sensitivity to Tegavivint than the ALDH1-low cells in vitro and subsequent IV injection of ALDH1-high PDX84 or ALDH1-low PDX84 cells demonstrated significantly more lung lesions in the ALDH1-high group than the ALDH1-low group. The number of ALDH1-high derived lung metastases was significantly suppressed by Tegavivint. Taken together, our preclinical findings demonstrate that Tegavivint has promising therapeutic potential for primary and metastatic OS through the blockade of Wnt signaling /ALDH1 axis. Citation Format: Motonari Nomura, Nino C. Rainusso, Ruolan Han, Jeff Larson, Ryan L. Shuck, Lyazat Kurenbekova, Jason T. Yustein. Tegavivint suppresses progression and metastasis of osteosarcoma via blockade of Wnt signaling/ALDH1 axis: Preclinical study of a novel Wnt/β-catenin pathway inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3186.

Osteosarcoma cell proliferation and survival requires mGluR5 receptor activity and is blocked by Riluzole.

Osteosarcomas are malignant tumors of bone, most commonly seen in children and adolescents. Despite advances in modern medicine, the poor survival rate of metastatic osteosarcoma has not improved in two decades. In the present study we have investigated the effect of Riluzole on a human and mouse metastatic osteosarcoma cells. We show that LM7 cells secrete glutamate in the media and that mGluR5 receptors are required for the proliferation of LM7 cells. Riluzole, which is known to inhibit glutamate release, inhibits proliferation, induces apoptosis and prevents migration of LM7 cells. This is also seen with Fenobam, a specific blocker of mGluR5. We also show that Riluzole alters the phosphorylation status of AKT/P70 S6 kinase, ERK1/2 and JNK1/2. Thus Riluzole is an effective drug to inhibit proliferation and survival of osteosarcoma cells and has therapeutic potential for the treatment of osteosarcoma exhibiting autocrine glutamate signaling.

Combination of Interleukin-11Rα chimeric antigen receptor T-cells and programmed death-1 blockade as an approach to targeting osteosarcoma cells In vitro

Aim: To test whether combining interleukin (IL)-11Rα chimeric antigen receptor (CAR) T-cells with an anti-programmed death (PD-1) antibody (an immune checkpoint inhibitor) is an effective therapeutic approach in osteosarcoma (OS), allowing improved tumor eradication. Methods: IL-11Rα-CAR T-cells were cocultured in vitro with human LM7 OS tumor cells (with and without anti-PD-1 antibody). Coculture of LM7 cells with purified T-cells served as the control. Cytotoxicity and surface PD-1 expression were analyzed in all groups. Results: PD-1 expression increased during expansion of CAR T-cells. Exposure of immune cells to tumor cells in vitro subsequently decreased surface PD-1 expression on the CAR T-cells. Addition of an anti-PD-1 antibody (Clone J110) to further decrease surface PD-1 expression on CAR T-cells before coculture did not enhance cytotoxic effects of the CAR T-cells against LM7 cells. Conclusion: This combination of IL-11Rα-CAR T-cells and an anti-PD-1 antibody did not provide any additional cytotoxic benefit over IL-11Rα-CAR T-cell therapy alone in this setting. Further studies are needed as simple interference with surface PD-1 expression alone may not be sufficient to inhibit this immune checkpoint pathway to then enhance IL-11Rα-CAR T-cell therapeutic effects.

Abstract 2905: HSP27 as a potential factor to determine the fate of Gemcitabine- induced autophagy in osteosarcoma: Survival vs. death

Abstract Lung metastasis constitutes the main cause of death in osteosarcoma (OS) patients. Survival has not improved in the past ten years. Therefore, new therapeutic strategies are needed. We previously reported that aerosol Gemcitabine (GCB) has a significant therapeutic effect against OS lung metastases. There was however, a subset of cells that fail to respond to GCB treatment resulting in persistent small isolated lung metastases that led to relapse and death. The mechanisms underlying OS resistance to GCB have remained elusive. Autophagy, a catabolic process involved in cellular homeostasis, has emerged as an important mechanism involved in cancer cells response to treatment. However, whether autophagy plays a role in OS resistance to GCB is unknown. Thus, the purpose of this study is to investigate the role of autophagy in OS lung metastases resistance to GCB. In this study, LM7 and CCH-OS-D human OS cells were treated with GCB and induction of autophagy was evaluated. GCB-induced autophagy was demonstrated by formation of acidic vesicular organelles (AVOs), conversion of microtubule-associated light chain 3 (LC3I/LC3II), increase in Beclin 1 (BECN1) and decrease in p62/SQSTM1 protein expression. Additionally, formation of autophagic vacuoles was confirmed by electron microscopy. GCB-induced autophagy also resulted in the inhibition of AKT /mTOR phosphorylation, suggesting this signaling pathway as a possible underlying mechanism responsible for GCB-induced autophagy in OS. To test whether autophagy contributes to OS cells survival to GCB, we determined the impact of autophagy inhibition on the sensitivity of OS cells to GCB. Sensitivity of LM7 cells to GCB was greatly enhanced after autophagy inhibition by the pharmacologic inhibitor hydroxychloroquine (HCQ) and shRNA targeting BECN1, suggesting autophagy as a pro-survival mechanism. Interestingly, inhibition of autophagy in CCH-OS-D cells decreased cell sensitivity to GCB, suggesting that GCB-induced autophagy contributed to cell death. Better understanding of the molecular pathways that govern the process of autophagy will allow identification of new strategies to enhance chemotherapeutic efficacy. Our preliminary work showed that the phosphorylation of Heat Shock protein 27 (HSP27) was significantly higher in GCB-treated LM7 cells when compared to CCH-OS-D GCB-treated cells, indicating a potential role of HSP27 in outlining the role of autophagy in osteosarcoma cells. Taken together, these results suggest that autophagy can be induced by GCB in OS cells and that inhibition of autophagy can lead to either increase or decrease OS cells sensitivity to Gemcitabine. Citation Format: Janice M. Santiago-O'Farrill, Mario Hollomon, Eugenie Kleinerman, Nancy Gordon. HSP27 as a potential factor to determine the fate of Gemcitabine- induced autophagy in osteosarcoma: Survival vs. death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2905. doi:10.1158/1538-7445.AM2015-2905

Abstract 1622: The AKT inhibitor reverses protein trafficking and inhibits tumor cell motility of p27-mislocalized osteosarcoma cells

Abstract Osteosarcoma is the most common malignant bone tumor in children. The development of metastasis remains to be the leading cause of death in osteosarcoma. This clinical challenge urges the development of a novel therapeutic strategy to improve the survival of the metastatic patients. mTOR has recently been regarded as a potential target for metastatic osteosarcoma. However, because of its feedback loop in the pathway, it has become clear that inhibition of mTOR alone would not yield effective therapeutics. More importantly, it is still not clear if the mTOR upstream kinases phosphorylate other important molecules that can also promote metastasis. Our lab has recently demonstrated that a vast majority of osteosarcoma cases harbor cytoplasmic mislocalization of p27. p27 is a tumor suppressor gene that regulates normal cell cycle progression when it is localized in the nucleus. We and others have indicated that p27 acquires a new pro-metastatic function in the cytoplasm of osteosarcoma and other tumors. To evaluate if this observation has any therapeutic significance, our lab has created a set of p27 phosphomutants and found that specific phosphorylations (S10 and T198) are important for the pro-metastatic function in the cytoplasm. Furthermore, shRNA-mediated gene silencing experiments showed that knocking down the expression of p27 decreased the motility of metastatic LM7 cells. Since AKT, the upstream kinase of mTOR, has shown to phosphorylate p27, we tested if AKT inhibitors have any effect on the migration/invasion ability of three osteosarcoma cell lines (LM7, MG63 and G292) that harbor the p27 mislocalization. The results showed that both the AKT1/2 inhibitor and the PI3K/mTOR dual inhibitor PP242, but not mTORC1 inhibitor rapamycin, reduced the tumor cell migration/invasion ability. Interestingly, the inhibitors could also reverse the protein trafficking of p27 into the nucleus. These results suggest that the inhibitors may be able to inhibit the pro-metastatic function and restore the cell cycle inhibitory function of p27. Our long-term goal is to develop a precision medicine approach that will ultimately improve the outcome of metastatic osteosarcoma. By targeting the p27 mislocalization, we believe that we will develop a novel and clinically applicable therapeutic strategy that would eradicate metastatic osteosarcoma and improve the outcome of children affected by this tumor. Citation Format: Xiang Chen, Yiting Li, Manjula Nakka, Tsz Kwong Man. The AKT inhibitor reverses protein trafficking and inhibits tumor cell motility of p27-mislocalized osteosarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1622. doi:10.1158/1538-7445.AM2015-1622

S-adenosylmethionine blocks osteosarcoma cells proliferation and invasion in vitro and tumor metastasis in vivo: therapeutic and diagnostic clinical applications.

Osteosarcoma (OS) is an aggressive and highly metastatic form of primary bone cancer affecting young children and adults. Previous studies have shown that hypomethylation of critical genes is driving metastasis. Here, we examine whether hypermethylation treatment can block OS growth and pulmonary metastasis. Human OS cells LM-7 and MG-63 were treated with the ubiquitous methyl donor S-adenosylmethionine (SAM) or its inactive analog S-adenosylhomocystine (SAH) as control. Treatment with SAM resulted in a dose-dependent inhibition of tumor cell proliferation, invasion, cell migration, and cell cycle characteristics. Inoculation of cells treated with 150 μmol/L SAM for 6 days into tibia or via intravenous route into Fox Chase severe combined immune deficient (SCID) mice resulted in the development of significantly smaller skeletal lesions and a marked reduction in pulmonary metastasis as compared to control groups. Epigenome wide association studies (EWAS) showed differential methylation of several genes involved in OS progression and prominent signaling pathways implicated in bone formation, wound healing, and tumor progression in SAM-treated LM-7 cells. Real-time polymerase chain reaction (qPCR) analysis confirmed that SAM treatment blocked the expression of several prometastatic genes and additional genes identified by EWAS analysis. Immunohistochemical analysis of normal human bone and tissue array from OS patients showed significantly high levels of expression of one of the identified gene platelet-derived growth factor alpha (PDGFA). These studies provide a possible mechanism for the role of DNA demethylation in the development and metastasis of OS to provide a rationale for the use of hypermethylation therapy for OS patients and identify new targets for monitoring OS development and progression.

Autophagy in osteosarcoma.

Osteosarcoma (OS) metastatic disease is resistant to conventional chemotherapy. Tumor resistance to chemotherapy has been one of the major areas of concern to clinicians and the topic of many laboratory investigators. Evaluation of mechanisms implicated in OS lung metastasis resistance to chemotherapy has been the focus of some of our most recent work. We have previously demonstrated the therapeutic efficacy of aerosol gemcitabine (GCB) in OS lung metastases. However, a subset of cells fails to respond to GCB treatment and persists as isolated lung metastases in vivo. Autophagy, a physiological mechanism that supports nutritional deprivation under stressful conditions, has been implicated in tumor resistance to chemotherapy. We demonstrated the induction of autophagy by GCB in LM7 metastatic human OS cells and K7M3 metastatic murine OS cells. Inhibition of autophagy resulted in increased sensitivity to GCB in LM7 cells. By contrast, inhibiting autophagy in K7M3 cells decreased GCB sensitivity. Defining the role autophagy plays in chemotherapy response in different tumor types has become of greater importance in order to identify the best suitable therapeutic approach. In this chapter, we summarize some of the most recent work related to autophagy in OS, identify some of the known mechanisms, and address the different roles autophagy plays in chemotherapy response.

Mitochondrial Dysfunction and Permeability Transition in Osteosarcoma Cells Showing the Warburg Effect

Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.

Abstract A18: Autophagy as a mechanism implicated in osteosarcoma resistance to gemcitabine.

Abstract The purpose of this study is to demonstrate the role of autophagy in osteosarcoma (OS) lung metastases resistance to aerosol Gemcitabine (GCB). Despite many advances in chemotherapy and successful control of the primary tumor, osteosarcoma overall survival has remained the same in the last 15 years. Pulmonary metastases remain the main cause of death in patients with OS underscoring the need for new therapeutic strategies. Using a human OS mouse model, we had previously demonstrated that aerosol Gemcitabine (GCB) has a significant therapeutic effect in OS lung metastases. Nevertheless, a subset of cells failss to respond to GCB treatment and persist as small isolated lung metastases in vivo. Resistance to GCB is also evidenced by survival of OS cells after treatment in vitro. Therefore, understanding the underlying mechanism of OS resistance to GCB will allow the implementation of new therapeutic strategies. Autophagy has been suggested as a mechanism induced by a variety tumor cells after chemotherapy treatment. It is a process involved in the degradation of organelles and damaged proteins, providing the cells amino acids to sustain survival under stressful conditions. Recently, it has also been associated with tumor cells resistance to chemotherapy. GCB-induced autophagy in OS primary and metastatic disease has not been studied. Therefore, we characterized the role of autophagy in OS cells after treatment with GCB. We first demonstrated GCB-induced autophagy in LM7 OS cells as indicated by an increase in autophagy markers: microtubule-associated light chain 3 (LC3I/LC3II) conversion, Beclin 1 (BECN1) expression and development of acidic vesicular organelles. GCB also caused a decrease in p62/SQSTM1 and Blc-2 expression, further indicating autophagy induction. Blockage of autophagy with the pharmacologic inhibitor Hydroxychroloquine (HCQ) coupled with GCB treatment decreased cell viability of LM7 OS cells. Furthermore, knockdown of BECN1 in LM7 cells coupled with GCB treatment also decreased cell viability of LM7 OS cells, indicating that autophagy plays an important role in OS cells resistance to GCB treatment. In conclusion, we demonstrated that autophagy is one of the mechanisms implicated in the resistance of OS cells to GCB. Autophagy modulation may shed new light towards metastatic OS therapy, possibly by implementing the use of combination therapy HCQ-aerosol GCB in the adjuvant setting. Current studies are underway to test our hypothesis in vivo and to address these results using other OS cells in vitro. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A18. Citation Format: Janice M. Santiago-O'Farrill, Mario G. Hollomon, Eugenie S. Kleinerman, Nancy Gordon. Autophagy as a mechanism implicated in osteosarcoma resistance to gemcitabine. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A18.

MiR-20a Encoded by the miR-17–92 Cluster Increases the Metastatic Potential of Osteosarcoma Cells by Regulating Fas Expression

The ability of osteosarcoma cells to form lung metastases has been inversely correlated to cell surface Fas expression. Downregulation of Fas allows osteosarcoma cells to circumvent FasL-mediated apoptosis upon entrance into the FasL(+) lung microenvironment. However, the mechanism of Fas regulation remains unclear. Here, we show that miRNA plays a role in the downregulation of Fas expression in osteosarcoma. Expression levels of several members of the miR-17-92 cluster including miR-20a and miR-19a were found to be higher in metastatic low-Fas-expressing LM7 cells than in the parental nonmetastatic high-Fas-expressing SAOS-2 cells. We also found an inverse correlation between Fas and miR-20a expression in all 8 cell lines derived from patient samples. Overexpression of miR-20a consistently resulted in the downregulation of Fas expression in SAOS-2 cells and thus in decreased sensitivity to FasL. Conversely, inhibiting miR-20a in LM7 cells increased Fas expression and their sensitivity to FasL. Mice injected with LM7 stably transfected with anti-miR-20a had fewer metastases than those with control plasmids. Taken together, our findings suggest that miR-20a, encoded by miR-17-92, downregulates Fas expression in osteosarcoma, thus contributing to the metastatic potential of osteosarcoma cells by altering the phenotype and allowing survival in the FasL(+) lung microenvironment.

Abstract 2348: Frzb/sFRP3 inhibits lung metastasis of osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignancy of bone. In spite of the recent advances in chemotherapy and surgery, 40-50% of the adolescent patients develop lung metastasis, 80% of patients with metastasis at diagnosis will relapse. Survival rate has shown no significant change over past 20 years. The molecular mechanism of metastasis of OS needs to be further explored and the novel anti-metastatic target needs to be developed. Our previous data showed that Frzb (also known as sFRP3), a secreted Fz-related protein, suppressed metastasis of fibrosarcoma. In this study, we examined the anti-metastatic effects of Frzb on OS, using the 143B and LM7 cell lines as a model. Frzb expression construct was stably transfected into 143B cells and LM7 cells. Over-expression of Frzb in OS cells was confirmed by real time PCR and western-blot. In a lung metastasis nude mice model, osteosarcoma LM7 cells expressing Frzb formed much fewer and much smaller lung metastatic nodules than control cells. Frzb also decreased in vitro cellular motility in a scratch wound assay. In order to determine the potential molecular mechanism underlying the anti-metastatic effect of Frzb on osteosarcoma, we examined the expression of c-Met, S100A4 and reversal of EMT (Epithelial Mesenchymal Transition). By real time PCR and western-blot assay, c-Met and S100A4, which were extensively reported to be involved in metastasis of different cancers, were both down-regulated by Frzb. However, most of epithelial markers (E-cadherin, keratin 8, keratin18, etc) and mesenchymal markers (N-cadherin, Vimentin, fibronectin) remained unchanged. Our study suggests that Frzb has strong anti-metastatic effect on osteosarcoma. The potential molecular mechanism may be through down-regulation of c-Met and S100A4 in OS cells by Frzb, instead of reversing the EMT. Therefore, strategies to over-express or deliver Frzb in osteosarcoma may lead to a reduction of lung metastasis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2348.

Abstract 2194: Novel receptor tyrosine kinases in osteosarcoma identified by phosphoproteomic and functional genomic screening

Abstract Osteosarcoma is the most common primary bone malignancy and is largely attributed to rapidly growing bones of adolescents. Approximately one-third of patients succumb to pulmonary metastasis despite high-dose neoadjuvant chemotherapy protocols. Survival rates have not improved over the last 30 years therefore novel therapeutic agents are needed. Tyrosine kinases have emerged as targets for the development of new cancer therapies since their activation leads to enhanced proliferation, survival, cell adhesion and metastasis. Unlike other cancers, the role of tyrosine kinases has not been well established in osteosarcoma. In this study, we used two families of genetically related human osteosarcoma cell lines with varying potentials for tumorigenesis and metastasis in mice. Phosphoproteomic and siRNA screening were performed to identify specific receptor tyrosine kinases that are activated and contribute to the in vitro behavior of the metastatic osteosarcoma cell lines. In the metastatic LM7 cells, 12 of the 42 RTKs on the phospho-receptor tyrosine kinase antibody array were phosphorylated. All except Axl had increased levels of phosphorylation compared to the parental, non-metastatic Saos-2 cells. Nine of the 12 RTKs were also phosphorylated in the metastatic 143B cells. Of these, FGFR3, PDGFRα and c-Ret had increased levels of phosphorylation when compared to the parental, non-metastatic TE85 cells. siRNAs targeting the 12 RTKs found to be activated in the metastatic cell lines were screened to identify those RTKs that are important for motility, invasion, colony formation and/or cell growth. In the 143B cell line, 9 of the 12 siRNAs inhibited at least one assay by ≥ 35%. Eph Kinase B2 (EphB2) knockdown had the greatest effect resulting in > 85% inhibition of motility, invasion, colony formation and cell growth compared to cells treated with luciferase control siRNA. In the LM7 cell line, 7 of the 12 siRNAs inhibited at least one of the assays by ≥ 35%. IGF-1R silencing had the greatest effect by inhibiting motility by 85%, colony formation by 58% and cell growth by 32%. Validation of the siRNA screening results have so far indicated that the effects from IGF-1R, EphB2, FGFR2, FGFR3 and c-Ret knockdown are consistent with on-target silencing in LM7 cells. Studies are underway to validate the remaining siRNAs. From our screens, we identified specific activated receptor tyrosine kinases that contribute to the in vitro behavior of osteosarcoma and may be valuable therapeutic targets. Furthermore, unlike IGF-1R, which has been widely studied in osteosarcoma, this is the first known report demonstrating that EphB2, FGFR2, FGFR3 and c-Ret may be important to the progression of osteosarcoma. Further studies will enable us to identify the underlying role of these receptor tyrosine kinases in ostesoarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2194.

Abstract 5570: An affinity lectin chromatography approach to characterize N-linked glycoproteome in metastatic osteosarcoma

Abstract Osteosarcoma (OS) is the most common bone cancer in children and adolescents. Approximately 20% of OS patients have clinically detectable metastasis at the time of diagnosis, which is the most consistent indicator of poor outcome. Therefore, an easy-to-use and sensitive method is urgently needed to detect and monitor metastatic patients in order to offer an intensified or personalized therapy. By analyzing glycoproteins in OS cells, we may identify biomarkers that would help detect early metastasis and shed light on how this cancer develops. Thus, we used a tumor-targeted glycoproteomic approach in order to identify metastasis-associated glycoproteins in OS cells, and test if these can be used as blood biomarkers to indicate metastasis or spread of disease at diagnosis. In this pilot study, we have analyzed a previously established human OS metastatic cell line (LM7). We captured a specific subset of N-linked glycoproteins from LM7 cells by Wheat Germ Agglutinin (WGA) affinity chromatography. Subsequently, we identified the captured glycoproteins by a multidimensional protein identification technology (MudPIT). In addition, two samples with or without PNGase treatment were tested to estimate the impact of protein glycosylation on the MS/MS analysis. A total of 121 and 368 unique peptides were identified in the non-treated and PNGase treated samples, respectively. All of the proteins identified with two or more distinct peptides were predicted to have potential N-linked glycosylation sites, and 57% of these were found to have one or more typical Asn-X-Ser/Thr N-glycosylation sites. Many of the identified glycoproteins are known to be involved in tumorigenesis. To this end, we have optimized the affinity lectin chromatography and mass spectrometry to capture and identify N-linked glycoproteins in metastatic osteosarcoma cells. Differentially expressed glycoproteins in metastatic OS will be further identified by comparing LM7 with a non-metastatic control. The candidate biomarkers will be validated in the plasma and tumor samples collected from OS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5570.

Abstract 2064: Down-regulation of miR-31 is associated with metastatic potential of human and canine osteosarcoma

Abstract Osteosarcoma is the most common primary bone tumor in humans and dogs. The primary cause for the poor survival of patients with osteosarcoma in both species is the emergence of metastases in organs distant from the primary tumor, especially in the lung. Therefore, identification of molecular determinants of osteosarcoma metastasis is highly warranted. In order to identify microRNAs (miRNAs) that regulate osteosarcoma metastasis, we examined, using qRT-PCR, levels of 10 miRNAs commonly associated with cancer metastasis (miR-10b, miR-146a, miR182, miR183, miR-206, miR-335, miR373, miR-520c-5p, miR-21 and miR-31) in total RNA samples prepared from canine parental osteosarcoma (POS) and highly metastatic POS (HMPOS) cells. Of the 10 miRNAs, only miR-31 showed differential expression between the two cell lines (reduced by 7.8-fold in HMPOS cells versus POS cells). Exposure of HMPOS cells to various chemopreventive agents (myo-inositol, indole-3-carbinol, diindolylmethane, deguelin, phenethyl isothiocyanate, cisplatin, and aza-2′-deoxycytidine) increased the level of miR-31 at least by two-fold, the most potent agents being deguelin and phenethyl isothiocyanate. Subsequently, we assessed the expression of miR-31 in additional canine osteosarcoma cell lines (three cell lines established from primary tumors and three cell lines developed from pulmonary metastases) and human osteosarcoma cell lines differing in metastatic potential (parental SaOS2, low- (LM2), and high- (LM7) metastatic cells). miR-31 levels decreased by 18-fold in canine cell lines derived from pulmonary metastatic tumors relative to that obtained from primary tumors and by 2-fold and 3-fold in LM2 and LM7 cells, respectively, compared to the expression in parental Saos-2 cells. Also, analysis of 3 primary human osteosarcoma tissues and 3 pulmonary metastatic tissues revealed a 13-fold reduction of miR-31 in metastatic tissues relative to primary tumor tissues. In preliminary experiments where attempts were made to identify potential targets of miR-31, exogenous over-expression of a synthetic miR-31 cassette in HMPOS cells negatively regulated a tight junction protein claudin-2, which is incriminated in cancer metastasis. In summary, our studies indicate that miR-31 is a promising marker for the metastatic potential of osteosarcoma and for disease prognosis. Moreover, reactivation of silenced miR-31 by chemopreventive/chemotherapeutic agents could reverse osteosarcoma metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2064.