Lyazat Kurenbekova

NKD2, a negative regulator of Wnt signaling, suppresses tumor growth and metastasis in osteosarcoma

Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor that has a high propensity for metastases. Through osteoblast-specific alteration of p53 status, we developed a genetically engineered mouse model of localized and metastatic OS to gain an understanding into the molecular pathogenesis of OS. Microarray analysis of both localized tumors and metastatic tumors identified the downregulation of the naked cuticle homolog 2 (NKD2) gene, a negative regulator of Wnt signaling. Overexpression of NKD2 in metastatic human and mouse OS cells significantly decreases cell proliferation, migration and invasion ability in vitro and drastically diminishes OS tumor growth and metastasis in vivo, whereas downregulation enhances migratory and invasive potential. Evaluation of NKD2-overexpressing tumors revealed upregulation of tumor-suppressor genes and downregulation of molecules involved in blood vessel formation and cell migration. Furthermore, assessment of primary human OS revealed downregulation of NKD2 in metastatic and recurrent OS. Finally, we provide biological evidence that use of small-molecule inhibitors targeting the Wnt pathway can have therapeutic efficacy in decreasing metastatic properties in OS. Our studies provide compelling evidence that downregulation of NKD2 expression and alterations in associated regulated pathways have a significant role in driving OS tumor growth and metastasis.

Cross-species identification of a plasma microRNA signature for detection, therapeutic monitoring, and prognosis in osteosarcoma.

Osteosarcoma (OS) is the primary bone tumor in children and young adults. Currently, there are no reliable, noninvasive biologic markers to detect the presence or progression of disease, assess therapy response, or provide upfront prognostic insights. MicroRNAs (miRNAs) are evolutionarily conserved, stable, small noncoding RNA molecules that are key posttranscriptional regulators and are ideal candidates for circulating biomarker development due to their stability in plasma, ease of isolation, and the unique expressions associated with specific disease states. Using a qPCR‐based platform that analyzes more than 750 miRNAs, we analyzed control and diseased‐associated plasma from a genetically engineered mouse model of OS to identify a profile of four plasma miRNAs. Subsequent analysis of 40 human patient samples corroborated these results. We also identified disease‐specific endogenous reference plasma miRNAs for mouse and human studies. Specifically, we observed plasma miR‐205‐5p was decreased 2.68‐fold in mice with OS compared to control mice, whereas, miR‐214, and miR‐335‐5p were increased 2.37‐ and 2.69‐fold, respectively. In human samples, the same profile was seen with miR‐205‐5p decreased 1.75‐fold in patients with OS, whereas miR‐574‐3p, miR‐214, and miR‐335‐5p were increased 3.16‐, 8.31‐ and 2.52‐fold, respectively, compared to healthy controls. Furthermore, low plasma levels of miR‐214 in metastatic patients at time of diagnosis conveyed a significantly better overall survival. This is the first study to identify plasma miRNAs that could be used to prospectively identify disease, potentially monitor therapeutic efficacy and have prognostic implications for OS patients.

Secreted Frizzled-Related Protein 2 (sFRP2) promotes osteosarcoma invasion and metastatic potential

BackgroundOsteosarcoma (OS), which has a high potential for developing metastatic disease, is the most frequent malignant bone tumor in children and adolescents. Molecular analysis of a metastatic genetically engineered mouse model of osteosarcoma identified enhanced expression of Secreted Frizzled-Related Protein 2 (sFRP2), a putative regulator of Wnt signaling within metastatic tumors. Subsequent analysis correlated increased expression in the human disease, and within highly metastatic OS cells. However, the role of sFRP2 in osteosarcoma development and progression has not been well elucidated.MethodsStudies using stable gain or loss-of-function alterations of sFRP2 within human and mouse OS cells were performed to assess changes in cell proliferation, migration, and invasive ability in vitro, via both transwell and 3D matrigel assays. In additional, xenograft studies using overexpression of sFRP2 were used to assess effects on in vivo metastatic potential.ResultsFunctional studies revealed stable overexpression of sFRP2 within localized human and mouse OS cells significantly increased cell migration and invasive ability in vitro and enhanced metastatic potential in vivo. Additional studies exploiting knockdown of sFRP2 within metastatic human and mouse OS cells demonstrated decreased cell migration and invasion ability in vitro, thus corroborating a critical biological phenotype carried out by sFRP2. Interestingly, alterations in sFRP2 expression did not alter OS proliferation rates or primary tumor development.ConclusionsWhile future studies further investigating the molecular mechanisms contributing towards this sFRP2-dependent phenotype are needed, our studies clearly provide evidence that aberrant expression of sFRP2 can contribute to the invasive and metastatic potential for osteosarcoma.

Metabolic modulation of Ewing sarcoma cells inhibits tumor growth and stem cell properties

Ewing sarcoma (EWS) is a highly aggressive and metabolically active malignant tumor. Metabolic activity can broadly be characterized by features of glycolytic activity and oxidative phosphorylation. We have further characterized metabolic features of EWS cells to identify potential therapeutic targets. EWS cells had significantly more glycolytic activity compared to their non-malignant counterparts. Thus, metabolic inhibitors of glycolysis such as 2-deoxy-D-glucose (2DG) and of the mitochondrial respiratory pathway, such as metformin, were evaluated as potential therapeutic agents against a panel of EWS cell lines in vitro. Results indicate that 2DG alone or in combination with metformin was effective at inducing cell death in EWS cell lines. The predominant mechanism of cell death appears to be through stimulating apoptosis leading into necrosis with concomitant activation of AMPK-α. Furthermore, we demonstrate that the use of metabolic modulators can target putative EWS stem cells, both in vitro and in vivo, and potentially overcome chemotherapeutic resistance in EWS. Based on these data, clinical strategies using drugs targeting tumor cell metabolism present a viable therapeutic modality against EWS.

miR-130b directly targets Arhgap1 to drive activation of a metastatic CDC42-PAK1-AP1 positive feedback loop in Ewing sarcoma

Ewing Sarcoma (ES) is a highly aggressive bone tumor with peak incidence in the adolescent population. It has a high propensity to metastasize, which is associated with dismal survival rates of approximately 25%. To further understand mechanisms of metastasis we investigated microRNA regulatory networks in ES. Our studies focused on miR‐130b due to our analysis that enhanced expression of this microRNA has clinical relevance in multiple sarcomas, including ES. Our studies provide insights into a novel positive feedback network involving the direct regulation of miR‐130b and activation of downstream signaling events contributing toward sarcoma metastasis. Specifically, we demonstrated miR‐130b induces proliferation, invasion, and migration in vitro and increased metastatic potential in vivo. Using microarray analysis of ES cells with differential miR‐130b expression we identified alterations in downstream signaling cascades including activation of the CDC42 pathway. We identified ARHGAP1, which is a negative regulator of CDC42, as a novel, direct target of miR‐130b. In turn, downstream activation of PAK1 activated the JNK and AP‐1 cascades and downstream transcriptional targets including IL‐8, MMP1 and CCND1. Furthermore, chromatin immunoprecipitation of endogenous AP‐1 in ES cells demonstrated direct binding to an upstream consensus binding site within the miR‐130b promoter. Finally, small molecule inhibition of PAK1 blocked miR‐130b activation of JNK and downstream AP‐1 target genes, including primary miR‐130b transcripts, and miR‐130b oncogenic properties, thus identifying PAK1 as a novel therapeutic target for ES. Taken together, our findings identify and characterize a novel, targetable miR‐130b regulatory network that promotes ES metastasis.

Abstract C80: Characterization of metastatic cancer stem cells in osteosarcoma

Pulmonary metastasis is the leading cause of death in osteosarcoma (OS), the most common malignant bone tumor in children and young adults. The molecular mechanisms that regulate the formation of metastases in OS remain mostly unknown. A subset of cancer stem cells (CSCs) with metastatic potential has been identified in breast, colon and pancreatic cancers. Therefore, we hypothesize that CSCs drive the metastatic process in OS by their ability to migrate, adapt to a different microenvironment, and subsequently generate distal metastatic lesions in the lungs. Using a novel mutant p53 mouse model of metastatic OS, which has been developed in Dr. Jason Yustein9s laboratory, we have identified putative OS CSCs in primary bone tumors and metastatic lesions. Cancer stem cells have been cultured from solid tumors growing as sphere-like structures under serum-free conditions. This method has been validated as a surrogate of their self-renewal capacity. We have observed that murine tumor cells obtained from both primary bone tumors and pulmonary nodules developed sarcospheres. Notably, sarcophere-forming efficiency was significantly increased (2-3 times) in tumor cells obtained from metastatic lesions. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in numerous solid tumors. We have found that both primary bone tumors and metastatic pulmonary nodules contain ALDH Hi (stem-like cells). The high expression of ALDH1 in murine OS tumors ranged from 0.7 to 17%. Further analysis of derived cell lines generated from primary OS tumor, circulating tumor cells (CTCs) and metastatic tumors showed that distal metastases have 4-5 times more ALDH Hi cells than CTCs and up to 10 times more ALDH Hi cells than the primary bone tumors. The isolation of OS CSCs by fluorescent activated cell sorting (FACS) facilitates the analysis of the molecular pathways and biological process that regulates this tumor cell compartment. Our initial data has shown that the Insulin-like Growth Factor (IGF) family members were differentially expressed between OS primary and metastatic cells. Furthermore, we are actively utilizing these mouse derived cell lines to perform orthotopic transplants in immunocompetent (syngeneic) mice aiming to better understand the biology and genetics of CSCs in OS. Subsequent studies will be performed to determine the functional significance of the unique OS CSC genetic alterations. In summary, we have identified putative metastatic OS CSCc in a mutant p53 mouse model of metastatic OS. Our preliminary data has shown that CSC content seems to be increased in metastases. Our studies using an OS mouse model that parallels the biological behavior of human disease in addition to provide freshly isolated tumor specimens constitute a strong pre-clinical model to test the effect of novel anti-cancer therapies in the CSC compartment. Citation Format: Nino Rainusso, Lyazat Kurenbekova, Lawrence Donehower, Jeffrey Rosen, Jason Yustein. Characterization of metastatic cancer stem cells in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C80.

Abstract 197: miR-130b targets Arhgap1 increasing Cdc42 activity and metastic potential in Ewing sarcoma cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Osteosarcoma (OS) and Ewings sarcoma (EWS) are the most common bone tumors in the pediatric population. While significant progress has been made with towards increasing patient outcomes, our success in eradicating these two malignances is significantly less than most other pediatric malignancies. One reason for continued treatment failure is due to high risk disease states such as the presence of metastatic lesions. Metastasis is of the utmost clinical relevance as it accounts for more than 90% of solid tumor deaths. Improvements in treatment regimens have been stagnant over the past 20-30 years most likely due to our lack of understanding their molecular pathogenesis. Overall survival rates are approximately 65-70% for localized disease and less than 20% for aggressive, disseminated states. Thus, the clinical need to understand high risk disease, such as metastasis is significant in order to develop novel therapeutics. miRNAs are highly conserved short 18-22 single stranded non-coding RNAs that have the ability to repress target translation and affect key cell signaling pathways often deregulated in cancer. Recently, miRNAs have been implicated in numerous cancers; however the functional role of miRNAs in sarcomas is limited. In order to identify novel alterations in miRNAs, we have utilized our analysis of microRNA expression from localized and metastatic tumors derived from novel genetically engineered mouse models of osteosarcoma. Among the miRNAs most significantly dysregulated, miR-130b was shown to be significantly upregulated in the metastatic lesions. Furthermore, it has been shown that miR-130b is overexpressed in OS and EWS patient samples, and this event is significantly associated with poor patient outcome in the latter sarcoma. Furthermore, we have verified miR-130b overexpression in both mouse and human sarcoma cell lines and in metastatic EWS tumor samples obtained from Texas Childrens Hospital. Functional analysis shows overexpression of miR-130b increases migration and invasion in vitro. In order to identify novel targets of miR-130b contributing to metastasis, we performed gene expression analysis. We identified Cdc42GAP as a novel target of miR-130b. Cdc42GAP is a negative regulator of Cdc42 which hydrolyzes the active GTPase. ELISA analysis of Cdc42 activity reveals that Cdc42 activity is increased with overexpression of miR-130b.Further microarray and pathway analysis suggest a mechanism by which activation of Cdc42 leads to positive regulation of the MAPK cascade. Subsequent western blot analysis reveals that overexpression of miR-130b leads to the activation of SAPK/JNK and subsequent phosphorylation of c-JUN suggesting activation of AP-1 transcription factor. Taken together, these findings suggest that overexpression of miR-130b promotes sarcoma cell migration and invasion through targeting of Cdc42GAP and modulation of the Cdc42 and SAPK pathways. Citation Format: Laura Satterfield, Lyazat Kurenbekova, Ryan Shuck, Lawrence Donehower, Jason Yustein. miR-130b targets Arhgap1 increasing Cdc42 activity and metastic potential in Ewing sarcoma 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 197. doi:10.1158/1538-7445.AM2015-197

Abstract 5278: Circulating microRNAs profile as a novel biomarker for osteosarcoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Osteosarcoma (OS) is the primary bone tumor in children, with approximately 400 cases each year diagnosed in patients under the age of twenty. Current therapies can achieve long-term relapse free survival rates near 75% for those with localized disease, while survival rates for patients with overt metastatic disease at diagnosis are often less than 20%. Without adjuvant chemotherapy, over 80% of children will develop metastatic or recurrent disease and all patients require long term monitoring. However, there are no reliable, non-invasive biological markers to detect the presence or progression of disease, assess therapy response, or provide upfront risk stratification. The present method for detecting and monitoring disease status in OS is radiographic imaging which is often difficult to interpret. MicroRNAs (miRNAs) are evolutionarily conserved, stable, small non-coding RNA molecules that are key post-transcriptional regulators and are ideal candidates for circulating biomarker development due to their stability in plasma, ease of isolation with high sensitivity, and the unique expressions associated with specific disease states. As such, we have developed a signature profile of circulating miRNAs utilizing a novel genetically engineered mouse model of OS that can discriminate between mice with and without OS. Specifically, we have determined that circulating miR-205 is decreased 3.2-fold in mice with disease compared to disease-free mice, whereas miR-574-3p, miR-214, and miR-335-5p are increased 1.4, 1.9, and 2.9 fold respectively in the diseased mice. Finally, based upon prior studies utilizing other cancer models, we believe that we will be able to directly translate these findings from the mouse model to patients afflicted with osteosarcoma. Citation Format: Wendy Allen-Rhoades, Lyazat Kurenbekova, Susan Hilsenbeck, Tsz-Kwong Man, Lawrence Donehower, Jason T. Yustein. Circulating microRNAs profile as a novel biomarker for osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5278. doi:10.1158/1538-7445.AM2013-5278

Abstract 3867: Novel mouse models to investigate the molecular pathogenesis of metastatic osteosarcoma.

Background: Osteosarcoma (OS) is the primary bone tumor in the pediatric population with the main determinant for patient prognosis being the presence of metastatic disease. Approximately 25-30% of patients will present with metastatic osteosarcoma. Patients with metastatic disease have long-term survival rates often Objectives: We hypothesize that the molecular pathogenesis of metastatic osteosarcoma is different from localized disease. We intend this work to provide a relevant, endogenous model of metastatic OS that can be utilized to advance our understanding of the molecular pathogenesis of the disease, insights into novel therapeutic targets and as a pre-clinical model for investigating the efficacy of novel therapies. Design: We have developed a tissue-specific alteration of the p53 status by using osteoblast specific Cre-recombinase expressing mice to generate progeny that spontaneously form endogenous osteosarcomas. Through the use of a mutated, gain of function form of p53, shown previously to be associated with metastatic disease, we have developed a novel immunocompetent model that significantly enhances the endogenous development of metastatic OS. Results: Tumor analysis has revealed genetic insights in the metastatic progression of osteosarcoma. These include the significant downregulation of Wnt-signaling inhibitors, such as NKD2, APCDD1 and Wnt5a in the metastatic tumors. Functional studies have determined that overexpression of NKD2, also downregulated in several human OS metastatic cell lines, in metastatic mouse OS cell lines leads to a significant decrease in metastatic lung lesions upon transplantation into immunodeficient mice. Possible NKD2-dependent functions include regulation of not only the Wnt signaling pathway, but also blood vessel formation, regulation of cell migration and cell adhesion. We have also noted the dysregulation of several critical microRNAs in metastatic OS, including the upregulation of mir-130b in the metastatic lesions. This particular microRNA has recently been shown to have clinical correlation in Ewing9s sarcoma, with higher levels of tumor mir-130b having a significantly poorer outcome. We are actively pursuing the role of this (and other) microRNA(s) in the molecular pathogenesis of metastatic osteosarcoma. Conclusion: This novel model has enabled valuable molecular insights into the development and progression of metastatic OS that can lead to the identification of novel therapeutic targets. Citation Format: Shuying Zhao, Lyazat Kurenbekova, Lawrence A. Donehower, Jason T. Yustein. Novel mouse models to investigate the molecular pathogenesis of metastatic osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A85.