Jyotika Varshney

MicroRNAs as potential target in human bone and soft tissue sarcoma therapeutics

Sarcomas are highly aggressive heterogeneous tumors that are mesenchymal in origin. There have been vast advancements on identifying diagnostic markers for sarcomas including chromosomal translocations, but very little progress has been made to identify targeted therapies against them. The tumor heterogeneity, genetic complexity and the lack of drug studies make it challenging to recognize the potential targets and also accounts for the inadequate treatments in sarcomas. In recent years, microRNAs that are a part of small non-coding RNAs have shown promising results as potential diagnostic and prognostic biomarkers in multiple sarcoma types. This review focuses on the current knowledge of the microRNAs that are deregulated in sarcomas, and an insight on the strategies to target these microRNAs that are essential for developing improved therapies for various human sarcomas.

Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma

Background: Gene expression signatures define prognostically significant osteosarcoma phenotypes. Results: Deregulation of the RB-E2F pathway establishes more aggressive phenotype. Inhibitors of DNA and chromatin remodeling promote comparable transcriptional changes as genetic restoration of RB. Conclusion: Aberrant RB-E2F pathway alters epigenetic landscape and biological behavior of osteosarcoma. Significance: Epigenetic remodeling regulated by RB-E2F gives rise to patterns of gene expression that are associated with different biological behavior and progression of osteosarcoma. We previously identified two distinct molecular subtypes of osteosarcoma through gene expression profiling. These subtypes are associated with distinct tumor behavior and clinical outcomes. Here, we describe mechanisms that give rise to these molecular subtypes. Using bioinformatic analyses, we identified a significant association between deregulation of the retinoblastoma (RB)-E2F pathway and the molecular subtype with worse clinical outcomes. Xenotransplantation models recapitulated the corresponding behavior for each osteosarcoma subtype; thus, we used cell lines to validate the role of the RB-E2F pathway in regulating the prognostic gene signature. Ectopic RB resets the patterns of E2F regulated gene expression in cells derived from tumors with worse clinical outcomes (molecular phenotype 2) to those comparable with those observed in cells derived from tumors with less aggressive outcomes (molecular phenotype 1), providing a functional association between RB-E2F dysfunction and altered gene expression in osteosarcoma. DNA methyltransferase and histone deacetylase inhibitors similarly reset the transcriptional state of the molecular phenotype 2 cells from a state associated with RB deficiency to one seen with RB sufficiency. Our data indicate that deregulation of RB-E2F pathway alters the epigenetic landscape and biological behavior of osteosarcoma.

Understanding the Osteosarcoma Pathobiology: A Comparative Oncology Approach

Osteosarcoma is an aggressive primary bone tumor in humans and is among the most common cancer afflicting dogs. Despite surgical advancements and intensification of chemo- and targeted therapies, the survival outcome for osteosarcoma patients is, as of yet, suboptimal. The presence of metastatic disease at diagnosis or its recurrence after initial therapy is a major factor for the poor outcomes. It is thought that most human and canine patients have at least microscopic metastatic lesions at diagnosis. Osteosarcoma in dogs occurs naturally with greater frequency and shares many biological and clinical similarities with osteosarcoma in humans. From a genetic perspective, osteosarcoma in both humans and dogs is characterized by complex karyotypes with highly variable structural and numerical chromosomal aberrations. Similar molecular abnormalities have been observed in human and canine osteosarcoma. For instance, loss of TP53 and RB regulated pathways are common. While there are several oncogenes that are commonly amplified in both humans and dogs, such as MYC and RAS, no commonly activated proto-oncogene has been identified that could form the basis for targeted therapies. It remains possible that recurrent aberrant gene expression changes due to gene amplification or epigenetic alterations could be uncovered and these could be used for developing new, targeted therapies. However, the remarkably high genomic complexity of osteosarcoma has precluded their definitive identification. Several advantageous murine models of osteosarcoma have been generated. These include spontaneous and genetically engineered mouse models, including a model based on forward genetics and transposon mutagenesis allowing new genes and genetic pathways to be implicated in osteosarcoma development. The proposition of this review is that careful comparative genomic studies between human, canine and mouse models of osteosarcoma may help identify commonly affected and targetable pathways for alternative therapies for osteosarcoma patients. Translational research may be found through a path that begins in mouse models, and then moves through canine patients, and then human patients.

Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype.

Imprinting defects at human 14q32 locus alters gene expression and is associated with the pathobiology of osteosarcoma

Osteosarcoma is the most common primary bone malignancy affecting children and adolescents. Although several genetic predisposing conditions have been associated with osteosarcoma, our understanding of its pathobiology is rather limited. Here we show that, first, an imprinting defect at human 14q32-locus is highly prevalent (87%) and specifically associated with osteosarcoma patients < 30 years of age. Second, the average demethylation at differentially methylated regions (DMRs) in the 14q32-locus varied significantly compared to genome-wide demethylation. Third, the 14q32-locus was enriched in both H3K4-me3 and H3K27-me3 histone modifications that affected expression of all imprinted genes and miRNAs in this region. Fourth, imprinting defects at 14q32 - DMRs are present in triad DNA samples from affected children and their biological parents. Finally, imprinting defects at 14q32-DMRs were also observed at higher frequencies in an Rb1/Trp53 mutation-induced osteosarcoma mouse model. Further analysis of normal and tumor tissues from a Sleeping Beauty mouse model of spontaneous osteosarcoma supported the notion that these imprinting defects may be a key factor in osteosarcoma pathobiology. In conclusion, we demonstrate that imprinting defects at the 14q32 locus significantly alter gene expression, may contribute to the pathogenesis of osteosarcoma, and could be predictive of survival outcomes.

Comparative transcriptome analysis quantifies immune cell transcript levels, metastatic progression and survival in osteosarcoma

Overall survival of patients with osteosarcoma (OS) has improved little in the past three decades, and better models for study are needed. OS is common in large dog breeds and is genetically inducible in mice, making the disease ideal for comparative genomic analyses across species. Understanding the level of conservation of intertumor transcriptional variation across species and how it is associated with progression to metastasis will enable us to more efficiently develop effective strategies to manage OS and to improve therapy. In this study, transcriptional profiles of OS tumors and cell lines derived from humans (n = 49), mice (n = 103), and dogs (n = 34) were generated using RNA sequencing. Conserved intertumor transcriptional variation was present in tumor sets from all three species and comprised gene clusters associated with cell cycle and mitosis and with the presence or absence of immune cells. Further, we developed a novel gene cluster expression summary score (GCESS) to quantify intertumor transcriptional variation and demonstrated that these GCESS values associated with patient outcome. Human OS tumors with GCESS values suggesting decreased immune cell presence were associated with metastasis and poor survival. We validated these results in an independent human OS tumor cohort and in 15 different tumor data sets obtained from The Cancer Genome Atlas. Our results suggest that quantification of immune cell absence and tumor cell proliferation may better inform therapeutic decisions and improve overall survival for OS patients.Significance: This study offers new tools to quantify tumor heterogeneity in osteosarcoma, identifying potentially useful prognostic biomarkers for metastatic progression and survival in patients. Cancer Res; 78(2); 326-37. ©2017 AACR.

Abstract 1097: The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression

Osteosarcoma is the most common primary bone malignancy that affects adolescents. Around 30% of patients with localized osteosarcoma and 70% of patients with metastasis will experience treatment failure within 5 years of diagnosis. The complex biology of osteosarcoma and astounding genetic heterogeneity has made it challenging to identify effective new gene targets and therapeutic agents. Our studies found an overall overexpression of a microRNA cluster, miR-17-92 microRNAs in human primary osteosarcoma compared to normal bone. We learned that upregulation of this miR-17-92 cluster simultaneously silences a suite of key tumor suppressors. Using data from a novel spontaneous osteosarcoma mouse model and genetic screen, we discovered miR-17-92 targets, such as PTEN, PTRPD and SRGAP2, which are potential tumor suppressor genes. Specifically blocking miR-17-92 function in osteosarcoma cells reduced their migration and ability to grow larger tumors in immunodeficient mice compared to the controls. Also, knockdown of miR-17-92 cluster microRNAs led to increase in the levels of PTPRD and SRGAP2 in cells as well as tumors; further suggesting that miR-17-92 is targeting these genes. We also performed gain-of-function of miR-17-92 studies in a poorly aggressive osteosarcoma cell line and found that overexpression of miR-17-92 leads to ability to grow in an anchorage independent manner and form tumors in immunodeficient mice, both features that are lacking in the parental line. Ongoing RNA sequencing studies on miR-17-92 target transcripts in osteosarcoma cells, and functional analyses of miR-17-92 deletion mutants, will be presented. In an attempt to target miR-17-92 miRNA expression, we have tested small molecules. Our data suggests that triptolide, a diterpenoid epoxide, inhibits MYC expression and downregulates miR-17-92 miRNAs resulting in upregulation of several tumor suppressor driver proteins including PTEN, PTPRD, and SRGAP2. Together, our data suggests that upregulation of miR-17-92 miRNAs contributes to osteosarcoma progression and triptolide inhibits miR-17-92 expression. These data have implications for how sarcomas develop in general and suggest a new way to treat cancer by targeting microRNAs using small molecules. Citation Format: Jyotika Varshney, Nicholas J. Slipek, John Osborne, Adrian Chang, Anne E. Sarver, Ingrid Cornax, Gerry M. O’ Sullivan, Subbaya Subramanian, David A. Largaespada. The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1097.