Ryan Shuck

Effects of TP53 mutational status on gene expression patterns across 10 human cancer types

Mutations in the TP53 tumour suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumour progression remain only partially understood. Here, using data from the Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumours from 10 human cancer types. We show that tumours with TP53 mutations have altered gene expression profiles compared to tumours retaining two wild‐type TP53 alleles. Among 113 known p53‐up‐regulated target genes identified from cell culture assays, 10 were consistently up‐regulated in at least eight of 10 cancer types that retain both copies of wild‐type TP53. RPS27L, CDKN1A (p21CIP1) and ZMAT3 were significantly up‐regulated in all 10 cancer types retaining wild‐type TP53. Using this p53‐based expression analysis as a discovery tool, we used cell‐based assays to identify five novel p53 target genes from genes consistently up‐regulated in wild‐type p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2 and FOXM1 were disproportionately up‐regulated in many TP53 mutant cancer types. Finally, > 93% of tumours with a TP53 mutation exhibited greatly reduced wild‐type p53 messenger expression, due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumour suppressor. The data indicate that tumours with wild‐type TP53 retain some aspects of p53‐mediated growth inhibitory signalling through activation of p53 target genes and suppression of cell cycle regulatory genes. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

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.

Cancer’s Achilles’ Heel: Apoptosis and Necroptosis to the Rescue

Apoptosis, and the more recently discovered necroptosis, are two avenues of programmed cell death. Cancer cells survive by evading these two programs, driven by oncogenes and tumor suppressor genes. While traditional therapy using small molecular inhibitors and chemotherapy are continuously being utilized, a new and exciting approach is actively underway by identifying and using synergistic relationship between driver and rescue genes in a cancer cell. Through these synthetic lethal relationships, we are gaining tremendous insights into tumor vulnerabilities and specific molecular avenues for induction of programmed cell death. In this review, we briefly discuss the two cell death processes and cite examples of such synergistic manipulations for therapeutic purposes.

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.

Mouse tissues that undergo neoplastic progression after K-Ras activation are distinguished by nuclear translocation of phospho-ERK1/2 and robust tumor suppressor responses

Mutation of K-Ras is a frequent oncogenic event in human cancers, particularly cancers of lungs, pancreas, and colon. It remains unclear why some tissues are more susceptible to Ras-induced transformation than others. Here, we globally activated a mutant oncogenic K-Ras allele (K-RasG12D) in mice and examined the tissue-specific effects of this activation on cancer pathobiology, Ras signaling, tumor suppressor, DNA damage, and inflammatory responses. Within 5 to 6 weeks of oncogenic Ras activation, mice develop oral and gastric papillomas, lung adenomas, and hematopoietic hyperproliferation and turn moribund. The oral, gastric, and lung premalignant lesions display activated extracellular signal–regulated kinases (Erk)1/2 and NF-κB signaling as well as activated tumor suppressor and DNA damage responses. Other organs such as pancreas, liver, and small intestine do not exhibit neoplastic progression within 6 weeks following K-RasG12D activation and do not show a potent tumor suppressor response. Even though robust Erk1/2 signaling is activated in all the tissues examined, the pErk1/2 distribution remains largely cytoplasmic in K-RasG12D–refractory tissues (pancreas, liver, and intestines) as opposed to a predominantly nuclear localization in K-RasG12D–induced neoplasms of lung, oral, and gastric mucosa. The downstream targets of Ras signaling, pElk-1 and c-Myc, are elevated in K-RasG12D–induced neoplastic lesions but not in K-RasG12D–refractory tissues. We propose that oncogenic K-Ras–refractory tissues delay oncogenic progression by spatially limiting the efficacy of Ras/Raf/Erk1/2 signaling, whereas K-Ras–responsive tissues exhibit activated Ras/Raf/Erk1/2 signaling, rapidly form premalignant tumors, and activate potent antitumor responses that effectively prevent further malignant progression. Mol Cancer Res; 10(6); 845–55. ©2012 AACR.

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis.

Background:Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear.Methods:si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role.Results:Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways.Conclusions:Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS.

Enhanced inflammation and attenuated tumor suppressor pathways are associated with oncogene-induced lung tumors in aged mice

Aging is often accompanied by a dramatic increase in cancer susceptibility. To gain insights into how aging affects tumor susceptibility, we generated a conditional mouse model in which oncogenic KrasG12D was activated specifically in lungs of young (3–5 months) and old (19–24 months) mice. Activation of KrasG12D in old mice resulted in shorter survival and development of higher‐grade lung tumors. Six weeks after KrasG12D activation, old lung tissues contained higher numbers of adenomas than their young tissue counterparts. Lung tumors in old mice displayed higher proliferation rates, as well as attenuated DNA damage and p53 tumor suppressor responses. Gene expression comparison of lung tumors from young and old mice revealed upregulation of extracellular matrix‐related genes in young tumors, indicative of a robust cancer‐associated fibroblast response. In old tumors, numerous inflammation‐related genes such as Ccl7, IL‐1β, Cxcr6, and IL‐15ra were consistently upregulated. Increased numbers of immune cells were localized around the periphery of lung adenomas from old mice. Our experiments indicate that more aggressive lung tumor formation in older KrasG12D mice may be in part the result of subdued tumor suppressor and DNA damage responses, an enhanced inflammatory milieu, and a more accommodating tissue microenvironment.

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 A70: Targeting pediatric bone sarcoma stem cell with metabolic inhibitors

Our project tests whether metabolic inhibitors are able to target putative sarcoma stem cells in osteosarcoma and Ewing sarcoma. The cure rate for the two most common bone sarcomas affecting children and adolescents, osteosarcoma and Ewing sarcoma, has plateaued around 70% over the past twenty years, and the cure rate for patients with metastatic bone sarcomas has not significantly improved over 20% in several decades. The subpopulation of cells in these tumors referred to as sarcoma stem cells are thought to be responsible for resistance to current therapies, relapses and metastases. To date therapies targeting the sarcoma stem cell population have not been identified. We studied the ability of metabolic inhibitors to eliminate the sarcoma stem cell population. A panel of established and verified osteosarcoma (HOS, 143B, MNNG, SAOS2) and Ewing sarcoma (TC71, TC32, MHH, A4573, CHLA9, CHLA10) cell lines was grown in 2-dimensional culture and treated with metabolic inhibitors, including 2-deoxyglucose, dichloroacetate, oxamate, lonidamine, 3-Bromopyruvate, and metformin for 72hrs establishing dose response curves for each individual agent. Most agents showed IC50s in the millimolar range. Combinations of two metabolic inhibitors at their IC50 were then tested and evaluated for synergy and if the combination would allow for dosing at physiologically attainable concentrations. The combination of 2-deoxyglucose and metformin proved toxic to the osteosarcoma and Ewing sarcoma cell lines (p The cells were then grown in 3-dimensional cultures using Magnetic 3D Bioprinting kit(n3D Biosciences, Houston, TX) and exposed to physiologically attainable concentrations of 2-deoxyglucose (500uM) and metformin (5uM), based on Phase I studies of these individual agents in humans. The resulting 3-dimentional cell cultures were incubated for 10 days with 2-deoxyglucose and metformin and there was a significant reduction in cell proliferation as measured by trypan blue dye exclusion and reduced aldehyde dehydrogenase expression. Additionally, the functional assay of cancer stem cells, sphere-forming efficiency, was impeded in all cell lines treated with the combination of 2-deoxyglucose and metformin. Finally, the ability to establish orthotopic tumors in immune deficient mice was significantly reduced for intratibialy-injected cells pretreated with 2-deoxyglucose and metformin. In vivo testing of the combination of 2-deoxyglucose and metformin to treat established xenograft osteosarcoma and Ewing sarcomas is currently underway, and resulting tumors will be harvested and assessed for aldehyde dehydrogenase expression, sphere-forming efficiency, and further characterization of the stem cell properties and metabolic alterations. Our study will provide the preclinical basis for testing metabolic inhibitors, in particular the combination of 2-deoxyglucose and metformin, in clinical trials for pediatric bone sarcomas. Citation Format: Matteo Trucco, Nino Rainsusso, Piti Techavichit, Ronald Bernardi, Ryan Shuck, Laura Satterfield, Wendy Allen-Rhoades, Larry Donehower, David Loeb, Jason Yustein. Targeting pediatric bone sarcoma stem cell with metabolic inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A70.