The histone chaperone function of Daxx is dispensable for embryonic development.

Daxx functions as a histone chaperone for the histone H3 variant, H3.3, and is essential for embryonic development. Daxx interacts with Atrx to form a protein complex that deposits H3.3 into heterochromatic regions of the genome, including centromeres, telomeres, and repeat loci. To advance our understanding of histone chaperone activity in vivo, we developed two Daxx mutant alleles in the mouse germline, which abolish the interactions between Daxx and Atrx (DaxxY130A), and Daxx and H3.3 (DaxxS226A). We found that the interaction between Daxx and Atrx is dispensable for viability; mice are born at the expected Mendelian ratio and are fertile. The loss of Daxx-Atrx interaction, however, does cause dysregulated expression of endogenous retroviruses. In contrast, the interaction between Daxx and H3.3, while not required for embryonic development, is essential for postnatal viability. Transcriptome analysis of embryonic tissues demonstrates that this interaction is important for silencing endogenous retroviruses and for maintaining proper immune cell composition. Overall, these results clearly demonstrate that Daxx has both Atrx-dependent and independent functions in vivo, advancing our understanding of this epigenetic regulatory complex.

Blood-based Proteomic Signatures Associated With MEN1-related Duodenopancreatic Neuroendocrine Tumor Progression.

PURPOSE: Patients with multiple endocrine neoplasia type 1 (MEN1) are predisposed to develop duodenopancreatic neuroendocrine tumors (dpNETs), and metastatic dpNET is the primary cause of disease-related mortality. Presently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. In the current study, we aimed to establish novel circulating molecular protein signatures associated with disease progression. EXPERIMENTAL DESIGN: Mass spectrometry-based proteomic profiling was conducted on plasmas procured through an international collaboration between MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht from a cohort of 56 patients with MEN1 [14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)]. Findings were compared to proteomic profiles generated from serially collected plasmas from a mouse model of Men1-pancreatic neuroendocrine tumors (Men1fl/flPdx1-CreTg) and control mice (Men1fl/fl). RESULTS: A total of 187 proteins were found to be elevated in MEN1 patients with distant metastasis compared to controls, including 9 proteins previously associated with pancreatic cancer and other neuronal proteins. Analyses of mouse plasmas revealed 196 proteins enriched for transcriptional targets of oncogenic MYCN, YAP1, POU5F1, and SMAD that were associated with disease progression in Men1fl/flPdx1-CreTg mice. Cross-species intersection revealed 19 proteins positively associated with disease progression in both human patients and in Men1fl/flPdx1-CreTg mice. CONCLUSIONS: Our integrated analyses identified novel circulating protein markers associated with disease progression in MEN1-related dpNET.

Context matters - Daxx and Atrx are not robust tumor suppressors in the murine endocrine pancreas.

Genome sequencing has revealed the importance of epigenetic regulators in tumorigenesis. The genes encoding the chromatin remodeling complex DAXX:ATRX are frequently mutated in pancreatic neuroendocrine tumors; however, the underlying mechanisms of how mutations contribute to tumorigenesis are only partially understood, in part because of the lack of relevant preclinical models. Here, we used genetically engineered mouse models combined with environmental stress to evaluate the tumor suppressor functions of Daxx and Atrx in the mouse pancreas. Daxx or Atrx loss, alone or in combination with Men1 loss, did not drive or accelerate pancreatic neuroendocrine tumorigenesis. Moreover, Daxx loss did not cooperate with environmental stresses (ionizing radiation or pancreatitis) or with the loss of other tumor suppressors (Pten or p53) to promote pancreatic neuroendocrine tumorigenesis. However, owing to promiscuity of the Cre promoter used, hepatocellular carcinomas and osteosarcomas were observed in some instances. Overall, our findings suggest that Daxx and Atrx are not robust tumor suppressors in the endocrine pancreas of mice and indicate that the context of a human genome is essential for tumorigenesis. This article has an associated First Person interview with the first author of the paper.

EGFR suppresses p53 function by promoting p53 binding to DNA-PKcs: a noncanonical regulatory axis between EGFR and wild-type p53 in glioblastoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) amplification and TP53 mutation are the two most common genetic alterations in glioblastoma multiforme (GBM). A comprehensive analysis of the TCGA GBM database revealed a subgroup with near mutual exclusivity of EGFR amplification and TP53 mutations indicative of a role of EGFR in regulating wild-type-p53 (wt-p53) function. The relationship between EGFR amplification and wt-p53 function remains undefined and this study describes the biological significance of this interaction in GBM. METHODS: Mass spectrometry was used to identify EGFR-dependent p53-interacting proteins. The p53 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interaction was detected by co-immunoprecipitation. We used CRISPR-Cas9 gene editing to knockout EGFR and DNA-PKcs and the Edit-R CRIPSR-Cas9 system for conditional knockout of EGFR. ROS activity was measured with a CM-H2DCFDA probe, and real-time PCR was used to quantify expression of p53 target genes. RESULTS: Using glioma sphere-forming cells (GSCs), we identified, DNA-PKcs as a p53 interacting protein that functionally inhibits p53 activity. We demonstrate that EGFR knockdown increased wt-p53 transcriptional activity, which was associated with decreased binding between p53 and DNA-PKcs. We further show that inhibition of DNA-PKcs either by siRNA or an inhibitor (nedisertib) increased wt-p53 transcriptional activity, which was not enhanced further by EGFR knockdown, indicating that EGFR suppressed wt-p53 activity through DNA-PKcs binding with p53. Finally, using conditional EGFR-knockout GSCs, we show that depleting EGFR increased animal survival in mice transplanted with wt-p53 GSCs. CONCLUSION: This study demonstrates that EGFR signaling inhibits wt-p53 function in GBM by promoting an interaction between p53 and DNA-PKcs.

A Blood-based Polyamine Signature Associated With MEN1 Duodenopancreatic Neuroendocrine Tumor Progression.

CONTEXT: Duodenopancreatic neuroendocrine tumors (dpNETs) frequently occur in patients with multiple endocrine neoplasia type 1 (MEN1), and metastatic dpNET is the primary cause of disease-related mortality. There is a need for biomarkers that can identify patients with MEN1-related dpNETs that are at high risk of developing distant metastasis. Polyamines have tumor-promoting roles in several cancer types. OBJECTIVE: We hypothesized that MEN1-dpNET-related disease progression is associated with elevated levels of circulating polyamines. METHODS: Through an international collaboration between The University of Texas MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht, plasma polyamine levels were assessed using mass spectrometry in 84 patients with MEN1 (20 with distant metastatic dpNETs [patients] and 64 with either indolent dpNETs or no dpNETs [controls]). A mouse model of MEN1-pNET, Men1fl/flPdx1-CreTg, was used to test time-dependent changes in plasma polyamines associated with disease progression. RESULTS: A 3-marker plasma polyamine signature (3MP: N-acetylputrescine, acetylspermidine, and diacetylspermidine) distinguished patients with metastatic dpNETs from controls in an initial set of plasmas from the 3 participating centers. The fixed 3MP yielded an area under the curve of 0.84 (95% CI, 0.62-1.00) with 66.7% sensitivity at 95% specificity for distinguishing patients from controls in an independent test set from MDACC. In Men1fl/flPdx1-CreTg mice, the 3MP was elevated early and remained high during disease progression. CONCLUSION: Our findings provide a basis for prospective testing of blood-based polyamines as a potential means for monitoring patients with MEN1 for harboring or developing aggressive disease.

Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/ß-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and ß-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. SIGNIFICANCE: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis.This article is highlighted in the In This Issue feature, p. 1861.

p53 drives a transcriptional program that elicits a non-cell-autonomous response and alters cell state in vivo.

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion of Mdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which included Mdm2 but not p21 Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.

Daxx maintains endogenous retroviral silencing and restricts cellular plasticity in vivo.

Tumor sequencing studies have emphasized the role of epigenetics and altered chromatin homeostasis in cancer. Mutations in DAXX, which encodes a chaperone for the histone 3.3 variant, occur in 25% of pancreatic neuroendocrine tumors (PanNETs). To advance our understanding of physiological functions of Daxx, we developed a conditional Daxx allele in mice. We demonstrate that Daxx loss is well tolerated in the pancreas but creates a permissive transcriptional state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to alter gene expression and cell state, impairing pancreas recovery from inflammatory stress in vivo. The transcriptional changes are associated with dysregulation of endogenous retroviral elements (ERVs), and dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. Our results reveal a physiologic function of DAXX, provide a mechanism associated with impaired tissue regeneration and tumorigenesis, and expand our understanding of ERV regulation in somatic cells.

Men1 maintains exocrine pancreas homeostasis in response to inflammation and oncogenic stress.

A more comprehensive understanding of the molecular mechanisms underlying pancreatic diseases, including pancreatitis and cancer, is essential to improve clinical management. MEN1 has established roles in epigenetic regulation and tumor suppression in the endocrine pancreas; however, intriguing recent data suggest MEN1 may also function in the exocrine pancreas. Using physiologically relevant genetic mouse models, we provide direct evidence that Men1 is essential for exocrine pancreas homeostasis in response to inflammation and oncogenic stress. Men1 loss causes increased injury and impaired regeneration following acute caerulein-induced pancreatitis, leading to more severe damage, loss of the normal acinar compartment, and increased cytokeratin 19-positive metaplasias and immune cell infiltration. We further demonstrate the Men1 protein is stabilized in response to insult, and loss of Men1 is associated with the overexpression of proinflammatory Jund target genes, suggesting that loss of Men1-mediated repression of Jund activity is, at least in part, responsible for the impaired response. Finally, we demonstrate that Men1 loss significantly accelerates mutant Kras-dependent oncogenesis. Combined, this work establishes Men1 as an important mediator of pancreas homeostasis in vivo.

Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells.

BACKGROUND: Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). METHODS: Mice bearing an inducible EWS-FLI1 transgene were crossed to p53-/- mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. RESULTS: Primary murine MSCs with the genotype EWS-FLI1 p53-/- were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53-/- MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p < .001). Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001). It also increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. CONCLUSION: Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.

Transient enhancement of p53 activity protects from radiation-induced gastrointestinal toxicity.

Gastrointestinal (GI) syndrome is a serious side effect and dose-limiting toxicity observed in patients undergoing lower-abdominal radiotherapy. Previous mouse studies show that p53 gene dosage determines susceptibility to GI syndrome development. However, the translational relevance of p53 activity has not been addressed. Here, we used a knock-in mouse in which the p53-Mdm2 negative feedback loop is genetically disrupted. These mice retain biallelic p53 and thus, normal basal p53 levels and activity. However, due to the lack of p53-mediated Mdm2 transcription, irradiated Mdm2P2/P2 mice exhibit enhanced acute p53 activity, which protects them from GI failure. Intestinal crypt cells residing in the +4 and higher positions exhibit decreased apoptosis, increased p21 expression, and hyperproliferation to reinstate intestinal integrity. Correspondingly, pharmacological augmentation of p53 activity in wild-type mice with an Mdm2 inhibitor protects against GI toxicity without affecting therapeutic outcome. Our results suggest that transient disruption of the p53-Mdm2 interaction to enhance p53 activity could be a viable prophylactic strategy for alleviating GI syndrome in patients undergoing radiotherapy.

Loss of Menin Expression by Immunohistochemistry in Pancreatic Neuroendocrine Tumors: Comparison Between Primary and Metastatic Tumors.

OBJECTIVES: Molecular characterization of sporadic pancreatic neuroendocrine tumors (PanNETs) demonstrates frequent alterations in MEN1. As the role of menin immunohistochemistry as a potential biomarker is being developed, knowledge of whether the pattern of menin expression is the same in primary tumors and distant metastases may help in patient care. Therefore, we compared patterns of menin expression in matched primary tumors and metastases. METHODS: We evaluated loss of menin nuclear expression by immunohistochemistry in 44 matched samples of primary and metastatic PanNETs and concordance in staining pattern between primary and metastatic tumors. RESULTS: Menin nuclear expression was lost in 18 (41%) of 44 primary tumors and 17 (39%) of 44 metastases. Concordant loss of menin expression was observed in 41 cases (93%); discordance was observed in 3 cases (7%; 95% confidence interval, 1.4%-18.7%), including 2 with loss in the primary tumor but not the metastasis. CONCLUSIONS: The concordance of menin staining between primary tumor and metastasis in most cases suggests that menin loss is an early event in PanNET tumorigenesis. The discordant expression observed in a small subset may be a source of menin-directed therapy failure; thus, repeat assessment of metastases may be helpful for treatment selection.

Dicer1 Phosphomimetic Promotes Tumor Progression and Dissemination.

Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/- ). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion. SIGNIFICANCE: This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.

Constitutive Dicer1 phosphorylation accelerates metabolism and aging in vivo.

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

p53 Mediates Vast Gene Expression Changes That Contribute to Poor Chemotherapeutic Response in a Mouse Model of Breast Cancer.

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse.

Daxx Functions Are p53-Independent In Vivo.

Mutations in the death domain-associated protein (DAXX) have been recently identified in a substantial proportion of human pancreatic neuroendocrine tumors (PanNETs). Remarkably, however, little is known about the physiologic role(s) of DAXX despite in vitro studies suggesting potential functions. Most prominently, and supported by tumor sequencing data, DAXX functions in concert with alpha thalassemia/mental retardation X-linked (ATRX) as a histone chaperone complex for the H3.3 variant. Studies have also identified potential roles in apoptosis, transcription, and negative regulation of the p53 tumor suppressor pathway. Herein, a mouse modeling approach was used to specifically address the latter and no significant genetic interaction between Daxx and the p53 pathway was determined. The embryonic lethal phenotype of Daxx loss is not p53-dependent. In addition, Daxx heterozygosity does not sensitize mice to a sublethal dose of ionizing radiation or alter the survival or tumor phenotype of Mdm2 transgenic mice. However, the data support a tumor suppressor role for DAXX as low-dose ionizing radiation produced a higher proportion of carcinomas in Daxx heterozygous mice than wild-type controls.Implications: While DAXX has important in vivo functions, they are independent of an inhibitory role on the p53 tumor suppressor pathway. Mol Cancer Res; 16(10); 1523-9. ©2018 AACR.

Loss of digestive organ expansion factor (Diexf) reveals an essential role during murine embryonic development that is independent of p53.

Increased levels of inhibitors of the p53 tumor suppressor such as Mdm2 and Mdm4 drive tumor development and thus serve as targets for therapeutic intervention. Recently, digestive organ expansion factor (Diexf) has been identified as a novel inhibitor of p53 in zebrafish. Here, we address the potential role of Diexf as a regulator of the p53 pathway in mammals by generating Diexf knockout mice. We demonstrate that, similar to Mdm2 and Mdm4, homozygous deletion of Diexf is embryonic lethal. However, unlike in Mdm2 and Mdm4 mice, loss of p53 does not rescue this phenotype. Moreover, Diexf heterozygous animals are not sensitive to sub-lethal ionizing radiation. Thus, we conclude that Diexf is an essential developmental gene in the mouse, but is not a significant regulator of the p53 pathway during development or in response to ionizing radiation.

Attenuating the p53 Pathway in Human Cancers: Many Means to the Same End.

The p53 pathway is perturbed in the majority of human cancers. Although this most frequently occurs through the direct mutation or deletion of p53 itself, there are a number of other alterations that can attenuate the pathway and contribute to tumorigenesis. For example, amplification of important negative regulators, MDM2 and MDM4, occurs in a number of cancers. In this work, we will review both the normal regulation of the p53 pathway and the different mechanisms of pathway inhibition in cancer, discuss these alterations in the context of the global genomic analyses that have been conducted across tumor types, and highlight the translational implications for cancer diagnosis and treatment.

PLA2G16 promotes osteosarcoma metastasis and drug resistance via the MAPK pathway.

The prognosis of metastatic osteosarcoma is dismal and a better understanding of the mechanisms underlying disease progression is essential to improve treatment options and patient outcomes. We previously demonstrated Pla2g16 overexpression in mouse osteosarcoma contributes to metastasis phenotypes and increased expression of PLA2G16 is associated with metastasis and poor prognosis in human tumors. To further examine the mechanisms through which PLA2G16 contributes to human osteosarcoma metastasis and explore the potential of PLA2G16 as a therapeutic target in osteosarcoma, we generated a panel of human osteosarcoma cell lines expressing different levels of PLA2G16. The functional analyses of these cell lines demonstrated high levels of PLA2G16 expression increased osteosarcoma cell migration, invasion, clonogenic survival, and anchorage-independent colony formation. Importantly, this activity was dependent on the phospholipase activity of PLA2G16. Additionally, PLA2G16 overexpression decreased the sensitivity of cells to a panel of chemotherapeutic agents. Analysis of downstream pathways revealed the pro-metastasis functions of PLA2G16 were mediated through the MAPK pathway, as knockdown of PLA2G16 decreased ERK1/2 phosphorylation and pharmacological inhibition of MEK significantly repressed PLA2G16 mediated cell migration and clonogenic survival. Furthermore, PLA2G16 overexpression promoted xenograft tumor growth in vivo, and these tumors exhibit increased ERK1/2 phosphorylation. Lastly, the expression of PLA2G16 is strongly correlated with the increased ERK1/2 phosphorylation in human osteosarcoma samples, and the combined lesions are associated with reduced overall and metastasis-free survival. Collectively, these results demonstrate increased PLA2G16 expression activates the MAPK pathway to enhance osteosarcoma metastasis and may be a novel therapeutic target for these cancers.