Chemokine signaling via the CXCR2 receptor reinforces senescence.

Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.

Intestinal microbiota enhances pancreatic carcinogenesis in preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States yet data are scant regarding host factors influencing pancreatic carcinogenesis. Increasing evidence support the role of the host microbiota in carcinogenesis but its role in PDAC is not well established. Herein, we report that antibiotic-mediated microbial depletion of KrasG12D/PTENlox/+ mice showed a decreased proportion of poorly differentiated tumors compared to microbiota-intact KrasG12D/PTENlox/+ mice. Subsequent 16S rRNA PCR showed that ~50% of KrasG12D/PTENlox/+ mice with PDAC harbored intrapancreatic bacteria. To determine if a similar observation in humans correlates with presence of PDAC, benign and malignant human pancreatic surgical specimens demonstrated a microbiota by 16S bacterial sequencing and culture confirmation. However, the microbial composition did not differentiate PDAC from non-PDAC tissue. Furthermore, murine pancreas did not naturally acquire a pancreatic microbiota, as germ-free mice transferred to specific pathogen-free housing failed to acquire intrapancreatic bacteria over time, which was not augmented by a murine model of colitis. Finally, antibiotic-mediated microbial depletion of Nod-SCID mice, compared to microbiota-intact, showed increased time to PDAC xenograft formation, smaller tumors, and attenuated growth. Interestingly, both xenograft cohorts were devoid of intratumoral bacteria by 16S rRNA PCR, suggesting that intrapancreatic/intratumoral microbiota is not the sole driver of PDAC acceleration. Xenografts from microbiota-intact mice demonstrated innate immune suppression by immunohistochemistry and differential regulation of oncogenic pathways as determined by RNA sequencing. Our work supports a long-distance role of the intestinal microbiota on PDAC progression and opens new research avenues regarding pancreatic carcinogenesis.

Inactivation of Patched1 in mice leads to development of gastrointestinal stromal-like tumors that express Pdgfrα but not kit.

BACKGROUND & AIMS: A fraction of gastrointestinal stromal tumor (GIST) cells overexpress the platelet-derived growth factor receptor (PDGFR)A, although most overexpress KIT. It is not known if this is because these receptor tyrosine kinases have complementary oncogenic potential, or because of heterogeneity in the cellular origin of GIST. Little also is known about why Hedgehog (HH) signaling is activated in some GIST. HH binds to and inactivates the receptor protein patched homolog (PTCH). METHODS: Ptch was conditionally inactivated in mice (to achieve constitutive HH signaling) using a Cre recombinase regulated by the lysozyme M promoter. Cre-expressing cells were traced using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, immunoblot, and quantitative reverse-transcriptase polymerase chain reaction analyses. Cell transformation was assessed by soft agar assay. RESULTS: Loss of Ptch from lysozyme M-expressing cells resulted in the development of tumors of GIST-like localization and histology; these were reduced when mice were given imatinib, a drug that targets KIT and PDGFRA. The Hh signaling pathway was activated in the tumor cells, and Pdgfrα, but not Kit, was overexpressed and activated. Lineage tracing revealed that Cre-expressing intestinal cells were Kit-negative. These cells sometimes expressed Pdgfrα and were located near Kit-positive interstitial cells of Cajal. In contrast to KIT, activation of PDGFRA increased anchorage-independent proliferation and was required for tumor formation in mice by cells with activated HH signaling. CONCLUSIONS: Inactivation of Ptch in mice leads to formation of GIST-like tumors that express Pdgfrα, but not Kit. Activation of Pdgfrα signaling appears to facilitate tumorigenesis.

Dual Pten/Tp53 suppression promotes sarcoma progression by activating Notch signaling.

Soft tissue sarcomas are a heterogeneous group of tumors associated with poor clinical outcome. Although a subset of soft tissue sarcomas is characterized by simple karyotypes and recurrent chromosomal translocations, the mechanisms driving cytogenetically complex sarcomas are largely unknown. Clinical evidence led us to partially inactivate Pten and Tp53 in the smooth muscle lineage of mice, which developed high-grade undifferentiated pleomorphic sarcomas, leiomyosarcomas, and carcinosarcomas that widely recapitulate the human disease, including the aberrant karyotype and metastatic behavior. Pten was found haploinsufficient, whereas the wild-type allele of Tp53 invariably gained point mutations. Gene expression profiles showed up-regulated Notch signaling in Pten(Δ/+)Tp53(Δ/+) tumors compared with Pten(+/+)Tp53(Δ/+) tumors. Consistently, Pten silencing exacerbated the clonogenic and invasive potential of Tp53-deficient bone marrow-derived mouse mesenchymal stem cells and tumor cells and activated the Notch pathway. Moreover, the increased oncogenic behavior of Pten(Δ/+)Tp53(Δ/+) and shPten-transduced Pten(+/+)Tp53(Δ/+) tumor cells was counteracted by treatment with a γ-secretase inhibitor, suggesting that the aggressiveness of those tumors can be attributed, at least in part, to enhanced Notch signaling. This study demonstrates a cooperative role for Pten and Tp53 suppression in complex karyotype sarcomas while establishing Notch as an important functional player in the cross talk of these pathways during tumor progression. Our results highlight the importance of molecularly subclassifying patients with high-grade sarcoma for targeted treatments.

Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis.

MicroRNA cluster miR-17-92 has been implicated in cardiovascular development and function, yet its precise mechanisms of action in these contexts are uncertain. This study aimed to investigate the role of miR-17-92 in morphogenesis and function of cardiac and smooth muscle tissues. To do so, a mouse model of conditional overexpression of miR-17-92 in cardiac and smooth muscle tissues was generated. Extensive cardiac functional studies identified a dose-dependent induction of dilated, hypertrophic cardiomyopathy, and arrhythmia inducibility in transgenic animals, which correlated with premature mortality (98.3 ± 42.5 d, P<0.0001). Expression analyses revealed the abundance of Pten transcript, a known miR-17-92 target, to be inversely correlated with miR-17-92 expression levels and heart size. In addition, we demonstrated through 3'-UTR luciferase assays and expression analyses that Connexin43 (Cx43) is a novel direct target of miR-19a/b and its expression is suppressed in transgenic hearts. Taken together, these data demonstrate that dysregulated expression of miR-17-92 during cardiovascular morphogenesis results in a lethal cardiomyopathy, possibly in part through direct repression of Pten and Cx43. This study highlights the importance of miR-17-92 in both normal and pathological functions of the heart, and provides a model that may serve as a useful platform to test novel antiarrhythmic therapeutics.

MiR-130b modulates the invasive, migratory, and metastatic behavior of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive, often poorly differentiated cancer of the smooth muscle (SM) lineage for which the molecular drivers of transformation and progression are poorly understood. In microRNA (miRNA) profiling studies, miR-130b was previously found to be upregulated in LMS vs. normal SM, and down-regulated during the differentiation of mesenchymal stem cells (MSCs) into SM, suggesting a role in LMS tumor progression. In the present study, the effects of miR-130b on human LMS tumorigenesis were investigated. Stable miR-130b overexpression enhanced invasion of LMS cells in vitro, and led to the formation of undifferentiated, pleomorphic tumors in vivo, with increased growth and metastatic potential compared to control LMS cells. TSC1 was identified as a direct miR-130b target in luciferase-3'UTR assays, and shRNA-mediated knockdown of TSC1 replicated miR-130b effects. Loss-of-function and gain-of-function studies showed that miR-130b levels regulate cell morphology and motility. Following miR-130b suppression, LMS cells adopted a rounded morphology, amoeboid mode of cell movement and enhanced invasive capacity that was Rho/ROCK dependent. Conversely, miR-130b-overexpressing LMS cells exhibited Rho-independent invasion, accompanied by down-regulation of Rho-pathway effectors. In mesenchymal stem cells, both miR-130b overexpression and TSC1 silencing independently impaired SM differentiation in vitro. Together, the data reveal miR-130b as a pro-oncogenic miRNA in LMS and support a miR-130b-TSC1 regulatory network that enhances tumor progression via inhibition of SM differentiation.

TYMS promotes genomic instability and tumor progression in Ink4a/Arf null background.

We previously showed that elevated TYMS exhibits oncogenic properties and promotes tumorigenesis after a long latency, suggesting cooperation with sequential somatic mutations. Here we report the cooperation of ectopic expression of human TYMS with loss of Ink4a/Arf, one of the most commonly mutated somatic events in human cancer. Using an hTS/Ink4a/Arf -/- genetically engineered mouse model we showed that deregulated TYMS expression in Ink4a/Arf null background accelerates tumorigenesis and metastasis. In addition, tumors from TYMS-expressing mice were associated with a phenotype of genomic instability including enhanced double strand DNA damage, aneuploidy and loss of G1/S checkpoint. Downregulation of TYMS in vitro decreased cell proliferation and sensitized tumor cells to antimetabolite chemotherapy. In addition, depletion of TYMS in vivo by TYMS shRNA reduced tumor incidence, delayed tumor progression and prolonged survival in hTS/Ink4a/Arf -/- mice. Our data shows that activation of TYMS in Ink4a/Arf null background enhances uncontrolled cell proliferation and tumor growth, supporting the development of new agents and strategies targeting TYMS to delay tumorigenesis and prolong survival.

First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival.

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

Corrigendum: Targeting thymidylate synthase enhances the chemosensitivity of triple-negative breast cancer towards 5-FU-based combinatorial therapy.

[This corrects the article DOI: 10.3389/fonc.2021.656804.].

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

A differentiation-based microRNA signature identifies leiomyosarcoma as a mesenchymal stem cell-related malignancy.

Smooth muscle (SM) is a spontaneously contractile tissue that provides physical support and function to organs such as the uterus. Uterine smooth muscle-related neoplasia comprise common well-differentiated benign lesions called leiomyomas (ULM), and rare, highly aggressive and pleomorphic tumors named leiomyosarcomas (ULMS). MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in normal cellular development and tissue homeostasis that can be used to accurately subclassify different tumor types. Here, we demonstrate that miRNAs are required for full smooth muscle cell (SMC) differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). We also report a miRNA signature associated with this process. Moreover, we show that this signature, along with miRNA profiles for ULMS and ULM, are able to subclassify tumors of smooth muscle origin along SM differentiation. Using multiple computational analyses, we determined that ULMS are more similar to hMSCs as opposed to ULM, which are linked with more mature SMCs and myometrium. Furthermore, a comparison of the SM differentiation and ULMS miRNA signatures identified miRNAs strictly associated with SM maturation or transformation, as well as those modulated in both processes indicating a possible dual role. These results support separate origins and/or divergent transformation pathways for ULM and ULMS, resulting in drastically different states of differentiation. In summary, this work expands on our knowledge of the regulation of SM differentiation and sarcoma pathogenesis.

Targeting Thymidylate Synthase Enhances the Chemosensitivity of Triple-Negative Breast Cancer Towards 5-FU-Based Combinatorial Therapy.

BACKGROUND: The ongoing treatment modalities for breast cancer (BC) primarily rely on the expression status of ER, PR and HER-2 receptors in BC tissues. Our strategy of chemosensitization provides new insights to counter chemoresistance, a major obstacle that limits the benefits of chemotherapy of mammary cancers. METHODS: By utilizing a murine breast cancer model employing NSG mice bearing orthotopic triple-negative breast cancer (TNBC) xenografts, we have evaluated the ability of phytochemical curcumin in chemosensitizing BC to 5-Fluorouracil (5-FU) chemotherapy and the differential modulations of cellular events in response to this strategy, independent of their receptor status. RESULTS: A significant synergistic antitumor potential was observed in the murine model with a sub-optimal dose treatment of 5-FU plus curcumin, as evaluated by a reduction in the tumor-related parameters. We authenticated the pivotal role of thymidylate synthase (TS) in regulating the 5-FU-curcumin synergism using the TNBC pre-clinical model. Our study also confirmed the pharmacological safety of this chemotherapeutic plus phytoactive combination using acute and chronic toxicity studies in Swiss albino mice. Subsequently, the molecular docking analysis of curcumin binding to TS demonstrated the affinity of curcumin towards the cofactor-binding site of TS, rather than the substrate-binding site, where 5-FU binds. Our concomitant in vivo and in silico evidence substantiates the superior therapeutic index of this combination. CONCLUSION: This is the first-ever pre-clinical study portraying TS as the critical target of combinatorial therapy for mammary carcinomas and therefore we recommend its clinical validation, especially in TNBC patients, who currently have limited therapeutic options.

Tsc1 Regulates the Proliferation Capacity of Bone-Marrow Derived Mesenchymal Stem Cells.

TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.

Oncolytic virotherapy for small-cell lung cancer induces immune infiltration and prolongs survival.

Oncolytic virotherapy has been proposed as an ablative and immunostimulatory treatment strategy for solid tumors that are resistant to immunotherapy alone; however, there is a need to optimize host immune activation using preclinical immunocompetent models in previously untested common adult tumors. We studied a modified oncolytic myxoma virus (MYXV) that shows high efficiency for tumor-specific cytotoxicity in small-cell lung cancer (SCLC), a neuroendocrine carcinoma with high mortality and modest response rates to immune checkpoint inhibitors. Using an immunocompetent SCLC mouse model, we demonstrated the safety of intrapulmonary MYXV delivery with efficient tumor-specific viral replication and cytotoxicity associated with induction of immune cell infiltration. We observed increased SCLC survival following intrapulmonary MYXV that was enhanced by combined low-dose cisplatin. We also tested intratumoral MYXV delivery and observed immune cell infiltration associated with tumor necrosis and growth inhibition in syngeneic murine allograft tumors. Freshly collected primary human SCLC tumor cells were permissive to MYXV and intratumoral delivery into patient-derived xenografts resulted in extensive tumor necrosis. We confirmed MYXV cytotoxicity in classic and variant SCLC subtypes as well as cisplatin-resistant cells. Data from 26 SCLC human patients showed negligible immune cell infiltration, supporting testing MYXV as an ablative and immune-enhancing therapy.

PPP1CA contributes to the senescence program induced by oncogenic Ras.

Ectopic expression of conditional murine p53 (p53val135) and oncogenic ras is enough to induce a senescent-like growth arrest at the restrictive temperature. We took advantage of this cellular system to identify new key players in the ras/p53-induced senescence. Applying a retroviral-based genetic screen, we obtained an antisense RNA fragment against PPP1CA, the catalytic subunit of protein phosphatase 1alpha, whose loss of function bypasses ras/p53-induced growth arrest and senescence. Expression of a specific short hairpin (sh)RNA against PPP1CA impairs the p53-dependent induction of p21 after DNA damage and blocks the subsequent pRb dephosphorylation, thus bypassing p53-induced arrest. We found that oncogenic ras promotes an increase in the intracellular level of ceramides together with an increase in the PPP1CA protein levels. Addition of soluble ceramide to the cells induced a senescence phenotype that is blocked through PPP1CA downregulation by specific shRNA. Analysis of human tumors suggests that one of the PPP1CA alleles might be lost in a high percentage of carcinomas such as kidney and colorectal. The overexpression of two out of five PPP1CA alternative spliced variants reduced tumor cell growth and the downregulation of the protein to hemizygosity increased the anchorage-independent growth. We propose that oncogenic stress induced by ras causes ceramide accumulation, therefore, increasing PPP1CA activity, pRb dephosphorylation and onset of the p53-induced arrest, contributing to tumor suppression.

MAP17 inhibits Myc-induced apoptosis through PI3K/AKT pathway activation.

MAP17 is a non-glycosylated membrane-associated protein that has been shown to be over-expressed in human carcinomas, suggesting a possible role of this protein in tumorigenesis. However, very little is known about the molecular mechanism mediating the possible tumor promoting properties of MAP17. To analyze the effect of MAP17 on cell survival, we used Rat1 fibroblasts model where Myc over-expression promotes apoptosis in low serum conditions. In the present work, we report that over-expression of MAP17 protects Rat1a fibroblasts from Myc-induced apoptosis through reactive oxygen species (ROS)-mediated activation of the PI3K/AKT signaling pathway. MAP17-mediated survival was associated with absence of Bax translocation to the mitochondria and reduced caspase-3 activation. We show that a fraction of PTEN undergoes oxidation in MAP17-over-expressing cells. Furthermore, activation of AKT by MAP17 as measured by Thr308 phosphorylation was independent of PI3K activity. Importantly, modulation of ROS by antioxidant treatment prevented activation of AKT, restoring the level of apoptosis in serum-starved Rat1/c-Myc fibroblasts. Finally, over-expression of a dominant-negative mutant of AKT in MAP17-expressing clones makes them sensitive to serum depletion. Our data indicate that MAP17 protein activates AKT through ROS and this is determinant to confer resistance to Myc-induced apoptosis in the absence of serum.

MAP17 enhances the malignant behavior of tumor cells through ROS increase.

Tumorigenesis occurs when the mechanisms involved in the control of tissue homeostasis are disrupted and cells stop responding to physiological signals. Therefore, genes capable of desensitizing tumoral cells from physiological signals may provide a selective advantage within the tumoral mass and influence the outcome of the disease. We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified by differential display being over-expressed in carcinomas. Tumor cells that over-express MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in presence or absence of contact inhibition, decreased apoptotic sensitivity and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 are associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of melanoma cells with inhibitors of Na+-coupled co-transporters lead to an inhibition of ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones. Finally, we show that MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain, since disruption of its sequence by point mutations abolishes its ability to enhance ROS production and tumorigenesis. Our work shows the tumorigenic capability of MAP17 through a connection between Na+-coupled co-transporters and ROS.

Genome-Wide miRNA Screening for Genes Bypassing Oncogene-Induced Senescence.

MicroRNAs are small noncoding RNAs that regulate gene expression by binding to sequences within the 3'-UTR of mRNAs. Genome-wide screens have proven powerful in associating genes with certain phenotypes or signal transduction pathways and thus are valuable tools to define gene function. Here we describe a genome-wide miRNA screening strategy to identify miRNAs that are required to bypass oncogene-induced senescence.

Limited miR-17-92 overexpression drives hematologic malignancies.

The overexpression of microRNA cluster miR-17-92 has been implicated in development of solid tumors and hematological malignancies. The role of miR-17-92 in lymphomagenesis has been extensively investigated; however, because of the developmental defects caused by miR-17-92 dysregulation, its ability to drive tumorigenesis has remained undetermined until recently. Here we demonstrate that overexpression of miR-17-92 in a limited number of hematopoietic cells is sufficient to cause B cell malignancies. In sum, our study provides a novel and physiologically relevant model that exposes the potent ability of miR-17-92 to act as a driver of tumorigenesis.

Osteosarcoma: mouse models, cell of origin and cancer stem cell.

Osteosarcoma (OS) is the most common non-hematologic primary tumor of bone in children and adults. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for non-metastatic disease approaching 70%. However, most OS tumors are high grade and tend to rapidly develop pulmonary metastases. Despite clinical advances, patients with metastatic disease or relapse have a poor prognosis. Here the cell biology of OS is reviewed with a special emphasis on mouse models as well as the roles of the cell of origin and cancer stem cells. A better understanding of the molecular pathogenesis of human OS is essential for the development of improved prognostic and diagnostic markers as well as targeted therapies for both primary and metastatic OS.