Osteosarcoma remains a leading cause of cancer death in adolescents. Treatment paradigms and survival rates have not improved in two decades. Driving the lack of therapeutic inroads, the molecular etiology of osteosarcoma remains elusive. MicroRNAs (miRNAs) have demonstrated far-reaching effects on the cellular biology of development and cancer. Their role in osteosarcomagenesis remains largely unexplored. Here we identify for the first time an miRNA signature reflecting the pathogenesis of osteosarcoma from surgically procured samples from human patients. The signature includes high expression of miR-181a, miR-181b, and miR-181c as well as reduced expression of miR-16, miR-29b, and miR-142-5p. We also demonstrate that miR-181b and miR-29b exhibit restricted expression to distinct cell populations in the tumor tissue. Further, higher expression of miR-27a and miR-181c* in pre-treatment biopsy samples characterized patients who developed clinical metastatic disease. In addition, higher expression of miR-451 and miR-15b in pre-treatment samples correlated with subsequent positive response to chemotherapy. In vitro and in vivo functional validation in osteosarcoma cell lines confirmed the tumor suppressive role of miR-16 and the pro-metastatic role of miR-27a. Furthermore, predicted target genes for miR-16 and miR-27a were confirmed as down-regulated by real-time PCR. Affymetrix array profiling of cDNAs from the osteosarcoma specimens and controls were interrogated according to predicted targets of miR-16, miR142-5p, miR-29b, miR-181a/b, and miR-27a. This analysis revealed positive and negative correlations highlighting pathways of known importance to osteosarcoma, as well as novel genes. Thus, our findings establish a miRNA signature associated with pathogenesis of osteosarcoma as well as critical pre-treatment biomarkers of metastasis and responsiveness to therapy.
The WW domain-containing oxidoreductase (WWOX) spans one of the most active common fragile sites (CFSs) involved in cancer, FRA16D. WWOX encodes a 46-kDa protein that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase (SDR) domain. Through its WW domain, Wwox interacts with its partners and modulates their functions. Our data indicate that Wwox suppresses the transactivation function of several transcription factors implied in neoplasia by sequestering them in the cytoplasm. Work from our laboratory and other research groups have demonstrated that Wwox participates in a number of cellular processes including growth, differentiation, apoptosis, and tumor suppression. Targeted deletion of the Wwox gene in mice causes increased spontaneous and chemically induced tumor incidence supporting bona fide tumor suppressor function of WWOX. Moreover, generation of the Wwox-deficient mice uncovers, at least in part, some of the physiological in vivo functions of the WWOX gene. This review focuses on recent progress that elucidates Wwox functions in biology and pathology.
The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P < 0.0001). Compared with the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorigenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas RUNX2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease. Cancer Res; 70(13); 5577-86. ©2010 AACR.
WW domain-containing oxidoreductase (WWOX) is highly conserved in both humans and murine. WWOX spans the second most common human chromosomal fragile site, FRA16D, and is commonly inactivated in multiple human cancers. Modeling WWOX inactivation in mice revealed a complex phenotype including postnatal lethality, defects in bone metabolism and steroidogenesis and tumor suppressor function resulting in osteosarcomas. For better understanding of WWOX roles in different tissues at distinct stages of development and in pathological conditions, Wwox conditional knockout mice were generated in which loxp sites flank exon 1 in the Wwox allele. We demonstrated that Cre-mediated recombination using EIIA-Cre, a Cre line expressed in germline, results in postnatal lethality by age of three weeks and decreased bone mineralization resembling total ablation of WWOX as in conventional null mice. This animal model will be useful to study distinct roles of WWOX in multiple tissues at different ages.
Osteosarcoma (OS) is a highly metastatic form of bone cancer in adolescents and young adults which is resistant to existing treatments. Development of an effective therapy has been hindered by very limited understanding of the mechanisms of osteosarcomagenesis. Here, we used genetically engineered mice to investigate the effects of deleting the tumor suppressor Wwox selectively in either osteoblast progenitors or mature osteoblasts. Mice with conditional deletion of Wwox in pre-osteoblasts (WwoxΔosx1) displayed a severe inhibition of osteogenesis accompanied by p53 upregulation, effects that were not observed in mice lacking Wwox in mature osteoblasts. Deletion of p53 in WwoxΔosx1 mice rescued the osteogenic defect. In addition, the Wwox;p53Δosx1 double knockout mice developed poorly differentiated osteosarcomas that resemble human OS in histology, location, metastatic behavior, and gene expression. Strikingly, the development of osteosarcomas in these mice was greatly accelerated compared to mice lacking p53 only. In contrast, combined WWOX and p53 inactivation in mature osteoblasts did not accelerate osteosarcomagenesis compared to p53 inactivation alone. These findings provide evidence that a WWOX-p53 network regulates normal bone formation and that disruption of this network in osteoprogenitors results in accelerated OS. The Wwox;p53Δosx1 double knockout establishes a new OS model with significant advancement over existing models.
MHC-class I tetramers technology enabled the characterization of peptide-specific T cells at the single cell level in a variety of studies. Several laboratories have also developed MHC-class II multimers to characterize Ag-specific CD4 1 T cells. However, the generation and use of MHC-class II multimers seems more problematic than that of MHC-I multimers. We have generated HLA-DR*1101 tetramers in a versatile empty form, which can be loaded after purification with peptides of interest. We discuss the impact of critical biological and structural parameters for the optimal staining of Agspecific CD41 T cells using HLA-DR*1101 tetramers, such as: (i) activation state of CD41 T cells; (ii) membrane trafficking in the target CD41 T cells; (iii) binding characteristics of the loaded CD4 epitope. Our data indicate that reorganization of TCR on the plasma membrane upon CD41 T cell activation, as well as an homogenous binding frame of the CD4 epitopes to the soluble HLA-DR monomer, are critical for a stable TCR/MHC-class II tetramer interaction. These factors, together with the low frequencies and affinities of specific CD41 T cells, explain the need for in vitro expansion or ex vivo enrichment of specific T cells for the optimal visualization with MHC-class II tetramers. ' International Society for Advancement of Cytometry Key terms CD4+ T cells; tetramers; HLA-DR1101THE MHC tetramer technology was developed to investigate the dynamic of T cell response at the single cell level. Until the advent of these reagents (1), identification of antigen-specific T cells was only possible using either limiting dilution analysis or by tracking the TCR V repertoire at the molecular level. However, none of these techniques allow the direct phenotypic and functional characterization of the antigenspecific T cells. In this scenario, MHC class I tetramers appeared immediately powerful, enabling the direct visualization of CD8 1 T cells and clarifying relevant aspects of antigen-specific response in viral infections and cancer (2-7) (Chattopadhyay et al., Cytometry, in press [this issue]). By contrast, the use of MHC class II tetramers turned out to be more problematic, both for the possibility to obtain stable peptide-MHC class II complexes and the ability to detect specific CD4 1 T cells directly ex vivo.We will try to highlight the advantages and potentials, as well as the limitations and problems that are still under solution, of MHC class II tetramer technology.
Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases’ seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease.
Detection of CD4 1 T cells specific for tumor-associated antigens is critical to investigate the spontaneous tumor immunosurveillance and to monitor immunotherapy protocols in patients. We investigated the ability of HLA-DR*1101 multimers to detect CD4 1 T cells specific for three highly promiscuous MAGE-A3 derived peptides: (p39), MAGE-A3 281-295 (p57) and MAGE-A3 286-300 (p58). Tetramers stained specific CD4 1 T cells only when loaded with p39, although all peptides activated the specific T cells when presented by plasticbound HLA-DR*1101 monomers. This suggested that tetramer staining ability was determined by the mode rather than the affinity of peptide binding to HLA-DR*1101. We hypothesized that peptides should bear a single P1 anchor residue to bind all arms of the multimer in a homogeneous register to generate peptide-HLA-DR conformers with maximal avidity. Bioinformatics analysis indicated that p39 contained one putative P1 anchor residue, whereas the other two peptides contained multiple ones. Designing p57 and p58 analogues containing a single anchor residue generated HLA-DR*1101 tetramers that stained specific CD4 1 T cells. Producing HLA-DR*1101 monomers linked with the optimized MAGE-A3 analogues, but not with the original epitopes, further improved tetramer efficiency. Optimization of CD4 1 T-cell epitope-binding registers is thus critical to generate functional HLA-DR tetramers.Key words: Anchor residues . HLA-DR Ã 1101 . MAGE-A3 . MHC tetramers . Tumor antigens Supporting Information available online IntroductionAnimal studies have clearly shown the critical role for CD4 1 T cells in anti-tumor immune response; nevertheless, little is known as yet about spontaneous tumor-specific CD4 1 T-cell responses in patients [1,2]. This knowledge would be instrumental to the definition of the mechanisms underlying tumor immunosurveillance and, possibly, tumor escape, as well as for the correct design of optimal vaccination strategies.CD4 1 T-cell responses specific for tumor associated antigens (TAA) can be investigated at the single cell level by using soluble [3][4][5][6]. The identification of primary antigen-specific CD4 1 T cells by peptide-pulsed MHC class II tetramers seems, however, less straightforward than that of CD8 1 T cells by peptide-loaded MHC class I tetramers [7,8]. Several biological and structural characteristics of the CD4 1 T-cell response that differ from those displayed by the CD8 1 one seem to affect the capacity of MHC class II tetramers to optimally stain specific CD4 1 T cells ex vivo: (i) the lower frequency of peptide-specific CD4 1 T cells, (ii) their apparent lower affinity for the peptide-MHC class II complexes [9][10][11][12][13], and (iii) the requirement for an activation-induced TCR reorganization to bind with sufficient avidity cognate peptide-MHC class II tetramers [12,14,15].Furthermore, the open structure of the MHC class II molecules allows peptides as long as 20 aa to extend out of the binding groove at both ends [16][17][18]. It is thus possible that a peptide,...
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