| IntroductionHuman mesenchymal stem cells (hMSCs) are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to osteoblast cells and contribute to bone development and fracture repair. Meanwhile, osteosarcoma transformation may be associated with osteoblastic differentiation of hMSCs, because osteosarcoma may be caused by genetic and epigenetic disruptions of osteoblastic differentiation pathway in hMSCs. Thus, this study focus on the cellular and molecular pathways involved in osteoblastic differentiation and osteosarcoma pathogenesis, investigated the role of ATRA in control of osteoblastic development of hMSCs, characterized the sub-population of cells within osteosarcoma possess cancer initiating properties, and analyzed potential links between defective osteogenic differentiation and bone tumorigenesis. Understanding the molecular pathogenesis of human osteosarcoma could ultimately lead to the development of diagnostic and prognostic markers, as well as targeted therapeutics for osteosarcoma patients.The current study include three parts:part 1 to evaluate the role of ATRA and its pathway in controlling of osteoblastic development of hMSCs; part 2 to identify and characterize cancer stem cells within osteosarcoma; part3 to analyzed potential links between defective osteogenic differentiation and osteosarcoma transformation using microarry analysis and Bioinformatics.Part 1 RARa-Bmp8-Fgf8 signaling in mediating osteoblastic lineage development of human mesenchymal stem cellsObjective:Retinoic acid (RA) and their receptors are present in differentiating tissues during development and are thought to play an important role in regulating various processes such as proliferation, differentiation and maturation of cells. ATRA is also known as the most potent differentiation inducer to treat malignant disease. We asked in this study whether retinoid signaling could have a role in the differentiation of human mesenchymal stem cells (hMSCs) to an osteoblastic lineage.Methods and results:ATRA effectively enhances the osteoblast-specific alkaline phosphatase (ALP) activities in both hMSCs and the supernatant and inhibits proliferation of early stage of hMSCs. The expression of osteoblastic differentiation marker genes, including OPN, osteocalcin, and RUNX2 are also up regulated in the presence of ATRA. In addition, DNA microarray analyses show that ATRA up regulate expression of multiply osteoblastic differentiation marker genes in hMSCs. Transduction of RARaS77A, a phosphorylation-defective form of RARa, mimics the effect of ATRA in mediating osteoblastic differentiation of hMSCs. Transduced hMSCs automatically activate ALP and enhance calcium deposition. RARαS77A also inhibit hMSCs'proliferation and induce OPN, Osteocalcin expression. Interestly, up-regulation of BMP8a is observered in ATRA, BDM and RARaS77A induced hMSCs osteoblastic differentiation. Overexpression of BMP8a in hMSCs shows calcium deposition and OPN up-regulation. Besides BMP8a, FGF8f is also up-regulated in RARaS77A transduced hMSCs, and FGF8f overexpression in hMSCs will cause calcium deposition. Conclusion:ATRA combine dexamethasone effectively enhance osteoblastic early differentiation of hMSCs. ATRA-induced RARa hypophosphorylation is involved in mediating osteoblastic lineage development of hMSC. BMP8a and FGF8f induction activated by ATRA or RARa hypophosphorylation are involved in regulation of the earlier and later stage of osteoblastic differentiation, respectively.Part 2 Identification of cells initiating human osteosarcoma and generating osteosarcoma-formation cellsObjective:Osteosarcoma, generally considered a differentiation disease, arises around the growth plate of long bones. Identification and understanding of OSIC are crucial to develop novel cancer stem cell-directed treatments against osteosarcoma, which should reduce therapy resistance, relapse, and the toxicity associated with current, non-selective chemotherapy in the treatment of osteosarcoma. In this study, we identified stem cell biomarkers of cancer stem cells within osteosarcoma, and isolated and characterized osteosarcoma initiating cells and osteosarcoma tumor forming cells.Methods and results:(1) Based on the idea that enhanced self-renewal of OSIC by serial transplantation will lead to an increasing generation of more differentiated progenies to accelerate tumor formation, we identify the progressively increased CD49f+ daughter cells that are responsible to constitute the bulk of osteosarcoma. By further seeking the lineage relationship between increasingly expanded CD49f+ progenies and their parental cells using lineage-tracking strategies both in vitro and in vivo, we determine that CD49f+cells are generated by CD49f-subpopulation. Those CD49f+ cells are weaker in tumorigenicity and drug resistance compared to their parental CD49f-subpopulation. Importantly, these CD49f-cells also generate CD 133+subpopulation. Among 30.47%CD49f_-subpopulation in UT2 cells, there are only 0.97%CD49f-CD133+cells. Such rare CD49f-CD133+subpopulation has higher capacity than CD49f-cells in generation of CD49f- cells. Whereas all those CD49f-CD133+, CD49f-, CD49f+,CD133+, and CD133-subpopulations can generate in vivo osteosarcoma formation, CD49f-CD133+cells exhibit the highest capacity of tumorigenicity (75%) than CD49f-(56%), CD133+(42%), CD49f+ (44%), CD133-(33%), or CD49f-CD133- (0%) subpopulation, respectively. (2) Our studies demonstrate that whereas all CD49f-, CD49f+, CD133+, or CD133- subpopulation show in vivo tumorigenicity, OSIC-enriched CD49f-CD133+cells possesses higher tumorigenicity than its direct progenies,CD49f+ cells, as well as other single subpopulations. Moreover, during the processes from progressively enhanced OSIC self-renewal by serial transplantation to increasingly production of their progenies forming the bulk of osteosarcoma, we observe that those CD49f+ daughter cells possess lower capacity of tumorigenicity but gain more osteogenic feature that can be induced by osteogenic reagent, compared to their parental cells. Correspondingly, while OSIC-enriched CD49f-CD133+cells retain similar capacity of adipocyte fate compared to CD49f-, CD49f+, CD133+, CD49f-, or CD49f-CD133- subset, their high tumorigenicity is associated with decreased osteogenic differentiation. These dynamic changes are also observed in the developmental processes from U2OS to UT2 cells induced by serial transplantation, in which high tumorigenicity of UT2 cells is associated with an inhibition in osteogenic differentiation but not in adipocyte fate, as shown by that there is a decreased expression of osteoblast genes than adipocyte genes in UT2 cells compared to U2OS cells. These data demonstrate that in CD49f-CD133+ subpopulation, the progressively increased activities of self-renewal and tumorigenicity from U2OS to UT2 cells by serial transplantation are associated with inhibited osteogenic fate. (3) To check whether CD117+and/or Stro-1+subpopulation share in vivo self-renewal and tumorigenicity with CD49f-CD133+subpopulation, we chose non-transformed UT2 cells first for xenotransplantion analysis. The same amount of 500 cells isolated by using CD117+, CD 117-, Stro-1+, Stro-1-, CD133+, CD 133-, CD49f-, or CD49f+ antibodies was engrafted into NOD/SCID mice. We found that CD117+, CD117-, Stro-1+, or Stro-1-cells show no or low tumorigenicity compared to CD133+, CD133-, CD49f-, or CD49f+cells. (4) Currently, the functions of CD49f- and/or CD133+in the properties of OSIC remain unknown beyond their roles in serving as selection markers. However, our initial data indicate that in UT2 cells generated by serial transplantation, the molecules that are crucial in mediating Wnt, Notch, and TGF-(3 signaling pathways have been significantly up-regulated compared to MSC and U2OS cells. This suggests that in UT2 cells, the enriched OSIC self-renewal and tumorigenicity represented by CD49f-CD133+subpopulation are related to activation of those pathways.Conclusion:In summary, we have identified that CD49f-CD133+subpopulation is enriched with OSIC. This OSIC-enriched CD49f-CD133+subset endures new phenotype of high tumorigenicity with inhibited osteogenic fate, compared to its progenies showing limited tumorigenicity with more differentiated osteogenic feature. Hence, these studies have not only laid down the foundation to further identify possible candidate partner(s) of CD49f-CD133+subpopulation and to determine the role of individual self-renewal pathways in OSIC vs. OSFC, but also provide the evidence for the first time that in the course of identifying cancer stem cells, a combined approaches of lineage tracking, tumorigenicity comparison, and differentiation evaluation are important to differentiate cancer stem cells from their progenies, cancer formation cells.Part 3 Study of gene expression profile regulating osteogenesis and osteosarcoma transformationObjective:With a rapid expansion of our knowledge about stem cell biology, emerging evidence suggests osteosarcoma should be regarded as a disease of cell differentiation caused by genetic and epigenetic changes that interrupt osteoblastic differentiation of human mesenchymal stem cells. We analyzed potential links between defective osteogenic differentiation and osteosarcoma transformation using microarry analysis and bioinformatics, trying to find the key step controlling osteosarcoma transformation and the potential diagnostic and prognostic markers for osteosarcoma.Methods and results:(1) Microarry was used to analysis the gene profiles of four osteosarcoma cells (TTC444, TTC606, UT2 and U2OS) vs. hMSCs.438 genes are sorted to show similar mRNA expression level change in these four cells with 133 genes up-regulated and 305 genes down-regulated; (2) A combination analysis of the gene profiles of ATRA-induced hMSCs differentiation, ATRA-induced U2OS differentiation, RARaS77A-induced U2OS differentiation, four osteosarcoma cells. The mRNA expression levels of 295 genes are inverse between osteogenesis and osteosarcoma transformation. These 295 genes are distributed in the protein family of cell junction, transmembrane region, transcriptional factor, kinase, ubiquitin like protein, collagen, metabolic enzyme, receptor, secreted extracellular signal, skeletal system development, Protease etc. (3) There are 13 transcriptional factors, including EPAS1, FOS, GTF2A1L, MXD1, CDKN2A, CDKN2B, CHURC1, GLIS3, NFKBIZ, HOXB6, FOXF1, BCL11A and EGR2. The mRNA level of EPAS1, FOS and GTF2A1L are up-regulated in osteosarcoma transformation and down-regulated in osteogenesis. The mRNA level of MXD1, CDKN2A, CDKN2B, CHURC1, GLIS3 and NFKBIZ are down-regulated in osteosarcoma transformation and stable in osteogenesis. Only HOXB6, FOXF1, BCL11A and EGR2 are up-regulated in osteosarcoma transformation and stable in osteogenesis. (4) ADAMTS2, ADRB2, B4GALT1, BTN3A1, CD14, CDKN2A, CDKN2B, CHI3L1, COL1A1, CYP1B1, DPP4, DPYSL3, ENG, EPAS1, FN1, FOS, HOXB6, IGFBP3, ITGA5, MCM7, OPTN, RARRES1, SNAP25 and SPARC are sorted as potential cancer biomarkers with inverse expression of mRNA between osteogenesis and osteosarcoma transformation. Only ADRB2, HOXB6 and MCM7 are recognized as best candidates for diagnostic and prognostic markers.Conclusion:It is concluded that 295 genes are inversely expressed in osteogenesis and osteosarcoma transformation. HOXB6, FOXF1, BCL11A and EGR2, as transcriptional factors, play important role in osteosarcoma transformation. Moreover, ADRB2, HOXB6 and MCM7 are powerful osteosarcoma diagnostic and prognostic markers. This study will provide insight into the pathogenesis of osteosarcoma, as well as targeted therapeutics for osteosarcoma patients. |
PDF Full Text Request