The role of Myc in bone metabolism

Various studies have proven the involvement of Myc in cell growth and proliferation but also cell transformation and tumorigenesis. Since regulation of cell proliferation and differentiation is mandatory in bone metabolism, we investigated the role of Myc in osteoblasts. Studies involving osteoblast specific deletion of c-Myc traced by EYFP revealed no difference in fluorescence comparing mice that have either two copies, one copy or that are lacking both copies of c-Myc under the microscope. FACS analysis of these same mice showed no significant difference in numbers of EYFP positive osteoblasts among these mice. Immunofluorescence stainings showed absence of c-Myc protein in the majority of EYFP positive cells. Studies involving osteoblast specific c-Myc and N-Myc deletion traced by EYFP were performed to analyze possible consequences of loss of both genes. Bones of newborn myc-deficient mice appeared EYFP fluorescent under the microscope. FACS analysis of myc-deficient mice demonstrated variable numbers of EYFP positive osteoblasts. However, numbers of EYFP positive osteoblasts were in the same range as numbers obtained by FACS for c-Myc deficient mice. Osteoblast specific overexpression of c-Myc was analyzed using a Tet-Off model in which human c-Myc is overexpressed. qRT-PCRs showed that markers of mid to late differentiation of osteoblasts are less expressed in mice overexpressing c-Myc compared to control mice. Since mutant mice develop osteosarcomas, the expression levels of osteoblast differentiation markers were tested by qRT-PCR. Tumors revealed to be composed of overall less differentiated osteoblasts. Using these two models we could show that myc is dispensable for osteoblasts in vivo, whereas ectopic expression of c-myc leads to severe changes in the transcription profile of osteoblasts with a specific loss of mature osteoblast markers, and to the development of osteosarcomas, which show a rather immature osteoblast phenotype

The role of MYC in bone and in the hematopoietic stem cell niche

Maike Jaworski

The nuclear protein c-Myc is one of the most potent proto-oncogenes described. Its expression is found to be deregulated in more than 50 percent of human cancers, where it has been shown to control a number of biological processes including driving proliferation, cell growth and angiogenesis while inhibiting differentiation. The effects of c-Myc are highly cell type specific and include cell autonomous and non-cell autonomous processes. In order to examine the effect of excess amounts of c-Myc in bone, we have generated a mouse line expressing the tTA transactivator under the control of the mouse 2.3 kb col1a1 promoter (named Col1a1::tTA), which directs transgene expression to mature osteoblasts. By combining this transgene with the Tet-O-Myc allele, we generated a Col1a1::tTA; Tet-O-Myc double transgenic mouse model in which it is possible to inducibly express human c-MYC in osteoblasts during development and in the adult. We document that the used 'tet-off' system directs transgene expression to bones and that c-MYC can be completely repressed by treatment with doxycycline. Using this mouse model, we demonstrate that overexpression of c-MYC leads to remarkable changes in mutant bones, which depend greatly in their severity on the developmental time point of c-MYC induction. Unrestricted c-MYC expression, starting with the first emergence of mature osteoblasts in the fetal skeleton (∼E14.0), leads to lethality of the mutants at birth; their bones are shortened and thicker than in controls. Later induction of c-MYC allows survival, but leads to significant changes in bone morphology. Mutant bones develop a very irregular bone structure with signs of excessive bone formation. Surprisingly, this also correlates with definite signs of excessive osteoclast activity, which explains the formation of cavities seen in the bone matrix. Osteoblasts not only produce bone matrix, they are also very important niche cells for hematopoietic cells, especially for hematopoietic stem cells and B cells. We find that c-MYC overexpression severely impairs B lymphopoiesis and also observe signs of extramedullary hematopoiesis in the spleen, which is indicative of a compromised HSC environment in the bone marrow. Most strikingly, these animals develop frank osteosarcomas leading to a much reduced life span. Tumors arise in various bones, with the ribs and hips being the most prominent sites. These c-MYC driven tumors are very aggressive as is evident by their capacity to spontaneously metastasize to lung and liver, a rather rare phenomenon in mouse models, indicating that these tumors share important aspects of human osteosarcomas. Molecular analysis of c-MYC overexpressing osteoblasts has revealed a defect in their terminal differentiation program, rendering them incapable to progress from a relatively immature proliferative to a quiescent, calcified matrix producing state. To our knowledge this is the first report of experimental osteosarcoma formation, originating from a mature and lineage committed osteoblast population indicating that c-MYC may be able to de-differentiate more mature cells. In summary, our novel conditional mouse model showed that c-MYC expression in mature osteoblasts can lead to the formation of highly aggressive and metastatic osteosarcomas, a lethal disease in humans. This model will be the basis for further studies elucidating the molecular effects of c-MYC on de-differentiation and metastasis formation, processes, which remain poorly understood but are critical events in highly malignant cancers.

EPFL2009

Platinum sensitivity of ovarian cancer cells does not influence their ability to induce M2-type macrophage polarization

Agata Mlynska, Mei-Wen Peng

ProblemDevelopment of platinum resistance in ovarian cancer is mediated by both cancer cells and tumor microenvironment. Activation of epithelial-mesenchymal transition program in cancer cells may lead to enrichment for resistant clones. These processes can be affected by tumor-associated macrophages, a highly plastic population of cells that participate in tumor progression and response to treatment by shaping the microenvironment. We aimed to study how platinum resistance influences the crosstalk between macrophages and ovarian cancer cells. Method of studyUsing cisplatin-sensitive ovarian cancer cell line A2780, we developed and characterized cisplatin-resistant A2780Cis and cisplatin and doxorubicin co-resistant A2780Dox cell lines. Next, we set up an indirect coculture system with THP-1 cell line-derived M0-type-, M1-type- and M2-type-like polarized macrophages. We monitored the expression of genes associated with cellular stemness, multidrug resistance, and epithelial-mesenchymal transition in cancer cells, and expression profile of M1/M2 markers in macrophages. ResultsDevelopment of drug resistance in ovarian cancer cell lines was accompanied by increased migration, clonogenicity, and upregulated expression of transcription factors, associated with cellular stemness and epithelial-mesenchymal transition. Upon coculture, we noted that the most relevant changes in gene expression profile occurred in A2780 cells. Moreover, M0- and M1-type macrophages, but not M2-type macrophages, showed significant transcriptional alterations. ConclusionOur results provide the evidence for bidirectional interplay between cancer cells and macrophages. Independent of platinum resistance status, ovarian cancer cells polarize macrophages toward M2-like type, whereas macrophages induce epithelial-mesenchymal transition and stemness-related gene expression profile in cisplatin-sensitive, but not cisplatin-resistant cancer cells.

WILEY2018

Platinum sensitivity of ovarian cancer cells does not influence their ability to induce M2-type macrophage polarization

Agata Mlynska, Mei-Wen Peng

WILEY2018

The role of MYC in bone and in the hematopoietic stem cell niche

Maike Jaworski

EPFL2009