Generation of Osteosarcomas from a Combination of Rb Silencing and c-Myc Overexpression in Human Mesenchymal Stem Cells

Abstract

Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c‐Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c‐Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum‐free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS‐like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient‐derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c‐Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS‐like cells by Rb knockdown and c‐Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512–526

Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells

“…However, when overexpression of c-Myc in this animal model was combined with gain-of-function of another oncogene, Her2 (other name Erbb2: erb-b2 receptor tyrosine kinase 2), tumor penetrance reached 100% [49]. Similar effects have been demonstrated for the cases when c-MYC overexpression was combined with a loss or silencing of tumor suppressor genes, e.g., CDKN2A locus [50], or with RB (Retinoblastoma) protein inactivation [51], when these led to transformation of MSC and acquisition of a malignant osteosarcoma phenotype.…”

“…Different studies on a role of c-MYC in malignant transformation suggest that high expression of MYC alone is not sufficient to induce malignant transformation, and only when MYC gene amplification is combined with other oncogenes overexpression, e.g., Her2 [49], or with a loss of tumor suppressors, either due to deletion, e.g., CDKN2A [50], or loss-of-function, e.g., RB protein [51], this leads to dramatic boost in neoplastic cell growth. Our finding that overexpression of c-MYC correlated with P16INK4A and P14ARF expression suggests that, in case of an absence of the concomitant mutations of other oncogenes and/or tumor suppressors, cells are able to counteract activating functions of c-MYC and induce safety mechanisms.…”

Methylation and PTEN activation in dental pulp mesenchymal stem cells promotes osteogenesis and reduces oncogenesis

“…These studies suggest that BM-MSCs and DP-MSCs exhibit differences in tumorigenic potential. A combination of retinoblastoma (Rb) silencing and cMyc overexpression has successfully transformed human MSCs into malignant cells 27 . We demonstrated that BM-MSCs from three individuals were successfully transformed with this protocol (Fig.…”

Osteosarcoma: Molecular Pathogenesis and iPSC Modeling

“…Moreover, transformed human MSCs engineered to deplete RB1 and overexpress c-MYC -- a combination observed in patients with poor survival-- acquire malignant osteosarcoma-like properties. These MSCs express osteosarcoma markers CD99, ALP, osteonectin, and osteocalcin (also known as bone gamma-carboxyglutamic acid-containing protein (BGLAP)) and form lung and liver metastases in immunocompromised mice, suggesting that MSCs constitute the cellular origin of osteosarcomas [14]. …”