Molecular characterization of spontaneous mesenchymal stem cell transformation

Abstract

Background: We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC).

Methodology/principal findings: Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection.

Conclusions/significance: In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell.

Figure 1. Cell cycle regulation.

(A) x-fold change, p-value and Z-score of cell cycle regulators expression measured by microarray analysis between pre- and post-senescence MSC and post-senescence MSC and TMC. (B) Relative mRNA expression of Cyclin D1 (CCND1), and cyclin-dependent kinases 2 (CDK2) and 6 (CDK6) in pre- and post-senescence MSC, TMC and met-TMC analyzed by qRT-PCR. (C) Western blot analysis of cell cycle regulator protein expression in pre- and post-senescence MSC and two TMC samples. α-tubulin was used as loading control.

Figure 2. DNA repair regulation.

(A) x-fold change, p-value and Z-score of DNA repair-related gene expression measured by microarray analysis between pre- and post-senescence MSC and post-senescence MSC and TMC. (B) Relative mRNA expression of ERCC3, DNA ligase IV (LIG IV), DNA polymerase β (POLβ) and μ (POLμ), RAD51, XPA and XRCC4 in pre- and post-senescence MSC, TMC and met-TMC analyzed by qRT-PCR. (C) Western blot analysis of DNA repair-related protein expression in pre- and post-senescence MSC and two TMC samples. α-tubulin was used as loading control.

Figure 3. Regulation of oncogenes and tumor suppressor genes.

(A) x-fold change, p-value and Z-score of oncogenes and tumor suppressor genes measured by microarray analysis between pre- and post-senescence MSC and post-senescence MSC and TMC. (B) Relative mRNA expression of p16 analyzed by qRT-PCR in different samples of pre-senescence MSC (n = 4), post-senescence MSC (n = 3) and TMC (n = 3). (C) Homozygous deletion analysis of p14, p15 (D) and p16 (E) genes. β-actin was used as internal PCR control. Control cell lines were normal lymphocytes (NL), HCT116 and MDA-MB231. (F) Western blot analysis of p21 expression in pre- and post-senescence MSC, TMC and met-TMC. α-tubulin was used as loading control. (G) Analysis of p53 activation following UV irradiation of cells. p53 levels and phosphorylation were tested in pre- and post-senescence MSC, in two TMC samples and a sample of met-TMC. α-tubulin was used as loading control. (H) Rb protein levels and phosphorylation tested in pre- and post-senescence MSC, two samples of TMC and a met-TMC sample. α-tubulin was used as loading control.

Figure 4. Model of spontaneous human MSC transformation.

Sequence of steps: morphologic, karyotypic, and molecular alterations during TMC generation. Black and blue boxes represent chromosomes, orange boxes represent telomeres.