Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Abstract

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second-line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB-OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB-OS cells have hypertriploid karyotype with 71-82 chromosomes. By comparing 3AB-OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan 1 Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let-7/98 and miR-29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets.

Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells

“…Moreover, the presence of OS stem-like cells has been reported in patient tumors [62] as well as in established human OS cell lines [63]. We have previously shown [11,13,14] that 3AB-OS cells highly express a large panel of stemness-related genes/proteins and that efficiently transdifferentiate in vitro into cells of all the three primary germ layers [12], whereas when 3AB-OS cells were engrafted in nude mice, they potently induced malignant tumors, although preserving multilineage commitment [13]. Here, we show that, after in vitro 3AB-OS differentiation, in each derived cell lineage, p53-R248W/P72R was profoundly down-regulated and after p53-R248W/P72R-knockdown the expression of pluripotent markers (Oct3/4, Nanog, Sox2, nucleostemin and CD133) markedly lowered, suggesting that p53-R248W/P72R might be responsible for 3AB-OS cells pluripotency and self-renewal.…”

“…Moreover, analyses of death receptors in OS samples and in OS cell lines-among which MG63 cells-demonstrated alterations only within the DR4 gene, suggesting that these genetic alterations may be implicated in OS formation [58]. We have previously shown that 3AB-OS cells highly express a great number of genes required for inhibiting apoptosis [11] and much lower levels of FAS and DR4 receptors than parental MG63 cells [14]. Here we show that in 3AB-OS cells p53-R248W/P72R-knockdown potently increases the expression of DR4 and DR5 receptors and sensitivity to TRAIL-induced apoptosis.…”

“…We have previously demonstrated that 3AB-OS cells express low levels of the death receptors FAS and DR4 [14] and show strong resistance to TRAIL (TNF-related apoptosis inducing ligand) (effects evaluated using TRAIL concentrations up to 100 ng/ml, unpublished data). Here, we evaluated whether the p53-R248W/P72R-knockdown modifies the expression levels of DR4 (TRAIL-R1), KILLER/DR5 (TRAIL-R2) and FAS/CD95 at 48 h post-transfection.…”

“…3AB-OS cells have been characterized at genetic and molecular level: compared to parental MG63 cells, which have a hypotriploid karyotype with chromosome number ranging from 61 to 66, they are hypertriploid with chromosome number ranging from 71 to 82; they also exhibit 49 copy number variations (gains/losses) spanning almost all the chromosomes, 3,512 dysregulated genes and 189 differentially expressed miRNAs. Moreover, bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness [14]. Remarkably, the abnormalities evidenced in 3AB-OS cells appear to be strongly congruent with abnormalities described in a large number of pediatric and adult OS patients, where karyotype ranging from haploid to near hexaploid with chromosome number ranging from 15 to 120 were described; in addition, a great number of chromosomal regions with structural abnormalities among which 17p11.2-13 that contains TP53 gene were found [15][16][17].…”

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

“…On the basis of these characteristics, such as acquired resistance to cytotoxic chemotherapy agents for the overexpression of ATP-binding cassette multidrug efflux transporters 28,32,33 , or for the upregulation of the expression of detoxification enzymes such as ALDH 32 , for the expression of a particular surface marker, such as CD133, CD44, CD34, CD90, and others 30,34,35,41 , several different methods to isolate CSCs have been developed [42][43][44] . One of these techniques is the sphere formation assay, which is based on the capacity of CSCs to grow under non-adherent conditions.…”