Temozolomide promotes genomic and phenotypic changes in glioblastoma cells

Abstract

BackgroundTemozolomide (TMZ) is a first-line drug for the treatment of glioblastoma. Long-term TMZ-treated tumour cells acquire TMZ resistance by profound reprogramming of the transcriptome, proteome, kinome, metabolism, and demonstrate versatile and opposite changes in proliferation, invasion, in vivo growth, and drug cross-resistance. We hypothesized that chromosomal instability (CIN) may be implicated in the generation of TMZ-driven molecular and phenotype diversity. CIN refers to the rate (cell-to-cell variability) with which whole chromosomes or portions of chromosomes are gained or lost.MethodsThe long-term TMZ-treated cell lines were established in vitro (U251TMZ1, U251TMZ2, T98GTMZ and C6TMZ) and in vivo (C6R2TMZ). A glioma model was achieved by the intracerebral stereotactic implantation of C6 cells into the striatum region of rats. Genomic and phenotypic changes were analyzed by conventional cytogenetics, array CGH, trypan blue exclusion assay, soft agar colony formation assay, scratch wound healing assay, transwell invasion assay, quantitative polymerase chain reaction, and Western blotting.ResultsLong-term TMZ treatment increased CIN-mediated genomic diversity in U251TMZ1, U251TMZ2 and T98GTMZ cells but reduced it in C6TMZ and C6R2TMZ cells. U251TMZ1 and U251TMZ2 cell lines, established in parallel with a similar treatment procedure with the only difference in the duration of treatment, underwent individual phenotypic changes. U251TMZ1 had a reduced proliferation and invasion but increased migration, whereas U251TMZ2 had an enhanced proliferation and invasion but no changes in migration. U251TMZ1 and U251TMZ2 cells demonstrated individual patterns in expression/activation of signal transduction proteins (e.g., MDM2, p53, ERK, AKT, and ASK). C6TMZ and C6R2TMZ cells had lower proliferation, colony formation efficiency and migration, whereas T98GTMZ cells had increased colony formation efficiency without any changes in proliferation, migration, and invasion. TMZ-treated lines demonstrated a differential response to a reduction in glucose concentration and an increased resistance to TMZ re-challenge but not temsirolimus (mTOR inhibitor) or U0126 (MEK1/2 inhibitor) treatment.ConclusionLong-term TMZ treatment selected resistant genotype-phenotype variants or generated novel versatile phenotypes by increasing CIN. An increase of resistance to TMZ re-challenge seems to be the only predictable trait intrinsic to all long-term TMZ-treated tumour cells. Changes in genomic diversity may be responsible for heterogeneous phenotypes of TMZ-treated cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0311-8) contains supplementary material, which is available to authorized users.

3D Mathematical Modeling of Glioblastoma Suggests That Transdifferentiated Vascular Endothelial Cells Mediate Resistance to Current Standard-of-Care Therapy

“…Experiments have suggested that intense hypoxia following anti-angiogenic treatments can select for invasive cell phenotypes as an adaptive response to hypoxia due to lack of vessels (19). In addition, new data suggest that long-term temozolomide treatment induces chromosomal instability, which in turn leads to potential increases in invasion and migration (38). Including such additional phenotypic changes in our model, as well as therapy-induced reprogramming of differentiated GBM cells to GSCs (35), would only add to the invasiveness we observed.…”

EMT‐ and MET‐related processes in nonepithelial tumors: importance for disease progression, prognosis, and therapeutic opportunities

“…Radiation was also found to activate NF-jB/STAT3 cooperative signaling that was linked to increased expression of intercellular adhesion molecule-1 (ICAM-1) and cell invasion (Kesanakurti et al, 2013). The standard chemotherapeutic TMZ was reported to select for GBM cells with different properties than the parental cells including an increase in migration and invasion and upregulation of EMT markers like SNAI1 and SNAI2 (Stepanenko et al, 2016). As earlier mentioned, treatment with the VEGF inhibitor causes MT in a hypoxia-dependent manner eventually establishing therapy-resistant cells (Piao et al, 2013).…”

Molecular Genetics of Secondary Glioblastoma

“…Stepaneko et al extended these findings with in vitro studies that demonstrated that long-term exposure of glioma cells to TMZ induces chromosomal instability, leading to alteration of cell growth, invasiveness, migration, and response to retreatment (69). Among the TMZ-resistant cell lines, some responded to temsirolimus, an mTOR inhibitor.…”