Author contributions: F.J.R.-J.: conception and design, data analysis and interpretation, manuscript writing, laboratory managing; V.M.-M.: conception and design, collection and assembly of data, data analysis; R.L.-D.: provision of study material, collection and assembly of data; J.-M.S.-P.: administrative support, financial support, conception and design, interpretation of data, manuscript writing, laboratory managing, final approval of manuscript.
An increasing body of research has shown that certain cancers contain multipotent neural stem cell-like cancer cells with tumor-initiating ability, which may originate this malignancy [1, –3]. Stem cells may undergo oncogenic transformation due to mutations, probably influenced by their environment, which leads them to become cancer stem cells, with uncontrolled and unlimited proliferation . Neural stem cells exist within a physiological hypoxia (1%–5% O2) in both embryonic and adult brains . Hypoxia-inducible factor-1 (HIF1) is a crucial regulator of tumor cell adaptation to hypoxic stress and is the key element responsible for the regulation, under low oxygen tension, of many genes involved in important biological processes, such as glycolysis, proliferation, and angiogenesis . HIF1 is a heterodimer composed of an oxygen-regulated α subunit and a constitutively expressed β subunit. Overexpression of HIF1α because of intratumoral hypoxia determines major physiological pathways in tumor growth and neovascularization . High HIF1α levels contribute to tumor progression and are a marker of aggressive disease in several tumor types [8, 9]. Moreover, hypoxia is responsible for diminished DNA repair and, therefore, high mutagenesis [10, 11], as well as for enrichment of mismatch repair (MMR)-deficient cells with augmented microsatellite instability (MSI) . MutS (MSH2, MSH3, MSH6) and two MutL homologues (MLH1, PMS2) make up the MMR family responsible for the DNA eukaryote repair process. The MutSα complex (MSH2–MSH6) is responsible for the repair of base-base mispairs, whereas MutSβ (MSH2–MSH3) repairs larger insertions/deletions. Both complexes recruit the MutL heterodimer to initiate downstream repair events . Mutation and loss of the MMR machinery is associated with increased genomic instability, shown in increased mutation rates, especially at microsatellite loci . MSI is considered a hallmark of nonpolyposis colorectal cancer  and is also present in a significant proportion of other cancer types [16, 17]. However, the role of MMR deficiency in the pathogenesis of brain tumors remains controversial . During the carcinogenic process, epigenetic alterations involving altered methylation and chromatin remodeling by histone modification lead to the functional loss of critical genes, such as tumor suppressor or DNA repair genes. Therefore, MLH1, but not MSH2, MSH3, or MSH6, can be silenced by promoter hypermethylation, a mechanism underlying the presence of the MSI in gastric  and endometrial carcinomas . Aberrant CpG island methylation in the promoter region is associated with transcriptionally repressive chromatin and seems to be linked to the deacetylation of histones. Deacetylation may be important in the initial silencing of transcription, usually accompanied by aberrant methylation . Both epigenetic events play a crucial role in stem cell identity and tumorigenesis . It has become increasingly clear that epigenetic mechanisms regulate the access of certain transcription factors to their binding sites. In fact, deacetylation and trimethylation of histone H3 on lysine 9 (H3K9Me3) impair SP1 occupancy . In addition, CpG island methylation prevents SP1 binding  and vice versa [25, 26]. There is a wide body of research concerning the epigenetic modifications that occur in tumor cells. However, little is known about the effects that altered epigenetic events may produce on the expression of DNA repair genes and the genomic integrity of stem cells under hypoxic conditions. In the research reported here, we used C17.2 cells, which are considered as operationally defined neural stem cells (NSC) [27, 28] and a promising vector for the treatment of regenerative diseases . C17.2 cells differentiate toward replacement of neurons , as a therapeutic tool in Parkinson's disease [29, 31] and in spinal cord regeneration . They migrate specifically toward an advancing neoplasm and trail islands of tumor cells migrating away from the tumor mass . Similar to C17.2, a human neural stem cell line (IhNSC) immortalized by v-myc, endowed with the properties of human NSC, has recently been reported as a suitable cell line for developing assays that are essential for diagnoses and cell therapy studies . Here we demonstrate, for the first time in the stem field, the repression of the MMR system in murine and human stem cells under hypoxia. We show that HIF1α is not directly involved in the repression of MMR in C17.2, as described for cancer cells. Our results show that MLH1 and MSH6 transcriptional downregulation in hypoxia are associated with hypoacetylated and hypermethylated histone H3 that impair SP1 binding in their promoter regions. The deregulated MMR system, caused by hypoxia, may contribute to generating genomic instability in stem cells, which leads to malignant transformation into cancer stem cells.