Igor Araújo Vieira

Combined Treatments with a Retinoid Receptor Agonist and Epigenetic Modulators in Human Neuroblastoma Cells

Neuroblastoma (NB) is the most common extracranial solid childhood tumor accounting for around 15% of pediatric cancer deaths and most probably originates from a failure in the development of embryonic neural crest cells. Retinoids can inhibit the proliferation and stimulate differentiation of NB cells. In addition, epigenetic events involving changes in chromatin structure and DNA methylation can mediate the effects of retinoids; hence, the scope of this study is to investigate the use of retinoids and epigenetic drugs in NB cell lines. Here, we demonstrate that the combination of retinoid all trans-retinoic acid (ATRA) with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase is more effective in impairing the proliferation of human SH-SY5Y and SK-N-BE(2) NB cells than any drug given alone. Treatments also induced differential changes on the messenger RNA (mRNA) expression of retinoid receptor subtypes and reduced the protein content of c-Myc, the neuronal markers NeuN and β-3 tubulin, and the oncoprotein Bmi1. These results suggest that the combination of retinoids with epigenetic modulators is more effective in reducing NB growth than treatment with single drugs.

Gastrin-Releasing Peptide Receptor Knockdown Induces Senescence in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, characterized by excessive cell proliferation, resistance to apoptosis, and invasiveness. Due to resistance to currently available treatment options, the prognosis for patients with GBM is very dismal. The activation of gastrin-releasing peptide receptors (GRPR) stimulates GBM cell proliferation, whereas GRPR antagonists induce antiproliferative effects in in vitro and in vivo experimental models of GBM. However, the role of GRPR in regulating other aspects of GBM cell function related to tumor progression remains poorly understood, and previous studies have not used RNA interference techniques as tools to examine GRPR function in GBM. Here, we found that stable GRPR knockdown by a lentiviral vector using a short hairpin interfering RNA sequence in human A172 GBM cells resulted in increased cell size and altered cell cycle dynamics consistent with cell senescence. These changes were accompanied by increases in the content of p53, p21, and p16, activation of epidermal growth factor receptors (EGFR), and a reduction in p38 content. These results increase our understanding of GRPR regulation of GBM cells and further support that GRPR may be a relevant therapeutic target in GBM.

p53 signaling pathway polymorphisms, cancer risk and tumor phenotype in TP53 R337H mutation carriers

Li-Fraumeni and Li-Fraumeni-like syndrome (LFS/LFL) are clinically heterogeneous cancer predisposition syndromes characterized by diagnosis of early-onset and often multiple cancers with variable tumor patterns and incomplete penetrance. To date, the genetic modifiers described in LFS/LFL have been shown to map to either TP53 or its main negative regulator, MDM2. Additionally, all studies were focused on families with different TP53 germline mutations. Hence, in this study we explored the effect of the most studied polymorphisms of p53 pathway genes on clinical manifestations of individuals carrying the founder TP53 mutation R337H (n = 136) and controls (n = 186). Cancer-affected carriers had been diagnosed either with adrenocortical carcinoma (ACC, n = 29) or breast cancer (BC, n = 43). Allelic discrimation using TaqMan assay was used for genotyping MDM2 SNP 309 (rs2279744) as well as MDM4 (rs1563828) and USP7 (rs1529916) polymorphisms. We found significantly higher MDM2 SNP 309 GG genotype and G allele frequencies in the LFS cohort than in controls. Furthermore, median age at first diagnosis was earlier in MDM2 SNP309 GG carriers when compared to other genotypes for both cancers (ACC: age 1 vs. 2 years; BC: age 35 vs. 43 years, respectively), although not statistically different. The allelic and genotypic frequencies for all SNPs did not differ between cancer affected and unaffected carriers, neither between patients with ACC or BC. In conclusion, our results suggest that MDM2 SNP 309 may contribute to the LFL phenotype and also to an earlier age at diagnosis of ACC and BC cancer in carriers of the R337H founder mutation.

Epidermal Growth Factor Receptor Regulation of Ewing Sarcoma Cell Function

Objective: Ewing sarcoma (ES) is a type of childhood cancer probably arising from stem mesenchymal or neural crest cells. The epidermal growth factor receptor (EGFR) acts as a driver oncogene in many types of solid tumors. However, its involvement in ES remains poorly understood. Methods: Human SK-ES-1 and RD-ES ES cells were treated with EGF, the EGFR inhibitor tyrphostin (AG1478), or phosphoinositide 3-kinase (PI3K) or extracellular-regulated kinase (ERK)/mitogen-activated kinase (MAPK) inhibitors. Cell proliferation survival, cycle, and senescence were analyzed. The protein content of possible targets of EGFR manipulation was measured by Western blot. Results: Cell proliferation and survival were increased by EGF and inhibited by AG1478. The EGFR inhibitor also altered the cell cycle, inducing arrest in G1 and increasing the sub-G1 population, reduced polyploidy and increased the population of senescent cells. In addition, AG1478 reduced the levels of phosphorylated AKT (p-AKT), ERK, p-ERK, cyclin D1, and brain-derived neurotrophic factor (BDNF), while enhancing p53 levels. Cell proliferation was also impaired by inhibitors of PI3K or ERK, alone or combined with AG1478. Conclusions: Our findings reveal novel aspects of EGFR regulation of ES cells and provide early evidence for antitumor activities of EGFR inhibitors in ES.

Abstract 804: Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome associated with TP53 germline mutations and characterized by increased risk to multiple early-onset cancers. Studies in families from Southern and Southeastern Brazil have identified a germline founder mutation in the TP53 gene, the p.R337H, in high population prevalence (∼0.3%). Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein, the TP53 p.R337H is located in the oligomerization domain (OD). Recently, we identified a new rare germline variant, the rs78378222 (A>C), in the 3′ UTR of the gene in 7/130 of the patients who tested negative for coding germline TP53 mutations. Based on the broad spectrum of p53 functions, which include metabolism and DNA damage response regulation, in the present study we aimed to characterize the functional impact of germline TP53 mutations identified in Brazilian families. Methods: Primary fibroblasts from p.R337H/WT (n = 2), p.R337H/p.R337H (n = 1), DBD mutations (n = 2), rs78378222[C] patients and from WT p53 controls (n = 2) were included. The DNA damage induction was performed using UVB (0.2W/m2) and ionizing irradiation (IR) (1 Gy). Results: First, we observed that all p53-mutated fibroblasts appeared to have lower p53 levels but higher levels of reactive oxygen species (ROS) and mitochondrial biomass compared to controls. Fibroblasts homozygous to p.R337H mutation presented more pronounced ROS production and, notably, higher expression of two antioxidant proteins, SOD2 and GPX1. On the other hand, the results of high-resolution respirometry showed higher oxygen consume in DBD-mutated fibroblasts. Second, using a specific multiplexed enzymatic DNA repair assay on biochip, we simultaneously investigated several DNA repair pathways. Overall, we observed a high DNA repair activation profile in p.R337H/p.R337H and poor DNA repair capacity in DBD-mutated fibroblasts. Interestingly, all p53-mutated fibroblasts showed higher repair activity of apurinic/pyrimidinic (AP) sites, a DNA lesion caused mainly by ROS attacks. Conclusions: Our data indicate important alterations in both metabolism and DNA damage response in normal cells (fibroblasts) from LFS patients, suggesting haploinsufficiency mechanism associated to these mutations. We were also able to demonstrate distinct phenotypes according to mutational status, which may explain, at least in part, the clinical variability in LFS. Citation Format: Gabriel Macedo, Sylvie Sauvaigo, Michele Silva Alves, Sylvain Caillat, Igor Araujo Vieira, Fernanda Timm, Cristina Brinckmann Oliveira Netto, Mauro Antonio Alves Castro, Ivi Bristot, Angela Fachel, Fabio Klamt, Patricia Ashton-Prolla. Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2015-804