Alpha2beta1 Integrin Polymorphism in Diffuse Astrocytoma Patients.

Integrins are heterodimeric transmembrane glycoproteins resulting from the non-covalent association of an α and ß chain. The major integrin receptor for collagen/laminin, α2ß1 is expressed on a wide variety of cell types and plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Integrin-triggered signaling pathways promote the invasion and survival of glioma cells by modifying the brain microenvironment. In this study, we investigated the association of a specific genetic polymorphism of integrin α2ß1 with the incidence of diffusely infiltrating astrocytoma and the progression of these tumors. Single-nucleotide polymorphism in intron 7 of the integrin ITGA2 gene was examined in 158 patients and 162 controls using polymerase chain reaction and restriction enzyme analysis. The ITGA2 genotype +/+ (with a BglII restriction site in both alleles) exhibited higher frequency in grade II astrocytoma compared to control (P = 0.02) whereas the genotype -/- (lacking the BglII site) correlated with the poorest survival rate (P = 0.04). In addition, in silico analyses of ITGA2 expression from low-grade gliomas (LGG, n = 515) and glioblastomas (GBM, n = 159) indicated that the higher expression of ITGA2 in LGG was associated with poor overall survival (P < 0.0001). However, the distribution of integrin ITGA2 BglII genotypes (+/+, +/-, -/-) was not significantly different between astrocytoma subgroups III and IV (P = 0.65, 0.24 and 0.33; 0.29, 0.48, 0.25, respectively) compared to control. These results suggest a narrow association between the presence of this SNP and indicate that further studies with larger samples are warranted to analyze the relation between tumor grade and overall survival, highlighting the importance of determining these polymorphisms for prognosis of astrocytomas.

Indisulam Reduces Viability and Regulates Apoptotic Gene Expression in Pediatric High-Grade Glioma Cells.

Pediatric high-grade glioma (pHGG) is one of the most aggressive brain tumors. Treatment includes surgery, radiotherapy, chemotherapy, or combination therapy in children older than 3−5 years of age. These devastating tumors are influenced by the hypoxic microenvironment that coordinatively increases the expression of carbonic anhydrases (CA9 and CA12) that are involved in pH regulation, metabolism, cell invasion, and resistance to therapy. The synthetic sulphonamide Indisulam is a potent inhibitor of CAs. The aim of this study was to evaluate the effects of Indisulam on CA9 and CA12 enzymes in pHGG cell lines. Our results indicated that, under hypoxia, the gene and protein expression of CA9 and CA12 are increased in pHGG cells. The functional effects of Indisulam on cell proliferation, clonogenic capacity, and apoptosis were measured in vitro. CA9 and CA12 gene and protein expression were analyzed by RT-PCR and western blot. The treatment with Indisulam significantly reduced cell proliferation (dose-time-dependent) and clonogenic capacity (p < 0.05) and potentiated the effect of apoptosis (p < 0.01). Indisulam promoted an imbalance in the anti-apoptotic BCL2 and pro-apoptotic BAX protein expression. Our results demonstrate that Indisulam contributes to apoptosis via imbalance of apoptotic proteins (BAX/BCL2) and suggests a potential to overcome chemotherapy resistance caused by the regulation these proteins.

The Carbonic Anhydrase Inhibitor E7070 Sensitizes Glioblastoma Cells to Radio- and Chemotherapy and Reduces Tumor Growth.

Glioblastomas (GBMs), the most common and lethal primary brain tumor, show inherent infiltrative nature and high molecular heterogeneity that make complete surgical resection unfeasible and unresponsive to conventional adjuvant therapy. Due to their fast growth rate even under hypoxic and acidic conditions, GBM cells can conserve the intracellular pH at physiological range by overexpressing membrane-bound carbonic anhydrases (CAs). The synthetic sulfonamide E7070 is a potent inhibitor of CAs that harbors putative anticancer properties; however, this drug has still not been tested in GBMs. The present study aimed to evaluate the effects of E7070 on CA9 and CA12 enzymes in GBM cells as well as in the tumor cell growth, migration, invasion, and resistance to radiotherapy and chemotherapy. We found that E7070 treatment significantly reduced tumor cell growth and increased radio- and chemotherapy efficacy against GBM cells under hypoxia. Our data suggests that E7070 has therapeutic potential as a radio-chemo-sensitizing in drug-resistant GBMs, representing an attractive strategy to improve the adjuvant therapy. We showed that CA9 and CA12 represent potentially valuable therapeutic targets that should be further investigated as useful diagnostic and prognostic biomarkers for GBM tailored therapy.

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer.

Although many patients with colorectal cancer initially respond to the chemotherapeutic agent oxaliplatin, acquired resistance to this treatment remains a major challenge to the long-term management of this disease. To identify molecular targets of oxaliplatin resistance in colorectal cancer, we performed an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of ataxia-telangiectasia mutated and RAD3-related (ATR), a serine/threonine protein kinase involved in the response to DNA stress, restored oxaliplatin sensitivity in a cellular model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin significantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer. SIGNIFICANCE: These findings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.

The aurora kinase inhibitor AMG 900 increases apoptosis and induces chemosensitivity to anticancer drugs in the NCI-H295 adrenocortical carcinoma cell line.

Adrenocortical tumor (ACT) is a malignancy with a low incidence rate and the current therapy for advanced disease has a limited impact on overall patient survival. A previous study from our group suggested that elevated expression of aurora-A and aurora-B is associated with poor outcome in childhood ACT. Similar results were also reported for adult ACTs. The present in-vitro study shows that AMG 900 inhibits aurora kinases in adrenocortical carcinoma cells. AMG 900 inhibited cell proliferation in NCI-H295 cells as well as in the ACT primary cultures and caused apoptosis in the cell line NCI-H295. Furthermore, it potentialized the mitotane, doxorubicin, and etoposide effects on apoptosis induction and acted synergistically with mitotane and doxorubicin in the inhibition of proliferation. In addition, we found that AMG 900 activated Notch signaling and rendered the cells sensitive to the combination of AMG 900 and Notch signaling inhibition. Altogether, these data show that aurora kinases inhibition using AMG 900 may be an adjuvant therapy to treat patients with invasive or recurrent adrenocortical carcinomas.

Novel histone deacetylase inhibitors for the treatment of pediatric brain tumors.

Pediatric brain tumors (BT) represent a broad group of malignancies that affect children, displaying different degrees of aggressiveness and prognosis. Current studies demonstrate a crosslink between genetic and epigenetic changes within these tumors. Histone modifications are key elements in the pathogenesis of cancer in general and in brain tumors in particular. It is well documented that at least two classes of enzymes control acetylation of histones: histone acetyltransferases (HATs) and histone deacetylase (HDACs). Transformed HAT or HDAC action was identified in a number of human tumors. It has been hypothesized that HDACs regulate gene expression by deacetylating important genes for cell maintenance. Several HDACs inhibitors have been characterized in the last years and have been shown to promote growth blockage, differentiation and apoptosis in various types of tumors, including glioblastomas, medulloblastomas, neuroblastomas, melanomas, and leukemias. Some of these inhibitors are currently under clinical investigation for different cancer treatments. This review summarizes important mechanisms of histone modifications and discusses recent discoveries with impact on the pre-clinical and clinical field of pediatric brain tumor treatment.

Update on the Use of l-Asparaginase in Infants and Adolescent Patients with Acute Lymphoblastic Leukemia.

Great improvements have been made in acute lymphoblastic leukemia (ALL) treatment in the past decades, especially due to the use of l-asparaginase (l-ASP). Despite the significant success rate, several side effects mainly caused by toxicity, asparaginase silent inactivation, and cellular resistance, encourage an open debate regarding the optimal dosage and formulation of l-ASP. Alternative sources of asparaginases have been constantly investigated in order to overcome hypersensitivity clinical toxicity. Additionally, genomic modulation as gene expression profiling, genetic polymorphisms, and epigenetic changes is also being investigated concerning their role in cellular resistance to l-ASP. Understanding the mechanisms that mediate the resistance to l-ASP treatment may bring new insights into ALL pathobiology and contribute to the development of more effective treatment strategies. In summary, this review presents an overview on l-ASP data and focuses on cellular mechanisms underlying resistance and alternative therapies for the use of asparaginase in childhood ALL treatment.

Hypoxia-related gene expression profile in childhood acute lymphoblastic leukemia: prognostic implications.

A cellular hypoxic condition is a key event in several human cancers, but knowledge about its role in childhood acute lymphoblastic leukemia (ALL) is very limited. In the present study, the gene expression profile of hypoxia-related genes (HIF1A, CA9, VEGF and SCL2A1) was evaluated in bone marrow samples of 113 pediatric patients. HIF1A mRNA up-regulation was significantly associated with higher 5-year event-free survival rates in patients with B-ALL as well as in the overall ALL population in both univariate and multivariate analysis (p = 0.023 and p = 0.041, respectively). In gene expression analysis, low oxygen levels promoted HIF1A activation in a time-dependent manner, in both ALL cell lines. In vitro cytotoxic assays suggested an initial trend toward hypoxia-related resistance in the first 24 h, but evaluation at later time points (48-72 h) clearly showed that there was no relevant difference in drug response when comparing hypoxic and normal oxygen level conditions. Modulation of mRNA expression of several hypoxia-related genes was also observed after hypoxic exposure in a cell specific manner, suggesting that HIF1A mRNA expression could play a different role in specific subtypes of leukemia. Despite the remaining questions regarding hypoxia-mediated mechanisms, these findings could be helpful to provide new insights into the role of hypoxia in childhood ALL.

Zebularine induces chemosensitization to methotrexate and efficiently decreases AhR gene methylation in childhood acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood. Despite the advances in treatment, about 20% of patients relapse and/or die, indicating the need for different therapies for this group. Zebularine (ZB) is a potent DNA methyltransferase (DNMT) inhibitor and has been associated with gene demethylation and enhancement of tumor chemosensitivity. This study aimed to evaluate the effects of ZB, alone or combined with chemotherapeutics (methotrexate and vincristine), on childhood ALL cell lines. Cell proliferation, apoptosis, and clonogenic capacity were studied in Jurkat and ReH cell lines. Bisulfite modification, followed by methylation-specific PCR was carried out to evaluate aryl hydrocarbon receptor (AhR) methylation status. Gene expression of DNMT1, DNMT3a, DNMT3b, and AhR was assessed using qRT-PCR. Both cell cultures were sensitive to ZB, showing a dose-dependent and time-dependent response (P<0.05). ZB induced apoptosis and decreased clonogenic capacity in both cell lines. Combination with methotrexate resulted in a strong synergistic effect, whereas combination with vincristine led to an antagonistic response in both cell lines. ZB treatment decreased gene expression of the three DNMTs and induced AhR gene promoter demethylation and its re-expression. These results indicate that ZB may be a promising drug for the adjuvant treatment of ALL, mainly when combined with methotrexate.