Marta M. Alonso

MicroRNA‐451 Is Involved in the Self‐renewal, Tumorigenicity, and Chemoresistance of Colorectal Cancer Stem Cells

Many antitumor therapies affect rapidly dividing cells. However, tumor proliferation may be driven by cancer stem cells (CSCs), which divide slowly and are relatively resistant to cytotoxic drugs. Thus, many tumors may progress because CSCs are not sensitive to the treatment. In this work, we searched for target genes whose expression is involved in proliferation and chemoresistance of CSCs. Both of these processes could be controlled simultaneously by cell regulators such as microRNAs (miRNAs). Therefore, colonospheres with properties of CSCs were obtained from different colon carcinoma cells, and miRNA profiling was performed. The results showed that miR‐451 was downregulated in colonspheres versus parental cells. Surprisingly, expression of miR‐451 caused a decrease in self‐renewal, tumorigenicity, and chemoresistance to irinotecan of colonspheres. We identified cyclooxygenase‐2 (COX‐2) as an indirect miR‐451 target gene involved in sphere growth. Our results indicate that miR‐451 downregulation allows the expression of the direct target gene macrophage migration inhibitory factor, involved in the expression of COX‐2. In turn, COX‐2 allows Wnt activation, which is essential for CSC growth. Furthermore, miR‐451 restoration decreases expression of the ATP‐binding cassette drug transporter ABCB1 and results in irinotecan sensitization. These findings correlate well with the lower expression of miR‐451 observed in patients who did not respond to irinotecan‐based first‐line therapy compared with patients who did. Our data suggest that miR‐451 is a novel candidate to circumvent recurrence and drug resistance in colorectal cancer and could be used as a marker to predict response to irinotecan in patients with colon carcinoma. STEM CELLS 2011;1661–1671

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi–histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Genetic and Epigenetic Modifications of Sox2 Contribute to the Invasive Phenotype of Malignant Gliomas

We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; N = 414), Sox2 gene amplification (8.5%; N = 492), and Sox 2 promoter hypomethylation (100%; N = 258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.

A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool

BACKGROUND Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. METHODS To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. RESULTS We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. CONCLUSIONS Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Delta-24-RGD in Combination With RAD001 Induces Enhanced Anti-glioma Effect via Autophagic Cell Death

Novel therapies are clearly needed for the treatment of gliomas, and strategies that involve combining oncolytic vectors with chemotherapy hold out significant hope for a more effective treatment of this malignancy. Whether chemotherapy acts directly on tumor cells by inducing cell arrest or cell death, or indirectly by blocking tumor angiogenesis, the resulting delay in tumor growth may provide the oncolytic virus with a wider window of opportunity to overcome the challenge imposed by the growth kinetics of the tumor. In this study we sought to determine whether the oncolytic adenovirus Delta-24-RGD, in combination with everolimus (RAD001), would result in an enhanced anti-glioma effect in vivo. Viability assays showed that Delta-24-RGD antitumoral activity is synergistically enhanced by combination with RAD001. Interestingly, combination treatment of Delta-24-RGD with RAD001 induced autophagy in vitro. We showed that Delta-24-RGD improved survival of tumor-bearing animals in a dose-dependent manner. A significant finding was that RAD001 enhanced the anti-glioma effect of Delta-24-RGD and resulted in the long-term survival of 80% of the experimental animals. Immunostaining of the treated tumors showed upregulation of Atg5, thereby indicating the therapeutic induction of autophagy. This is the first report showing that Delta-24-RGD plus RAD001 causes autophagic cell death, and dramatically increases long-term survival rates of glioma-bearing animals.

The RB-E2F1 Pathway Regulates Autophagy

Autophagy is a protective mechanism that renders cells viable in stressful conditions. Emerging evidence suggests that this cellular process is also a tumor suppressor pathway. Previous studies showed that cyclin-dependent kinase inhibitors (CDKI) induce autophagy. Whether retinoblastoma protein (RB), a key tumor suppressor and downstream target of CDKIs, induces autophagy is not clear. Here, we show that RB triggers autophagy and that the RB activators p16INK4a and p27/kip1 induce autophagy in an RB-dependent manner. RB binding to E2 transcription factor (E2F) is required for autophagy induction and E2F1 antagonizes RB-induced autophagy, leading to apoptosis. Downregulation of E2F1 in cells results in high levels of autophagy. Our findings indicate that RB induces autophagy by repressing E2F1 activity. We speculate that this newly discovered aspect of RB function is relevant to cancer development and therapy.

Adenovirus-Based Strategies Overcome Temozolomide Resistance by Silencing the O6-Methylguanine-DNA Methyltransferase Promoter

Currently, the most efficacious treatment for malignant gliomas is temozolomide; however, gliomas expressing the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) are resistant to this drug. Strong clinical evidence shows that gliomas with methylation and subsequent silencing of the MGMT promoter are sensitive to temozolomide. Based on the fact that adenoviral proteins directly target and inactivate key DNA repair genes, we hypothesized that the oncolytic adenovirus Delta-24-RGD could be successfully combined with temozolomide to overcome the reported MGMT-mediated resistance. Our studies showed that the combination of Delta-24-RGD and temozolomide induces a profound therapeutic synergy in glioma cells. We observed that Delta-24-RGD treatment overrides the temozolomide-mediated G(2)-M arrest. Furthermore, Delta-24-RGD infection was followed by down-modulation of the RNA levels of MGMT. Chromatin immunoprecipitation assays showed that Delta-24-RGD prevented the recruitment of p300 to the MGMT promoter. Importantly, using mutant adenoviruses and wild-type and dominant-negative forms of the p300 protein, we showed that Delta-24-RGD interaction with p300 was required to induce silencing of the MGMT gene. Of further clinical relevance, the combination of Delta-24-RGD and temozolomide significantly improved the survival of glioma-bearing mice. Collectively, our data provide a strong mechanistic rationale for the combination of oncolytic adenoviruses and temozolomide, and should propel the clinical testing of this therapy approach in patients with malignant gliomas.

ICOVIR-5 shows E2F1 addiction and potent antiglioma effect in vivo

During 2007, approximately 200,000 people in the United States will be diagnosed with brain tumors. Gliomas account for 77% of primary malignant brain tumors, and the prognosis has hardly changed in the past 20 years, with only 30% of patients with malignant glioma surviving 5 years after diagnosis. Oncolytic adenoviruses are promising therapies for the treatment of gliomas. Here, report the antiglioma activity of the tumor-selective ICOVIR-5 adenovirus, which encompasses an early 1A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, substitution of the E1A promoter for E2F-responsive elements, and an RGD-4C peptide motif inserted into the adenoviral fiber to enhance adenoviral tropism. Mechanistic studies showed a dramatic addiction of ICOVIR-5 to the E2F1 oncogene in vitro and in vivo. This addiction was mediated by the occupancy of the ectopic adenoviral E2F1-responsive elements by the endogenous E2F1 protein resulting in high level of E1A expression in cancer cells and potent antiglioma effect. Importantly, we showed for the first time the ability of oncolytic adenoviruses to enhance E2F transcriptional activity in vivo, and we provided direct evidence of the interaction of the E2F1 protein with native and ectopic adenovirus promoters. Restoration of Rb function led to the association of Rb/E2F1 repressor complexes with ICOVIR-5 ectopic E2F1 promoter and subsequent down-modulation of E1A, dramatically impairing adenoviral replication. In xenografted mice, intratumoral injection of ICOVIR-5 resulted in a significant improvement of the median survival (P < 0.0001), and furthermore, led to 37% of long-term survivors free of disease. The antitumor activity of ICOVIR-5 suggests that it has the potential to be an effective agent in the treatment of gliomas.

Expression of the receptor tyrosine kinase Tie2 in neoplastic glial cells is associated with integrin β1-dependent adhesion to the extracellular matrix

The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non–stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31− and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin β1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell–to–extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas. (Mol Cancer Res 2006;4(12):915–26)

A novel E1A–E1B mutant adenovirus induces glioma regression in vivo

Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 × 108 PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.