Lorea Manterola

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.

Oncogenicity of the Developmental Transcription Factor Sox9

SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer.

New Concepts in Cancer Biomarkers: Circulating miRNAs in Liquid Biopsies

The effective and efficient management of cancer patients relies upon early diagnosis and/or the monitoring of treatment, something that is often difficult to achieve using standard tissue biopsy techniques. Biological fluids such as blood hold great possibilities as a source of non-invasive cancer biomarkers that can act as surrogate markers to biopsy-based sampling. The non-invasive nature of these “liquid biopsies” ultimately means that cancer detection may be earlier and that the ability to monitor disease progression and/or treatment response represents a paradigm shift in the treatment of cancer patients. Below, we review one of the most promising classes of circulating cancer biomarkers: microRNAs (miRNAs). In particular, we will consider their history, the controversy surrounding their origin and biology, and, most importantly, the hurdles that remain to be overcome if they are really to become part of future clinical practice.

Targeted next-generation sequencing and non-coding RNA expression analysis of clear cell papillary renal cell carcinoma suggests distinct pathological mechanisms from other renal tumour subtypes

Clear cell tubulopapillary renal cell carcinoma (CCPRCC) is a recently described rare renal malignancy that displays characteristic gross, microscopic and immunohistochemical differences from other renal tumour types. However, CCPRCC remains a very poorly understood entity. We therefore sought to elucidate some of the molecular mechanisms involved in this neoplasm by carrying out targeted next‐generation sequencing (NGS) to identify associated mutations, and in addition examined the expression of non‐coding (nc) RNAs. We identified multiple somatic mutations in CCPRCC cases, including a recurrent [3/14 cases (21%)] non‐synonymous T992I mutation in the MET proto‐oncogene, a gene associated with epithelial‐to‐mesenchymal transition (EMT). Using a microarray approach, we found that the expression of mature (n = 1105) and pre‐miRNAs (n = 1105), as well as snoRNA and scaRNAs (n = 2214), in CCPRCC cases differed from that of clear cell renal cell carcinoma (CCRCC) or papillary renal cell carcinoma (PRCC) tumours. Surprisingly, and unlike other renal tumour subtypes, we found that all five members of the miR‐200 family were over‐expressed in CCPRCC cases. As these miRNAs are intimately involved with EMT, we stained CCPRCC cases for E‐cadherin, vimentin and β‐catenin and found that the tumour cells of all cases were positive for all three markers, a combination rarely reported in other renal tumours that could have diagnostic implications. Taken together with the mutational analysis, these data suggest that EMT in CCPRCC tumour cells is incomplete or blocked, consistent with the indolent clinical course typical of this malignancy. In summary, as well as describing a novel pathological mechanism in renal carcinomas, this study adds to the mounting evidence that CCPRCC should be formally considered a distinct entity. Microarray data have been deposited in the GEO database [GEO accession number (GSE51554)]. Copyright

The circulating transcriptome as a source of non-invasive cancer biomarkers: concepts and controversies of non-coding and coding RNA in body fluids.

The gold standard for cancer diagnosis remains the histological examination of affected tissue, obtained either by surgical excision, or radiologically guided biopsy. Such procedures however are expensive, not without risk to the patient, and require consistent evaluation by expert pathologists. Consequently, the search for non‐invasive tools for the diagnosis and management of cancer has led to great interest in the field of circulating nucleic acids in plasma and serum. An additional benefit of blood‐based testing is the ability to carry out screening and repeat sampling on patients undergoing therapy, or monitoring disease progression allowing for the development of a personalized approach to cancer patient management. Despite having been discovered over 60 years ago, the clear clinical potential of circulating nucleic acids, with the notable exception of prenatal diagnostic testing, has yet to translate into the clinic. The recent discovery of non‐coding (nc) RNA (in particular micro(mi)RNAs) in the blood has provided fresh impetuous for the field. In this review, we discuss the potential of the circulating transcriptome (coding and ncRNA), as novel cancer biomarkers, the controversy surrounding their origin and biology, and most importantly the hurdles that remain to be overcome if they are really to become part of future clinical practice.

mTOR inhibition decreases SOX2-SOX9 mediated glioma stem cell activity and temozolomide resistance

ABSTRACT Background: SOX2 and SOX9 are commonly overexpressed in glioblastoma, and regulate the activity of glioma stem cells (GSCs). Their specific and overlapping roles in GSCs and glioma treatment remain unclear. Methods: SOX2 and SOX9 levels were examined in human biopsies. Gain and loss of function determined the impact of altering SOX2 and SOX9 on cell proliferation, senescence, stem cell activity, tumorigenesis and chemoresistance. Results: SOX2 and SOX9 expression correlates positively in glioma cells and glioblastoma biopsies. High levels of SOX2 bypass cellular senescence and promote resistance to temozolomide. Mechanistic investigations revealed that SOX2 acts upstream of SOX9. mTOR genetic and pharmacologic (rapamycin) inhibition decreased SOX2 and SOX9 expression, and reversed chemoresistance. Conclusions: Our findings reveal SOX2-SOX9 as an oncogenic axis that regulates stem cell properties and chemoresistance. We identify that rapamycin abrogate SOX protein expression and provide evidence that a combination of rapamycin and temozolomide inhibits tumor growth in cells with high SOX2/SOX9.

MicroRNAs in Lymphoma: Regulatory Role and Biomarker Potential

Although it is now evident that microRNAs (miRNAs) play a critical regulatory role in many, if not all, pathological and physiological processes, remarkably they have only formally been recognized for less than fifteen years. These endogenously produced short non-coding RNAs have created a new paradigm of gene control and have utility as both novel biomarkers of cancer and as potential therapeutics. In this review we consider the role of miRNAs in lymphoid biology both under physiological (i.e. lymphopoiesis) and malignant (i.e. lymphomagenesis) conditions. In addition to the functional significance of aberrant miRNA expression in lymphomas we discuss their use as novel biomarkers, both as a in situ tumour biomarker and as a non-invasive surrogate for the tumour by testing miRNAs in the blood of patients. Finally we consider the use of these molecules as potential therapeutic agents for lymphoma (and other cancer) patients and discuss some of the hurdles yet to be overcome in order to translate this potential into clinical practice

Dysregulation of Mir-196b in Head and Neck Cancers Leads to Pleiotropic Effects in the Tumor Cells and Surrounding Stromal Fibroblasts

The miR-196 family members have been found dysregulated in different cancers. Therefore, they have been proposed as promising biomarkers and therapeutic targets. This study is the first to investigate the role of miR-196b in the development and progression of head and neck squamous cell carcinomas (HNSCC), and also the impact on the surrounding tumor microenvironment. Increased miR-196b levels were detected in 95% of primary tumors and precancerous lesions, although no significant differences were observed between non-progressing versus progressing dysplasias. Furthermore, increased levels of both miR-196a and miR-196b were successfully detected in saliva samples from HNSCC patients. The functional consequences of altered miR-196 expression were investigated in both HNSCC cell lines and cancer-associated fibroblasts (CAFs) by transfection with specific pre-miR precursors. Results showed that both miR-196a and miR-196b elicit cell-specific responses in target genes and downstream regulatory pathways, and have a distinctive impact on cell proliferation, migration and invasion. These data reveal the early occurrence and prevalence of miR-196b dysregulation in HNSCC tumorigenesis, suggesting its utility for early diagnosis and/or disease surveillance and also as a non-invasive biomarker in saliva. The pleiotropic effects of miR-196a/b in HNSCC cell subpopulations and surrounding CAFs may complicate a possible therapeutic application.

Aberrant expression of MicroRNAs in B-cell lymphomas.

B-cell lymphomas represent a heterogeneous collection of more than twenty-five different malignancies. Classification is often challenging as primarily based upon, sometimes subjective, histopathological criteria and misdiagnosis can result in inappropriate treatment decisions. MicroRNAs (miRNAs) hold great promise as novel biomarkers (diagnostic, prognostic and predictive) of B-cell lymphoma in addition to being potential therapeutic targets. The most promising of these miRNAs more often than not play key regulatory roles in lymphopoiesis (development of lymphocytes) when under physiological conditions, and in the pathology of lymphoid malignancies when aberrantly expressed. In this review we consider the identity and functional role of miRNAs in the most common forms of B-cell lymphomas, their role in lymphopoiesis and their potential as biomarkers for these malignancies.