Jesús García-Foncillas

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.

Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2

BackgroundThe p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway.ResultsHere we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor.ConclusionsOur results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

Hyperphosphorylation of PP2A in colorectal cancer and the potential therapeutic value showed by its forskolin-induced dephosphorylation and activation

BACKGROUND The tumor suppressor protein phosphatase 2A (PP2A) is frequently inactivated in human cancer and phosphorylation of its catalytic subunit (p-PP2A-C) at tyrosine-307 (Y307) has been described to inhibit this phosphatase. However, its molecular and clinical relevance in colorectal cancer (CRC) remains unclear. METHODS p-PP2A-C Y307 was determined by immunoblotting in 7 CRC cell lines and 35 CRC patients. CRC cells were treated with the PP2A activator forskolin alone or combined with the PP2A inhibitor okadaic acid, 5-fluorouracil and oxaliplatin. We examined cell growth, colonosphere formation, caspase activity and AKT and ERK activation. RESULTS PP2A-C was found hyperphosphorylated in CRC cell lines. Forskolin dephosphorylated and activated PP2A, impairing proliferation and colonosphere formation, and inducing activation of caspase 3/7 and changes in AKT and ERK phosphorylation. Moreover, forskolin showed additive effects with 5-fluorouracil and oxaliplatin treatments. Analysis of p-PP2A-C Y307 in primary tumors confirmed the presence of this alteration in a subgroup of CRC patients. CONCLUSIONS Our data show that PP2A-C hyperphosphorylation is a frequent event that contributes to PP2A inhibition in CRC. Antitumoral effects of forskolin-mediated PP2A activation suggest that the analysis of p-PP2A-C Y307 status could be used to identify a subgroup of patients who would benefit from treatments based on PP2A activators.

The challenge of blocking a wider family members of EGFR against head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) represent 95% of head and neck cancer with an incidence of over half a million people globally. The prognosis for patients with recurrent or metastatic HNSCC is generally poor with low 5-year survival rates despite treatment advances over the past few decades. Consequently, it is essential to search for new biomarkers and effective therapy options to optimize HNSCC treatment. Epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of tumours. EGFR has become one of most common targets for new therapies being investigated in HNSCC. In this way, multiple therapies targeting EGFR in HNSCC have been tested but response rates are still low especially in the recurrent or metastatic setting. This has been attributed to mechanisms of resistance to EGFR-targeted therapies. Afatinib, an oral small molecule ErbB Family Blocker that irreversibly binds to ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4), is being investigated in HNSCC treatment with encouraging phase II results and several ongoing phase III trials. Results of these trials will help to understand the place of afatinib in the HNSCC treatment armamentarium.

Up-regulation of c-Cbl suggests its potential role as oncogene in primary colorectal cancer

Dear Editor: Although progressive advances in early detection has been carried out in the last years, colorectal cancer (CRC) still represents the third cause of cancer death worldwide. This is because more than half of the patients progress to metastatic disease, which determines a very poor outcome. Therefore, it remains necessary to better understand the molecular basis that governs the progression from the early stages of CRC to metastatic development. In this context, the acquisition of invasiveness properties is a key molecular event that determines the progression from primary tumor to metastasis. Of importance, it has been recently reported that c-Cbl, an E3 ubuquitin ligase and a multifunctional adaptor protein, regulates invasion ability of cancer cells through matrix metalloproteinase 2. It is an unexpected observation since c-Cbl was largely reported to play a tumor suppressor role in several tumor types. However, in concordance with this novelproposed oncogenic role as regulator of cell adhesion, migration, and degradation, c-Cbl has been described as a marker of poor clinical outcome in prostate cancer. Therefore, it seems that this protein could be playing a dual role as a tumor suppressor or oncogene depending on the cancer model and stage of the disease. This novel-reported function for c-Cbl and the fact that its role in CRC remains unclear prompted us to analyze c-Cbl expression by western blot in a series of seven CRC cell lines. Interestingly, we observed increased c-Cbl levels in the DLD-1, RKO, LoVo, and SW620 cell lines whereas the WiDr, SW480, and HT-29 cell lines showed a c-Cbl expression similar to normal colonic mucosa. To further evaluate the importance of in CRC, we studied c-Cbl expression levels in 22 patients with primary CRC. Primary colorectal tissues were surgical resection specimens from CRC tumors obtained from Fundacion Jimenez Diaz Biobank (BFJD, Madrid). Tumor, node, metastases (TNM) staging was classified using the 7th American Joint Committee on Cancer (AJCC) staging system for colorectal cancer. Clinical data were collected from medical clinical records by an oncologist (J. G.-F.). Paired normal mucosa obtained from each patient was used as control. Moreover, a pathologist confirmed that primary tumor tissues used in this work contained greater than 70 % tumoral component (F. R.). All samples were taken anonymously and the ethical committee and institutional review board approved the project. Importantly, we found that 8 out of the 22 CRC cases showed c-Cbl overexpression in the tumor samples compared to their paired normal colonic mucosa. These results confirmed the previous observations made in CRC cell lines. Altogether, our findings suggest that c-Cbl deregulation is a recurrent event that could be playing a role in the acquisition of invasiveness properties of CRC cells and might serve as a novel potential therapeutic target in a subset of CRC patients. However, further studies are needed to clarify this potential role of c-Cbl as oncogene in primary colorectal cancer and to determine its biological and clinical significance in the pathogenesis of this disease.

Deregulation of miR-92a in locally advanced rectal cancer.

We read with interest the recent article by Pelossof et al. (2016), reporting a comparison between locally advanced rectal cancer (LARC) samples and normal rectal mucosa using an integrative genomic analysis that included microRNA and mRNA expression profiles and copy number alterations. Of importance, miR-92a was upregulated in LARC specimens and correlated inversely with the expression levels of its putative targets IQGAP2 and KLF4. These findings are consistent with previous work suggesting an oncogenic role of miR-92a in colorectal cancer (CRC) (Tsuchida et al., 2011; Yamada et al., 2013; Zhou et al., 2013). Despite the interesting data, the authors failed to perform a correct experimental validation and interpretation of their results. Western blot and luciferase assays, as also mentioned in their discussion, are required to demonstrate a direct regulation of IQGAP2 and KLF4 by miR-92a. Unfortunately, only changes in mRNA levels of these putative targets were analyzed after the ectopic modulation of miR92a expression. The authors claimed that a direct KLF4 regulation by miR-92a was not supported by their observations because they found no changes in KLF4 mRNA after miR-92a modulation. However, in the absence of a perfect match between microRNA and its seed region, the targeted mRNA is not degraded and changes in mRNA levels cannot be observed. Therefore, Western blot and luciferase assays have to be performed to evaluate KLF4 as a novel potential miR-92a target in forthcoming studies. Finally, it would be necessary to analyze the functional relevance of IQGAP2 and KLF4 deregulation compared to other previously described miR-92a targets such as BIM (Tsuchida et al., 2011), DKK-3 (Yamada et al., 2013), and PTEN (Zhang et al., 2014).

Analysis of Potential Alterations Affecting SETBP1 as a Novel Contributing Mechanism to Inhibit PP2A in Colorectal Cancer Patients

BackgroundThe functional loss of the tumor suppressor protein phosphatase 2A (PP2A) occurs in a wide variety of human cancers including colorectal cancer (CRC), and SET overexpression has been reported as a key contributing mechanism to inhibit PP2A. Although SET binding protein 1 (SETBP1) overexpression and gain of function mutations have been described in several hematological malignancies as common events that increase the expression levels of the PP2A inhibitor SET, thereby leading to PP2A inactivation, the potential existence of SETBP1 alterations in CRC still remains unexplored.MethodsWe studied the expression profile of SETBP1 by Western blot in a set of CRC cell lines and patient samples. Moreover, we performed co-immunoprecipitation assays to analyze the formation of the previously reported SETBP1–SET–PP2A inhibitory complex. Furthermore, we evaluated the mutational status of SETBP1 by pyrosequencing assays in a cohort of 55 CRC patients with metastatic disease after the immunohistochemical characterization of SET and p-PP2A expression in this cohort.ResultsWe found high SETBP1 expression in several CRC lines but only in two of the patients analyzed. In addition, we demonstrated the formation of the SETBP1–SET–PP2A heterotrimeric complex in CRC cells. However, we failed to detect SETBP1 mutations in any of the CRC patient samples included in the study.ConclusionsOur results suggest that SETBP1 expression is mainly similar o lower in colorectal cancer tissue compared to normal colonic mucosa. However, its overexpression is a low prevalent alteration which could contribute to inhibit PP2A in CRC through the formation of a SETBP1–SET–PP2A complex in some CRC patients. Moreover, SETBP1 mutations are, if exist, rare events in CRC patients.

Cancer and suicidal ideation and behaviours: protocol for a systematic review and meta-analysis.

Introduction Prevalence of suicidal ideation (SI) and behaviours are higher among patients with cancer than general population. No systematic review/meta-analysis investigated this topic; therefore, our aim will be to assess the relationship between cancer and SI and behaviours. Methods We will search PubMed/MEDLINE, EMBASE, SCOPUS, Web of Science, PsycINFO and Cochrane Library databases from their inception until 30 June 2018. Case–control and cohort studies focused on the association between cancer (any type) and suicidal outcomes (suicide, suicide attempt and SI) will be included. Two team members will independently: (A) perform the selection of the included studies and data extraction, with the supervision of a third member in case of discrepancies and (B) assess each study with: (1) Newcastle-Ottawa Scale (NOS); (2) Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement; (3) Grading of Recommendations Assessment, Development and Evaluation (GRADE). We will conduct a random-effects meta-analysis. Individual and pooled ORs and associated 95% CIs will be calculated as well as between-study heterogeneity. We will examine the potential for publication bias. If possible, we will explore reasons for potential between-study heterogeneity. Ethics and dissemination This study does not require ethical approval. The study will be submitted to a peer-reviewed journal, will be publicly disseminated and will be the topic of research presentations. PROSPERO registration number CRD42017072482.

Letter to the Editor: Is miR-199b-3p really involved in the migration ability of bone marrow mesenchymal stem cells?

Letter to the Editor: Is miR-199b-3p really involved in the migration ability of bone marrow mesenchymal stem cells? Blanca Torrejón,* Ion Cristóbal,* Juan Madoz-Gúrpide, Federico Rojo, and Jesús Garćıa-Foncillas* *Translational Oncology Division, Oncohealth Institute, IIS-Fundación Jiménez Dı́az, and Pathology Department, University Hospital “Fundación Jiménez Dı́az,” Autonomous University of Madrid, Madrid, Spain

Comment on Goldsworthy et al. Haploinsufficiency of the Insulin Receptor in the Presence of a Splice-Site Mutation in Ppp2r2a Results in a Novel Digenic Mouse Model of Type 2 Diabetes. Diabetes 2016;65:1434-1446.

We have read with great interest the recently published article by Goldsworthy et al. (1), which provides novel relevant findings about the role of protein phosphatase 2A (PP2A) in diabetes development. In this elegant work, the authors showed that the presence of an alternative splicing of the Ppp2r2a gene in mice heterozygous for a null allele of the insulin receptor resulted in a digenic mouse model of type 2 diabetes. Interestingly, the alternative splicing of Ppp2r2a reduced the protein levels of this PP2A regulatory …