Daniel Rueda

Involvement of the cAMP/protein kinase A pathway and of mitogen-activated protein kinase in the anti-proliferative effects of anandamide in human breast cancer cells.

Anandamide (ANA) inhibits prolactin‐ and nerve growth factor (NGF)‐induced proliferation of human breast cancer cells by decreasing the levels of the 100 kDa prolactin receptor (PRLr) and the high affinity trk NGF receptor, respectively, and by acting via CB1‐like cannabinoid receptors. However, the intracellular signals that mediate these effects are not known. Here, we show that, in MCF‐7 cells: (i) forskolin and the mitogen‐activated protein kinase (MAPK) kinase inhibitor PD098059 prevent, and the protein kinase A inhibitor RpcAMPs mimics, the inhibitory effects of ANA on cell proliferation and PRLr/trk expression and (ii) ANA inhibits forskolin‐induced cAMP formation and stimulates Raf‐1 translocation and MAPK activity, in a fashion sensitive to the selective CB1 antagonist SR141716A. ANA stimulation of MAPK was enhanced by inhibitors of ANA hydrolysis. Forskolin inhibited MAPK and ANA‐induced Raf‐1 translocation. These findings indicate that, in MCF‐7 cells, ANA inhibits adenylyl cyclase and activates MAPK, thereby exerting a down‐regulation on PRLr and trk levels and a suppression of cell proliferation.

Endocannabinoids : A new family of lipid mediators involved in the regulation of neural cell development

The endocannabinoids (eCBs) anandamide and 2-arachidonoylglycerol are important retrograde messengers that inhibit neurotransmitter release via presynaptic CB1 receptors. In addition, cannabinoids are known to modulate the cell death/survival decision of different neural cell types, leading to different outcomes that depend on the nature of the target cell and its proliferative/differentiation status. Thus, cannabinoids protect primary neurons, astrocytes and oligodendrocytes from apoptosis, whereas transformed glial cells are prone to apoptosis by cannabinoid challenge. Moreover, a potential role of the eCB system in neurogenesis and neural differentiation has been proposed. Recent research shows that eCBs stimulate neural progenitor proliferation and inhibit hippocampal neurogenesis in normal adult brain. Cannabinoids inhibit cortical neuron differentiation and promote glial differentiation. On the other hand, experiments with differentiated neurons have shown that cannabinoids also regulate neuritogenesis, axonal growth and synaptogenesis. These new observations support that eCBs constitute a new family of lipid signaling cues responsible for the regulation of neural progenitor proliferation and differentiation, acting as instructive proliferative signals through the CB1 receptor.

Age at Onset Should Be a Major Criterion for Subclassification of Colorectal Cancer

An important proportion of early-onset colorectal cancer (CRC) does not show a hereditary component with limited knowledge about its molecular basis and features. We analyzed a subset of patients with early-onset CRC and compared them with patients with late-onset CRC. We analyzed the microsatellite instability and CpG island methylator phenotype (CIMP) in both populations and classified them into four molecular subtypes. We analyzed the differential features between groups. Only 12 of 81 early-onset cases (15%) showed microsatellite instability, 10 of which (83%) were Lynch syndrome cases; microsatellite instability cases in elderly patients were sporadic. Early-onset microsatellite-stable cases showed different tumor locations and more family history of cancer than the elderly. Microsatellite instability/CIMP-high early-onset CRC was associated with Lynch syndrome, whereas the elderly cases were associated with BRAF mutations. Early-onset microsatellite-stable/CIMP-high CRCs were more frequently mucinous and right sided than elderly cases, with a high incidence of Lynch syndrome neoplasms; early-onset microsatellite stable/CIMP-low/0 differed from elderly cases in location, stages, incidence of multiple primary neoplasms, and the familial component. The clinical and familial differences observed between early- and late-onset CRC when considering the different carcinogenetic pathways underline that the age at onset criterion should be considered when classifying CRC.

Inhibition of related JAK/STAT pathways with molecular targeted drugs shows strong synergy with ruxolitinib in chronic myeloproliferative neoplasm

This study aimed to assess the antitumour effects, molecular mechanisms of action, and potential synergy of ruxolitinib with sorafenib, KNK437, dasatinib, and perifosine, in Philadelphia‐negative chronic myeloproliferative neoplasms (MPN). Cytotoxic and cytostatic effects of the different compounds were determined in the JAK2 V617F‐positive cell lines, HEL and Ba/F3 JAK2V617F EPOR, and in primary mononuclear and bone marrow CD34‐positive cells from 19 MPN patients. Ruxolitinib [50% inhibitory concentration (IC50)PV = 15 nmol/l], as well as sorafenib ( IC50 PV=8μmol/l ), KNK437 ( IC50 PV=100μmol/l ), and perifosine ( IC50 PV=15μmol/l ), were able to inhibit proliferation in cell line models and in primary cells from MPN patients. Dasatinib, KNK437, and sorafenib showed a strong synergistic effect in combination with ruxolitinib [combination index (CI)PV < 0·3]. Western blot confirmed that ruxolitinib blocked ERK, and consequently STAT5 activation, sorafenib inhibited ERK, P38 and STAT5, dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the JAK2 protein, reducing its expression. Inhibiting JAK2‐related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of ruxolitinib with inhibitors that target these pathways has a strong synergistic effect, which may be due to decreased activation of the common effector, STAT5.

Differential expression of JAK2 and Src kinase genes in response to hydroxyurea treatment in polycythemia vera and essential thrombocythemia

This study investigates the differential gene expression profile of JAK2V617F-positive myeloproliferative neoplasm (MPN) patients, with and without response to hydroxyurea (HU) treatment. Twenty-one polycythemia vera, 28 essential thrombocythemia, eight secondary erythrocytosis, and 30 controls were studied. Thirty-four genes were overexpressed in patients who did not respond to HU. Of these, some participate in proliferative pathways: MAPK, AKT, Src kinase (SFK), and JAK2 pathway. JAK2 allele burden was similar between groups of responders and nonresponder. A molecular fingerprint distinguishes JAK2V617F-positive MPN patients without response to HU treatment, with overexpression of JAK2, MAPK14, PIK3CA, and SFK genes.

DNA Copy Number Profiling Reveals Different Patterns of Chromosomal Instability Within Colorectal Cancer According to the Age of Onset

Chromosomal instability resulting in copy number alterations is a hallmark of colorectal cancer (CRC). However, few studies have attempted to characterize the chromosomal changes occurring in early‐onset CRC in order to compare them with those taking place within the more extensively studied late‐onset CRC subset. Our aim was to characterize the genomic profiles of these two groups of colorectal tumors and to compare them to each other. Array comparative genomic hybridization profiling of 146 colorectal tumors (60 early‐onset and 86 late‐onset) in combination with an unsupervised analysis was used to define common and specific copy number alterations. We found a number of important differences between the chromosomal instability profiles of each age subset. Thus, losses at 1p36, 1p12, 1q21, 9p13, 14q11, 16p13, and 16p12 were significantly more frequent in younger patients, whereas gains at 7q11 and 7q22 were more frequent in older patients. Moreover, the unsupervised analysis stratified the tumors into two clusters, each one of which was enriched in patients from one of the age subsets. Our findings confirm the existence of substantial differences between the chromosomal instability profiles of the two groups which are more important from a qualitative point of view. Further studies are needed to understand the clinicopathological implications of these dissimilarities.

Toward a Molecular Classification of Synchronous Colorectal Cancer: Clinical and Molecular Characterization

Background Two or more primary colorectal tumors coexisting at the time of diagnosis are considered to be synchronous tumors. It is estimated that synchronous colorectal cancer (SCRC) only accounts for 1.1% to 8.1% of all colorectal cancers (CRCs), and its molecular basis is still poorly understood. Patients and Methods We evaluated the microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP) statuses in a series of 49 patients (98 tumors) diagnosed with sporadic SCRC at the 12 de Octubre University Hospital with the aim of improving the molecular characterization of this type of tumor. We considered Lynch syndrome, familial adenomatous polyposis, and MUTYH‐associated polyposis (MAP) as exclusion criteria. Results Molecular subgrouping on the basis of MSI and CIMP enabled us to define 4 groups that corresponded to the molecular classification proposed for single‐tumor CRC. We observed a significant predominance of MSI tumors at the right side regardless of the methylation pattern, and a significant prevalence of microsatellite‐stable tumors either at the left side or throughout the entire colon (P = .026). Furthermore, we defined some molecular features frequently observed in sporadic SCRC such as a low‐frequency of MSI (8.2%). We observed a high concordance in terms of MSI between simultaneous tumors (93.9%) and a lower concordance in terms of CIMP (51%) between such tumors. Conclusion Our findings support the hypothesis that SCRC involves an environmental rather than a genetic component in which various etiologic factors might modify tumor progression. Further studies are required to refine the molecular characterization of SCRC. Micro‐Abstract On the basis of microsatellite instability (MSI) and CpG island methylator phenotype, we performed a molecular subgrouping of 49 patients diagnosed with synchronous colorectal cancer (SCRC). Our results suggest that MSI is an infrequent feature in sporadic SCRC (8.2%) commonly related with the right side of the colon regardless of the methylation pattern.

Use of Sorafenib as an effective treatment in an AML patient carrying a new point mutation affecting the Juxtamembrane domain of FLT3

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Use of exome sequencing to determine the full profile of genetic variants in the fluoropyrimidine pathway in colorectal cancer patients affected by severe toxicity

AIM To identify genetic variants associated with capecitabine toxicity in fluoropyrimidine pathway genes using exome sequencing. PATIENTS & METHODS Exomes from eight capecitabine-treated patients with severe adverse reactions (grade >2), among a population of 319, were sequenced (Ion Proton). SNPs in genes classified as potentially damaging (Sorting Intolerant from Tolerant and Polymorphism Phenotyping v2) were tested for association with toxicity in a validation cohort of 319 capecitabine-treated patients. RESULTS A total of 17 nonsynonymous genetic variants were identified. Of these, five putative damaging SNPs in DPYD, ABCC4 and MTHFR were genotyped in the validation cohort. DPYD rs1801160 was associated with the risk of toxicity (p = 0.029) and MTHFR rs1801133 with delayed administration of chemotherapy due to toxicity (p = 0.047). CONCLUSION Exome sequencing revealed two specific biomarkers of the risk of toxicity to capecitabine.

A Second Order Smooth Variational Principle on Riemannian Manifolds

We establish a second order smooth variational principle valid for functions defined on (possibly infinite- dimensional) Riemannian manifolds which are uniformly locally convex and have a strictly positive injectivity radius and bounded sectional curvature.