Santiago Ramón y Cajal

Phosphorylated 4E Binding Protein 1: A Hallmark of Cell Signaling That Correlates With Survival in Ovarian Cancer

Growth factor receptors and cell signaling factors play a crucial role in human carcinomas and have been studied in ovarian tumors with varying results. Cell signaling involves multiple pathways and a myriad of factors that can be mutated or amplified. Cell signaling is driven through the mammalian target of rapamycin (mTOR) and extracellular regulated kinase (ERK) pathways and by some downstream molecules, such as 4E binding protein 1 (4EBP1), eukaryotic initiation factor 4E, and p70 ribosomal protein S6 kinase (p70S6K). The objectives of this study were to analyze the real role that these pathways play in ovarian cancer, to correlate them with clinicopathologic characteristics, and to identify the factors that transmit individual proliferation signals and are associated with pathologic grade and prognosis, regardless specific oncogenic alterations upstream.

Role of the p38 MAPK pathway in cisplatin-based therapy

p38 MAPK has been implicated in the response to cancer therapy. To determine whether the activation of p38 MAPK could be specific to cancer therapy, we investigated the activation of p38 MAPK in response to several chemotherapeutic agents, such as cisplatin, doxorubicin and taxol in several human cell lines. Activation of p38 MAPK was measured after exposure to several chemotherapeutic agents, using specific phosphoantibodies. Only cisplatin was able to activate p38 MAPK in all the cell lines tested. Furthermore, other platinum compounds such as transplatin and platinum (IV) chloride can induce activation of p38 MAPK. The kinetics of this activation is a key event in the biological role of p38 MAPK in response to cisplatin, as we conclude from the differences observed after treatment with transplatin and cisplatin. The p38 MAPK activation is independent of the origin or genetic alterations of the cell lines and seems to be mediated through both upstream activators MKK6 and MKK3. Although the isoforms α/β are mainly activated, we also demonstrated that other members of the p38 MAPK family were susceptible to activation by cisplatin when they were overexpressed in 293 T. Finally, pretreatment with specific inhibitors (SB 203580 and SKF 86002) induces a resistant phenotype in response to cisplatin. Furthermore, low activation of this SAPK pathway correlates with a resistant phenotype as demonstrated in our experimental model of head and neck cancer. Therefore, we conclude that the p38 MAPK pathway is a specific target for cisplatin-based therapy with clinical implications.

Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma

Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis.

Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients

Background Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients. Methods NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored. Results ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative. Conclusions ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients.

Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer

Bacteria go the distance in cancer The bacterial species Fusobacterium nucleatum is associated with a subset of human colorectal cancers, but its role in tumorigenesis is unclear. Studying patient samples, Bullman et al. found that F. nucleatum and certain co-occurring bacteria were present not only in primary tumors but also in distant metastases. Preliminary evidence suggests that the bacterium is localized primarily within the metastatic cancer cells rather than in the stroma. Antibiotic treatment of mice carrying xenografts of F. nucleatum–positive human colorectal cancer slowed tumor growth, consistent with a causal role for the bacterium in tumorigenesis. Science, this issue p. 1443 The same bacteria present in primary tumors of patients with colorectal cancer are also present in liver metastases. Colorectal cancers comprise a complex mixture of malignant cells, nontransformed cells, and microorganisms. Fusobacterium nucleatum is among the most prevalent bacterial species in colorectal cancer tissues. Here we show that colonization of human colorectal cancers with Fusobacterium and its associated microbiome—including Bacteroides, Selenomonas, and Prevotella species—is maintained in distal metastases, demonstrating microbiome stability between paired primary and metastatic tumors. In situ hybridization analysis revealed that Fusobacterium is predominantly associated with cancer cells in the metastatic lesions. Mouse xenografts of human primary colorectal adenocarcinomas were found to retain viable Fusobacterium and its associated microbiome through successive passages. Treatment of mice bearing a colon cancer xenograft with the antibiotic metronidazole reduced Fusobacterium load, cancer cell proliferation, and overall tumor growth. These observations argue for further investigation of antimicrobial interventions as a potential treatment for patients with Fusobacterium-associated colorectal cancer.

Cell signaling in endometrial carcinoma: phosphorylated 4E-binding protein-1 expression in endometrial cancer correlates with aggressive tumors and prognosis☆

In the oncogenic process, cell growth control plays a crucial role, and growth factor receptors and their signaling pathways are known to be altered in endometrial cancer, mostly in type I carcinomas. Two main pathways are involved in transmitting the proliferative signal from the membrane receptors to the nucleus: phosphatydil-inositol-3-kinase-protein kinase B-mammalian target of rapamycin and RAS-RAF-ERK pathways. A final effector of these signaling cascades is the cap-dependent mRNA translation initiation complex, which is negatively regulated by 4E-BP1. The aim of our work was to study the relative importance of the factors involved in these pathways and to see their correlation with the clinicopathologic features of the tumors and their prognosis. We studied 120 endometrial carcinomas, including 93 type I and 27 type II carcinomas, and 18 control cases. Tissue microarrays were constructed and immunohistochemistry was performed for HER2, p53, and the phosphorylated forms of protein kinase B, extracellular signal-regulated kinase, and 4E-BP1. HER2 was overexpressed in 11% of carcinomas but not in control cases, and 30% of carcinomas showed activation of protein kinase B and extracellular signal-regulated kinase, mostly in type II carcinomas. The phosphorylated form of 4E-BP1 was found to be cytoplasmic in 31% of cases, and in 63% of cases it showed nuclear expression; the latter was only found in carcinomas. p53 positivity was found in type II and in grade 3 type I carcinomas. This nuclear expression of phospho-4E-BP1 and HER2 overexpression were the only characteristics with prognostic significance. The activation of the signaling pathways that control cell growth is a common event in endometrial carcinomas. 4E-BP1 is a downstream effector of these pathways whose activation status correlates with aggressive phenotypes and prognosis. This factor can reflect the activity of these pathways, regardless of the upstream molecular alterations, and, therefore, it can be a hallmark of the transmission of the oncogenic signal to the nucleus.

An association between viral genes and human oncogenic alterations: the adenovirus E1A induces the Ewing tumor fusion transcript EWS-FLI1.

Malignant transformation of human cells requires the accumulation of multiple genetic alterations, such as the activation of oncogenes and loss of function of tumor suppressor genes or those related to genomic instability. Among the genetic alterations most frequently found in human tumors are chromosomal translocations that may result in the expression of chimeric products with transforming capability or are able to change the expression of oncogenes. We show here that the adenovirus early region 1A (E1A) gene can induce a specific human fusion transcript (EWS–FLI1) that is characteristic of Ewing tumors. This fusion transcript was detected by RT–PCR in normal human fibroblasts and keratinocytes after expression of the adenovirus E1A gene, as well as in human cell lines immortalized by adenoviruses. Cloning and sequencing of the RT–PCR product showed fusion points between EWS and FLI1 cDNA identical to those detected in Ewing tumors. In addition, we detected a chimeric protein by western blot analysis and immunoprecipitation and a t(11,22) by fluorescent in situ hybridization. This association between a single viral gene and a specific human fusion transcript indicates a direct link between viral genes and chromosome translocations, one of the hallmarks of many human tumors.

Epstein-Barr virus associated B-cell lymphoma after autologous bone marrow transplantation for T-cell acute lymphoblastic leukaemia

Epstein‐Barr virus associated lymphoproliferative disease after autologous bone marrow transplantation (ABMT) has rarely been reported. We report a case of B‐cell lymphoma following ABMT for T‐acute lymphoblastic leukaemia; bone marrow was purged in vitro with monoclonal antibodies to remove T cells. Immunoglobulin and T‐cell receptor gene rearrangement studies were used to demonstrate clonality and to show that this patient developed a second neoplasm after ABMT. EBV proteins and genome (type A) were present in post‐transplantation lymphoma, suggesting a causative role in its development.

Modulation of PI3K/Akt pathway by E1a mediates sensitivity to cisplatin.

In order to investigate the molecular mechanisms implicated in the induction of chemo sensitivity by adenovirus E1a gene expression, we decided to investigate which signal transduction pathways could be affected by the E1a gene in Human Normal Fibroblast (IMR90). No effect was observed in SAPK pathways (p38MAPK and JNK), but E1a was able to affect the Akt activation mediated by insulin. This result was confirmed by transient transfection experiments performed in Cos-7 cells and also observed in other transformed cell lines such as A431. Furthermore, E1a expression induces a decrease in the basal status of Akt activity. Finally we demonstrated that E1a is able to block the Akt activation mediated by cisplatin and correlates with a sensitive phenotype. In summary, our data demonstrate that specific inhibition of the PI3K/Akt pathway mediates some of the biological properties of E1a such as induction of chemosensitivity.

Internal translation of the connexin 43 transcript.

BackgroundConnexin 43 (Cx43), the most widely expressed gap junction protein, is associated with a number of physiological and pathological conditions. Many functions of Cx43 have been shown to be independent of gap junction formation and only require the expression of Cx43 C-terminal fragments. Recent evidence demonstrated that naturally occurring C-terminal isoforms can be generated via internal translation.FindingsHere, we confirm that C-terminal domains of Cx43, particularly the major 20-kDa isoform, can be independently generated and regulated by internal translation of the same single GJA1 gene transcript that encodes full-length Cx43. Through direct RNA transfection experiments, we provide evidence that internal translation is not due to a bona fide cap-independent IRES-mediated mechanism, as upstream ribosomal scanning or translation is required. In addition to the mTOR pathway, we show for the first time, using both inhibitors and cells from knockout mice, that the Mnk1/2 pathway regulates the translation of the main 20-kDa isoform.ConclusionsInternal translation of the Cx43 transcript occurs but is not cap-independent and requires translation upstream of the internal start codon. In addition to the PI3K/AKT/mTOR pathway, the major 20-kDa isoform is regulated by the Mnk1/2 pathway. Our results have major implications for past and future studies describing gap junction-independent functions of Cx43 in cancer and other pathological conditions. This study provides further clues to the signalling pathways that regulate internal mRNA translation, an emerging mechanism that allows for increased protein diversity and functional complexity from a single mRNA transcript.