IDENTIFICATION OF INTRONIC MICRORNAS ALTERED IN BREAST CANCER THROUGH MICROARRAY HOST GENE EXPRESSION ANALYSIS

MicroRNA (miRNAs) are endogenous non-coding RNAs of 3c 22 nucleotides in length that function as post-transcriptional regulators of gene and pro- tein expression through degradation or translation inhibition of the target messenger RNAs. MiRNAs show altered expression profiles in several hu- man pathologies, including cancer. They can act as tumour suppressors or as oncogenes, depending on the characteristics of their target genes. More than half of the mammalian miRNAs, including several of the miR- NAs implicated in breast cancer, are localized within the introns of protein- coding genes, often organized in clusters, and usually transcribed together with their host gene. It is therefore possible, at least in principle, to iden- tify novel intronic cancer-regulated miRNAs by examining the expression profile of their host genes by means of microarrays. For this purpose, we analyzed the regulation of 253 miRNA host genes in five large breast cancer microarray data sets comprising more than 950 samples, examining their association with different clinical and pathological parameters such as tu- mour grade, estrogen and progesterone receptor status, p53 status, survival, and occurrence of relapse or metastasis. We found that MCM7 and SMC4 were the most frequently and significantly overexpressed genes in high grade tumours. These genes contain two well known cancer-associated miRNA clusters: miR25-93-106b and miR-15b/16- 2 respectively. In addition we identified six other miRNA host genes that were significantly downregulated in high grade tumours in all the data sets. Much less evidence is available in the literature about the involvement in cancer of the miRNAs contained in these genes (i.e., miR-218-1, miR-342, miR-483, miR-548f-2, miR-1245 and miR-1266 ). We measured the expression of the selected miRNAs by Real Time PCR on an independent cohort of 36 formalin-fixed paraffin-embedded (FFPE) samples, and we observed reduced expressions level of such miRNAs in high grade tumours. In particular, we found miR-342-3p, miR-342-5p, miR-483- 3p, and miR-483-5p to be the most significantly downregulated miRNAs. These miRNAs were also found to correlate with bad prognosis in grade 2 tumours. Finally we provided initial evidence that increased expression of miR-342-5p, but not miR-342-3p, induces apoptosis in the highly metastatic MDA-MB-231 breast cancer cell line

Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under Accession Nos E-MTAB-990 and E-MTAB-1026. © 2013 Swiss Institute of Bioinformatics. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Abrogation of Junctional Adhesion Molecule-A Expression Induces Cell Apoptosis and Reduces Breast Cancer Progression

Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A) is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A−/− tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target

3577: Thymidylate synthase (TS) expression as a prognostic molecular marker in stage II/III colon cancer.

Background: Studies on the association between colorectal cancer (CC) outcome and thymidylate synthase expression have provided inconsistent results. In this study we attempted to resolve the issue by assessing the associations between TS expression and outcome in a population of primary CC patients (pts), who after resection were randomized to 5-FU/FA vs. FOLFIRI adjuvant therapy. Methods: Immunohistochemical staining for TS protein was successfully performed for 1211 pts in the PETACC3 trial. TS immunoreactivity was scored as high expression (≥75% positive) and low expression (<75% positive). Gene expression respectively copy number data were available for 853 respectively 306 of these samples. Twelve single nucleotide polymorphisms (SNPs) close to the TYMS gene were assessed in 923 pts. Association of variables with relapse-free (RFS) and overall survival (OS) was assessed using Cox regression models. Results: High TS expression and RNA level were strongly associated (log fold change 0.65, p<0.001). Both were significantly higher in proximal CC. As expected, both were associated with other characteristics of proximal CC: MSI, BRAF mutation, high tumor grade. RNA was significantly correlated with gene copy number, distal CC showing more frequent allelic loss. Three SNPs were associated with gene expression which was validated in data from the 1000 genomes project, but none with survival. High TS expression was more strongly associated with better OS in pts receiving FOLFIRI (HR 0.4, 95% CI 0.3-0.6, p<0.001), than 5-FU/FA (HR 0.8, 95% CI 0.5-1.1, p=0.13), with a significant interaction (p=0.05). Similar results were observed for RFS (HR 0.5, p<0.001 vs. HR 0.7, p=0.07; interaction p=0.11). TS expression is still highly prognostic in multivariate models adjusting for factors associated with risk or proximal tumors in FOLFIRI treated pts (OS: HR 0.5, p=0.008; RFS: HR 0.6, p=0.02), but not in F-FU/FA treated pts (OS and RFS: HR=1, p=1). Conclusions: TS expression is lower in distal CC, partly due to deletion of the TYMS locus. Pts with high TS expression have longer RFS and OS, notably when treated with FOLFIRI. For these pts addition of irinotecan to 5-FU/FA adjuvant chemotherapy might be considered

Alterations of the Notch pathway in lung cancer

Notch signaling regulates cell specification and homeostasis of stem cell compartments, and it is counteracted by the cell fate determinant Numb. Both Numb and Notch have been implicated in human tumors. Here, we show that Notch signaling is altered in approximately one third of non-small-cell lung carcinomas (NSCLCs), which are the leading cause of cancer-related deaths: in 4830% of NSCLCs, loss of Numb expression leads to increased Notch activity, while in a smaller fraction of cases (around 10%), gain-of-function mutations of the NOTCH-1 gene are present. Activation of Notch correlates with poor clinical outcomes in NSCLC patients without TP53 mutations. Finally, primary epithelial cell cultures, derived from NSCLC harboring constitutive activation of the Notch pathway, are selectively killed by inhibitors of Notch (\u3b3-secretase inhibitors), showing that the proliferative advantage of these tumors is dependent upon Notch signaling. Our results show that the deregulation of the Notch pathway is a relatively frequent event in NSCLCs and suggest that it might represent a possible target for molecular therapies in these tumors

3526: Proximal and distal colon tumors as distinct biologic entities with different prognoses.

Background: It has been shown that tumors arising in the proximal and distal colon, defined by the embryological midgut and hindgut, have distinctive clinical and molecular features, but very little is known concerning the differences in the mechanism of tumorigenesis and the effect that this could have on therapy. Methods: The distribution of clinico-pathological and molecular features was evaluated between proximal (N = 1110, Caecum to hepatic flexure) and distal colon (N = 1728, splenic flexure down to sigmoid) in patients included in the PETACC3 trial. Gene expression profile was also available from 783 tumors and 32 normal colon. A further set of 473 metastatic patients treated with cetuximab combined with chemotherapy (De Roock Lancet Oncol. 2010) was used to test tumor location with response. Results: Pathological features, such as tumor differentiation, and mucinous histology as well as molecular characteristics, such as MSI status, BRAF, PIK3Ca mutations and LOH18q loss show higher frequency in proximal compared to distal colon (N = 1214; Fisher test, P<0.001). Proximal tumors showed a significantly worse overall survival (N= 2838; HR=1.4 [1.18 - 1.64] P<0.001) and survival after relapse (N = 861; HR=1.97 [1.65 - 2.35] P <0.001) only if they were stage III at diagnosis, while no difference was observed for relapse free survival. Microarray profiling identified 997 genes differentially expressed between the two anatomical sites, after adjustment for age, gender, mucinous histology, BRAF, KRAS and MSI status. Only 20 of those were present in normal colon site comparison indicating tumor specificity. Data mining analysis of the differentially expressed genes showed that the distal colon is characterized by an enrichment for MAPK activated pathways as well as for the cetuximab response gene signature (Khambata-Ford JCO 2007). In fact, cetuximab treated KRAS/BRAF wild-type tumors in distal colon had prolonged PFS and a 2-fold higher response rate then proximal. Conclusions: Proximal and distal colon tumors have distinctive patterns of clinical-pathological and molecular features. These biological differences likely have significant prognostic and therapeutic implications

Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features.

BACKGROUND: Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications. MATERIALS AND METHODS: Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR). RESULTS: Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis. CONCLUSIONS: Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis

A Snapshot of the Physical and Functional Wiring of the Eps15 Homology Domain Network in the Nematode

<div><p>Protein interaction modules coordinate the connections within and the activity of intracellular signaling networks. The Eps15 Homology (EH) module, a protein-protein interaction domain that is a key feature of the EH-network, was originally identified in a few proteins involved in endocytosis and vesicle trafficking, and has subsequently also been implicated in actin reorganization, nuclear shuttling, and DNA repair. Here we report an extensive characterization of the physical connections and of the functional wirings of the EH-network in the nematode. Our data show that one of the major physiological roles of the EH-network is in neurotransmission. In addition, we found that the proteins of the network intersect, and possibly coordinate, a number of “territories” of cellular activity including endocytosis/recycling/vesicle transport, actin dynamics, general metabolism and signal transduction, ubiquitination/degradation of proteins, DNA replication/repair, and miRNA biogenesis and processing.</p> </div

In vitro binding assays.

<p>Sixteen interactors, identified by Y2H (listed at the bottom), were expressed as GST-fusion proteins and used for <i>in vitro</i> binding assays with FLAG-EH proteins expressed in Phoenix cells. Results are the average of three independent experiments (examples are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383.s003" target="_blank">Figure S3</a>), and are expressed in arbitrary units on a scale 0–100, in which 100 represents the efficiency of the pull-down for the strongest interacting protein in each panel.</p