Identification of small open reading frames (sm ORF s) encoding small proteins (≤ 100 amino acids; SEP s) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a novel bioinformatics tool (Ran SEP s) with “‐omics” approaches, we were able to describe 109 bacterial small ORF omes. Predictions were first validated by performing an exhaustive search of SEP s present in Mycoplasma pneumoniae proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, Ran SEP s predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEP s from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEP s. Integration of Ran SEP s predictions with transcriptomics data showed that some annotated non‐coding RNA s could in fact encode for SEP s. A functional study of SEP s highlighted an enrichment in the membrane, translation, metabolism, and nucleotide‐binding categories. Additionally, 9.7% of the SEP s included a N‐terminus predicted signal peptide. We envision Ran SEP s as a tool to unmask the hidden universe of small bacterial proteins.

Activation of the small GTPase RHOA has strong oncogenic effects in many tumor types, although its role in colorectal cancer remains unclear. Here we show that RHOA inactivation contributes to colorectal cancer progression/metastasis, largely through the activation of Wnt/β-catenin signaling. RhoA inactivation in the murine intestine accelerates the tumorigenic process and in human colon cancer cells leads to the redistribution of β-catenin from the membrane to the nucleus and enhanced Wnt/β-catenin signaling, resulting in increased proliferation, invasion and de-differentiation. In mice, RHOA inactivation contributes to colon cancer metastasis and reduced RHOA levels were observed at metastatic sites compared to primary human colon tumors. Therefore, we have identified a new mechanism of activation of Wnt/β-catenin signaling and characterized the role of RHOA as a novel tumor suppressor in colorectal cancer. These results constitute a shift from the current paradigm and demonstrate that RHO GTPases can suppress tumor progression and metastasis.

The loss of the epithelial architecture and cell polarity/differentiation is known to be important during the tumorigenic process. Here we demonstrate that the brush border protein Myosin Ia (MYO1A) is important for polarization and differentiation of colon cancer cells and is frequently inactivated in colorectal tumors by genetic and epigenetic mechanisms. MYO1A frame-shift mutations were observed in 32% (37 of 116) of the colorectal tumors with microsatellite instability analyzed, and evidence of promoter methylation was observed in a significant proportion of colon cancer cell lines and primary colorectal tumors. The loss of polarization/differentiation resulting from MYO1A inactivation is associated with higher tumor growth in soft agar and in a xenograft model. In addition, the progression of genetically and carcinogeninitiated intestinal tumors was significantly accelerated in Myo1a knockout mice compared with Myo1a wild-type animals. Moreover, MYO1A tumor expression was found to be an independent prognostic factor for colorectal cancer patients. Patients with low MYO1A tumor protein levels had significantly shorter disease-free and overall survival compared with patients with high tumoral MYO1A (logrank test P = 0.004 and P = 0.009, respectively). The median time-to-disease recurrence in patients with low MYO1A was 1 y, compared with >9 y in the group of patients with high MYO1A. These results identify MYO1A as a unique tumor-suppressor gene in colorectal cancer and demonstrate that the loss of structural brush border proteins involved in cell polarity are important for tumor development.

Colorectal cancer is the second cause of cancer-related death in the western world, and although the genetic and molecular mechanisms involved in the initiation and progression of these tumors are among the best characterized, there are significant gaps in our understanding of this disease. The role of EPHB signaling in colorectal cancer has only recently been realized. Here, we use animal models to investigate the role of EphB4 in intestinal tumorigenesis. Modulation of EPHB4 levels in colon cancer cell lines resulted in significant differences in tumor growth in a xenograft model, with low levels of EPHB4 associated with faster growth. In addition, using a genetic model of intestinal tumorigenesis where adenomatous polyposis coli (Apc) mutations lead to initiation of the tumorigenic process (Apc min mice), we show that inactivation of a single allele of EphB4 results in higher proliferation in both the normal epithelium and intestinal tumors, significantly larger tumors in the small intestine, and a 10-fold increase in the number of tumors in the large intestine. This was associated with a 25% reduction in the lifespan of Apc min mice (P < 0.0001). Gene expression analysis showed that EphB4 mutations result in a profound transcriptional reprogramming, affecting genes involved in cell proliferation, remodeling of the extracellular matrix, and cell attachment to the basement membrane among other functional groups of genes. Importantly, in agreement with the expression profiling experiments, using an in vitro assay, we show that loss of EPHB4 in colon cancer cells results in a significantly increased potential to invade through a complex extracellular matrix. Collectively, these results indicate that EphB4 has tumor suppressor activities and that regulation of cell proliferation, extracellular matrix remodeling, and invasive potential are important mechanisms of tumor suppression. [Cancer Res 2009;69(18):7430-8]

Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immuno-histochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs. Europe PMC Funders GroupAuthor Manuscript Am J Pathol. Author manuscript; available in PMC 2017 May 11. Europe PMC Funders Author Manuscripts Europe PMC Funders Author ManuscriptsColorectal cancer (CRC) can be broadly classified as those displaying microsatellite instability (MSI) or chromosomal instability (CIN); herein referred to as microsatellite stable (MSS).1 MSI colon cancers can be further separated into familial (Lynch syndrome) or sporadic MSI. Patients with Lynch syndrome have inherited mutations in one of six DNA mismatch repair genes, although mutations in MLH1 and MSH2 account for the majority (∼90%) of cases.1,2 In Lynch syndrome, colonic tumors typically arise in the fourth decade of life, following loss of heterozygosity of the wild-type allele.1 In comparison, sporadic MSI is driven largely by epigenetic silencing of the MLH1 locus with significantly later tumor onset.3 Inactivation of DNA mismatch repair genes results in acquisition of a mutator phenotype, which in turn drives tumorigenesis through mutation of key oncogenic and tumor suppressor signaling pathways including Wnt, Ras/BRAF, transforming growth factor-β (TGF-β), and PI3K.Several cytogenetic and epigenetic differences exist between MSS/CIN and MSI tumors. In particular, MSS/CIN tumors display gains and losses of large chromosomal regions and multiple chromosomal rearrangements, whereas MSI tumors are largely diploid. Conv...

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II’ (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

Adipogenic differentiation is driven by a complex cascade of transcription factors (TFs) 1 and cell signaling molecules that lead to the expression of the master regulators CCAAT/enhancer-binding protein (C/EBP) (1) and peroxisome proliferator-activated receptor (PPAR) (2) family proteins. In a sequential process, C/EBP␦ and C/EBP␤ are initially induced and followed by C/EBP␣ and PPAR␥ expression. These two master TFs induce the final program of gene expression for adipocyte differentiation.The transcription factor Snail1 is a major inducer of the epithelial-mesenchymal transition (EMT) during embryonic development and cancer progression (3, 4). Snail1 expression is very restricted in adult individuals (5), but reappears to drive the EMT process that confers promigratory, invasive, and stem cell properties to cancer epithelial cells (4). During this process, Snail represses the expression of E-cadherin and promotes the expression of mesenchymal genes like vimenFrom the ‡Department

Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes.