Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence

Proteomic analysis of the secretome of p95HER2-induced senescence. a MCF7 Tet-Off p95HER2 cells were cultured with or without doxycycline for 1 week and stained for senescence-associated β-galactosidase. Representative images of the stained cultures are shown. b The secretomes of the same cells as in a were analyzed by label-free quantitative proteomics. The results are shown as unsupervised hierarchical clustering analysis corresponding to three technical replicas (a-c) of two independent experiments (1 and 2). c The proteins identified in b were classified according to the presence of transmembrane or glycophosphatidylinositol domains (cell membrane), signal peptide but not transmembrane domain (secreted, canonical), or the lack of these domains (secreted, unknown). See also Additional file 2: Table S1. Doxy doxycycline. (JPEG 585 kb

PVT1 long non-coding RNA in gastrointestinal cancer

Whole genome and transcriptome sequencing technologies have led to the identification of many long non-coding RNAs (lncRNAs) and stimulated the research of their role in health and disease. LncRNAs participate in the regulation of critical signaling pathways including cell growth, motility, apoptosis, and differentiation; and their expression has been found dysregulated in human tumors. Thus, lncRNAs have emerged as new players in the initiation, maintenance and progression of tumorigenesis. PVT1 (plasmacytoma variant translocation 1) lncRNA is located on chromosomal 8q24.21, a large locus frequently amplified in human cancers and predictive of increased cancer risk in genome-wide association studies (GWAS). Combined, colorectal and gastric adenocarcinomas are the most frequent tumor malignancies and also the leading cause of cancer-related deaths worldwide. PVT1 expression is elevated in gastrointestinal tumors and correlates with poor patient prognosis. In this review, we discuss the mechanisms of action underlying PVT1 oncogenic role in colorectal and gastric cancer such as MYC upregulation, miRNA production, competitive endogenous RNA (ceRNA) function, protein stabilization, and epigenetic regulation. We also illustrate the potential role of PVT1 as prognostic biomarker and its relationship with resistance to current chemotherapeutic treatments.This study was partially funded by grants of the Spanish Ministry for Economy and Competitiveness (PI16/00540, AC15/00066, AC19/00095, and PI19/00993) and the Spanish Association Against Cancer (AECC GCA15152966ARAN) to DA

PVT1 Long Non-coding RNA in Gastrointestinal Cancer

Whole genome and transcriptome sequencing technologies have led to the identification of many long non-coding RNAs (lncRNAs) and stimulated the research of their role in health and disease. LncRNAs participate in the regulation of critical signaling pathways including cell growth, motility, apoptosis, and differentiation; and their expression has been found dysregulated in human tumors. Thus, lncRNAs have emerged as new players in the initiation, maintenance and progression of tumorigenesis. PVT1 (plasmacytoma variant translocation 1) lncRNA is located on chromosomal 8q24.21, a large locus frequently amplified in human cancers and predictive of increased cancer risk in genome-wide association studies (GWAS). Combined, colorectal and gastric adenocarcinomas are the most frequent tumor malignancies and also the leading cause of cancer-related deaths worldwide. PVT1 expression is elevated in gastrointestinal tumors and correlates with poor patient prognosis. In this review, we discuss the mechanisms of action underlying PVT1 oncogenic role in colorectal and gastric cancer such as MYC upregulation, miRNA production, competitive endogenous RNA (ceRNA) function, protein stabilization, and epigenetic regulation. We also illustrate the potential role of PVT1 as prognostic biomarker and its relationship with resistance to current chemotherapeutic treatments

The Receptor CMRF35-Like Molecule-1 (CLM-1) Enhances the Production of LPS-Induced Pro-Inflammatory Mediators during Microglial Activation.

CMRF35-like molecule-1 (CLM-1) belongs to a receptor family mainly expressed in myeloid cells that include activating and inhibitory receptors. CLM-1 contains two ITIMs and a single immunoreceptor tyrosine-based switch motif (ITSM), although also displays a binding site for p85α regulatory subunit of PI3K. By using murine primary microglial cultures, we show the presence of all CLM members in microglial cells and characterize the expression of CLM-1 both in basal conditions and during microglial activation. The TLR4 agonist lipopolysaccharide (LPS) and the TLR3 agonist polyinosinic-polycytidylic acid (Poly I:C) induce an increase in microglial CLM-1 mRNA levels in vitro, whereas the TLR2/6 heterodimer agonist peptidoglycan (PGN) produces a marked decrease. In this study we also describe a new soluble isoform of CLM-1 that is detected at mRNA and protein levels in basal conditions in primary microglial cultures. Interestingly, CLM-1 engagement enhances the transcription of the pro-inflammatory mediators TNFα, COX-2 and NOS-2 in microglial cells challenged with LPS. These results reveal that CLM-1 can acts as a co-activating receptor and suggest that this receptor could play a key role in the regulation of microglial activation

Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

EPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

Tyrosine kinase; EPHA3; Colorectal cancerTirosina-cinasa; EPHA3; Càncer colorectalTirosina quinasa; EPHA3; Cáncer colorrectalEPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Effect of Specific Mutations in Cd300 Complexes Formation; Potential Implication of Cd300f in Multiple Sclerosis.

Herein, we have used bioinformatics tools to predict five clusters defining ligand-binding sites on the extracellular domain of human CD300b receptor, presumably involved in the formation of both homodimers and heterodimers with other CD300 family members. Site-directed mutagenesis revealed residues glutamic acid 28 and glutamine 29 in cluster 5 to be necessary for the formation of CD300b complexes. Surprisingly, the disruption of cluster 2 and 4 reconstituted the binding capability lost by the mutation of residues glutamic acid 28 to alanine, glutamine 29 to alanine (E28A-Q29G). We identified a missense mutation arginine 33 to glutamine (R33Q) in CD300f by direct sequencing of exon 2 in peripheral blood samples from 50 patients with multiple sclerosis (MS). Levels of expression of CD300f were almost undetectable on monocytes from the patient bearing the R33Q mutation compared with healthy individuals. Whereas R33Q mutation had no effect in the formation of CD300f complexes, the inhibition of protein synthesis with cycloheximide indicated that CD300f R33Q is less stable than native CD300f. Finally, we report that the levels of expression of CD300f on the surface of classical and intermediate monocytes from MS patients are significantly lower when compared to the same cell populations in healthy individuals

Mechanisms of inactivation of the tumour suppressor gene RHOA in colorectal cancer

Reduced RHOA signalling has been shown to increase the growth/metastatic potential of colorectal tumours. However, the mechanisms of inactivation of RHOA signalling in colon cancer have not been characterised. A panel of colorectal cancer cell lines and large cohorts of primary tumours were used to investigate the expression and activity of RHOA, as well as the presence of RHOA mutations/deletions and promoter methylation affecting RHOA. Changes in RHOA expression were assessed by western blotting and qPCR after modulation of microRNAs, SMAD4 and c-MYC. We show here that RHOA point mutations and promoter hypermethylation do not significantly contribute to the large variability of RHOA expression observed among colorectal tumours. However, RHOA copy number loss was observed in 16% of colorectal tumours and this was associated with reduced RHOA expression. Moreover, we show that miR-200a/b/429 downregulates RHOA in colorectal cancer cells. In addition, we found that TGF- β /SMAD4 upregulates the RHOA promoter. Conversely, RHOA expression is transcriptionally downregulated by canonical Wnt signalling through the Wnt target gene c-MYC that interferes with the binding of SP1 to the RHOA promoter in colon cancer cells. We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms

Additional file 8: Figure. S3. of Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence

a Short-term cholesterol depletion activates the shedding of AP-tagged TGF-α and Areg. MCF7 cells expressing AP-tagged TGF-α or Areg were cultured with or without doxycycline and treated with MβCD or vehicle for 1 h as indicated. AP was quantified in serum-free conditioned media and cell lysates. Data shown represent the averages and standard deviations of three independent experiments. **P < 0.01 using the two-sided Student’s t test. b The secretome of p95HER2-induced senescent cells contains factors that activate the EGFR. A431 cells were stimulated with conditioned media obtained from culturing MCF7 Tet-Off p95HER2 cells shNT or shADAM17 in serum-free media for 48 h, after 5 days of plating with or without doxycycline (see schematic drawing). Then A431 cell lysates were analyzed by Western blot by using the indicated antibodies. Quantification of densitometric data is shown. AP alkaline phosphatase, Areg amphiregulin, Doxy doxycycline, MβCD methyl-beta-cyclodextrin, TGF-α transforming growth factor-alpha. (JPEG 275 kb