Biology subjects

Raposo, T. P.

Publications and source records attributed to Raposo, T. P..

4 recordsLinked to original sources

bioRxiv2021

N-glycosylation of CD24 mediates cell motility but inhibits cell proliferation in colorectal cancer

CD24 confers features of stemness and enhances cell motility in colorectal cancer. It contains numerous O-glycosylation sites but just two putative N-glycosylation sites. The study aimed to mutate N-glycosylation as it thought to be important in mediating many glycoproteins function. Site-directed mutagenesis performed to mutate N-glycosylation sites by converting asparagine residues to glutamine. Each site was mutated individually (CD24N36Q and CD24N52Q) and in combination (CD24N[36,52]Q) and were overexpressed in HCT116 and SW480 cell lines. The effect of the mutant CD24 on cell viability, cell motility and induction of downstream targets was compared to wild-type CD24 and empty vector control (EVC). Overexpression of the individually mutated clones increased cell motility compared to EVC but was significantly less than that induced by CD24WT. Overexpression of the double mutant clone resulted in near-complete abrogation of cell motility induction. Unexpectedly, the expression of mutant clones (single and double) increased cell viability. Analysis of downstream targets and F-actin staining demonstrated reduced induction of known CD24 targets by mutant vectors and reduced epithelial-mesenchymal transition. In conclusion, each N-glycosylation site on CD24 contributes partially to induce motility by CD24, and these are additive. Loss of the N-glycosylation results in an unexpected gain of proliferative function. HighlightsO_LICD24 is a heavily glycosylated molecule that contains two N-glycosylation sites, and these contribute to the activation of downstream targets and induction of cell motility C_LIO_LIEach N-glycosylation site contributes partially, and their effects appear to be additive C_LIO_LIThe N-glycosylation sites may exert an inhibitory effect on cell proliferation C_LIO_LIMuch of the CD24 activity is mediated through post-transcriptional effects C_LI

cancer biology↗

bioRxiv2020

Investigating TNS4 in the Colorectal Tumour Microenvironment Using 3D Spheroid Models of Invasion

TNS4 (Tensin 4 or Cten) is a putative oncogene in colorectal cancer (CRC) with a role in regulating cell adhesion, motility, invasion and epithelial to mesenchymal transition (EMT). Our objective was to study TNS4 role in CRC using more realistic models of the tumour microenvironment. CRC cells expressing TdTomato protein and shTNS4/shLUC hairpin oligos were grown in 3D spheroids with and without cancer-associated fibroblasts (CAFs). Adhesiveness to collagen I and CAFs was assessed in 2D and cell proliferation, volume and invasion were assessed in 3D conditions. The role of TNS4 knockdown in Gefitinib chemosensitivity and EGFR and Ras protein levels were also tested. In general, TNS4 knockdown increased cell proliferation in cell lines producing compact spheroids. The addition of CAFs in spheroids supported CRC cells proliferation, whereas CAFs themselves did not proliferate, but increased ECM degradation. TNS4 knockdown reduced adhesiveness and 3D invasion and disrupted EGFR signalling which resulted in increased sensitivity to Gefitinib. In conclusion, in a 3D spheroid model, TNS4 inhibits cell proliferation and promotes cell invasion into the ECM, possibly by adhesion to the ECM and stromal cells. TNS4 knockdown enhances sensitivity to the EGFR inhibitor Gefitinib and may be helpful for KRAS mutant CRC patients.

cancer biology↗

bioRxiv2018

The Cten signalling pathway stabilises Src protein to promote Epithelial-Mesenchymal Transition (EMT) in colorectal cancer

Cten is an oncogene which promotes epithelial-mesenchymal transition (EMT) in many signalling pathways. Having previously shown that Cten promotes EMT through Snail, we investigated whether Cten function could be mediated through Src (a known regulator of Snail).\n\nCten levels were modulated by forced expression in colorectal cancer (CRC) cell lines with low Cten expression (HCT116 and RKO) and gene knockdown in a cell line with high Cten expression (SW620). In all cell lines, Cten was a positive regulator of Src expression. The functional importance of Src was tested by forcibly expressing Cten and simultaneously knocking down Src. This resulted in abrogation of Cten motility-inducing activity (cell migration, cell invasion, wound healing - each p<0.001) and abrogation of the promotion of colony formation by Cten (p<0.001) together with failure to induce the Cten targets - Snail and ROCK1. To complement these experiments, Cten expression was restored by forced expression in a subclone of SW620 in which the Cten gene had been deleted (SW620{Delta}Cten). SW620{Delta}Cten showed reduced expression of Src which increased following restoration of Cten by forced expression. In SW620{Delta}Cten, restoration of Cten increased cell motility (cell migration, cell invasion, wound healing) and colony formation (each p<0.001) which were all lost if Src was concomitantly knocked down. Quantitative Reverse-Transcription PCR (qRT-PCR) showed that modulation of Cten had no effect on Src mRNA levels. However, a cycloheximide (CHX) pulse chase assay demonstrated stabilisation of Src protein by Cten. Finally, the expression of Cten and Src was tested in a series of 84 primary CRCs and there was significant correlation between Cten and Src expression (p=0.001).\n\nWe conclude that Src is a novel and functionally important target of the Cten signalling pathway and that Cten protein causes post-transcriptional stabilisation of Src protein in order to promote EMT and possibly metastasis in CRC.

cancer biology↗

bioRxiv2018

TGFβ1-induced cell motility is mediated through Cten in colorectal cancer

Cten is a tensin which promotes epithelial-mesenchymal transition (EMT) and cell motility. The precise mechanisms regulating Cten are unknown, although Cten could be regulated by several cytokines and growth factors. Since Transforming growth factor beta 1 (TGF-{beta}1) regulates integrin function and promotes EMT / cell motility, we investigated whether this happens through Cten signalling in colorectal cancer (CRC).\n\nTGF-{beta}1 signalling was modulated by either stimulation or knockdown in the CRC cell lines SW620 and HCT116. The effect of this modulation on expression of Cten, EMT markers and cellular function was tested. Cten role as a direct mediator of TGF-{beta}1 signalling was investigated in a CRC cell line with a deleted Cten gene (SW620{Delta}Cten).\n\nWhen TGF-{beta}1 was stimulated or inhibited, this resulted in, respectively, upregulation and downregulation of Cten expression and EMT markers. Cell migration and invasion were significantly increased following TGF-{beta}1 stimulation and lost by TGF-{beta}1 knockdown. TGF-{beta}1 stimulation in SW620{Delta}Cten resulted in selective loss of the effect of TGF-{beta}1 signalling on EMT and cell motility whilst the stimulatory effect on cell proliferation was retained.\n\nThese data suggested Cten may play an essential role in mediating TGF-{beta}1-induced EMT and cell motility and may play a role in metastasis in CRC.

cancer biology↗