TGFβ1-induced cell motility but not cell proliferation is mediated through Cten in colorectal cancer

Cten (C-terminal tensin-like) is a member of the tensin protein family found in complex with integrins at focal adhesions. It promotes epithelial‐mesenchymal transition (EMT) and cell motility. The precise mechanisms regulating Cten are unknown, although we and others have shown that Cten could be under the regulation of several cytokines and growth factors. Since Transforming growth factor beta 1 (TGF-β1) regulates integrin function and promotes EMT / cell motility, we were prompted to investigate whether TGF-β1 induces EMT and cell motility through Cten signalling in colorectal cancer. TGF-β1 signalling was modulated by either stimulation with TGF-β1 or knockdown of TGF-β1 in the CRC cell lines SW620 and HCT116. The effect of this modulation on expression of Cten, EMT markers and on cellular function was tested. The role of Cten as a direct mediator of TGF-β1 signalling was investigated in a CRC cell line in which the Cten gene had been deleted (SW620ΔCten). When TGF-β1 was stimulated or inhibited, this resulted in, respectively, upregulation and downregulation of Cten expression and EMT markers (Snail, Rock, N-Cadherin, Src). Cell migration and cell invasion were significantly increased following TGF-β1 stimulation and lost by TGF-β1 knockdown. TGF-β1 stimulation of the SW620ΔCten cell line resulted in selective loss of the effect of TGF-β1 signalling pathway on EMT and cell motility whilst the stimulatory effect on cell proliferation was retained. These data suggested Cten may play an essential role in mediating TGF-β1 induced EMT and cell motility and may therefore play a role in metastasis in CRC

Role and regulation of CTEN in colorectal cancer

C-terminal tensin-like (CTEN), also called Tensin4 (TNS4), is the fourth member of the tensin family. The function of CTEN is tissue dependent; it acts as an oncogene in numerous cancers, including colorectal cancer (CRC) but appears to be a tumour suppressor in prostate cancer. While it is barely detectable in normal colorectal tissues, CTEN is upregulated in early-stage colorectal cancer (CRC). CTEN induces invasion and migration in CRC cell lines; however, the mechanisms by which CTEN exerts these effects are poorly understood. This thesis investigated (i) the regulation of CTEN and (ii) the biological function of specific protein domains in CTEN using CRC cell lines and induced pluripotent stem cells (iPSCs). CTEN has been reported to interact with β-catenin in CRC cell lines and form a complex in the nucleus. The oncogenic activity of CTEN and β-catenin, independently, in CRC is well established. However, the biological function of the CTEN/β- interaction is unknown. Firstly, CTEN deletion mapping was used to identify the molecular sites responsible for the CTEN/β- catenin interaction. Co-immunoprecipitation (Co-IP) assays revealed that the N-terminus of CTEN binds β-catenin. Moreover, functional assays demonstrated that the CTEN/β- catenin interaction was not required for CTEN-induced migration and invasion in CRC cells. Upregulation of CTEN and Wnt pathway activation are early events in CRC and induce cancer stemness. β-catenin interacts with CTEN and is a downstream target of the Wnt pathway. To study the involvement of CTEN in the Wnt pathway, the levels of CTEN and β-catenin were modulated in CRC cells and in normal fibroblasts. Western blotting for CTEN and active β- catenin showed that β-catenin positively regulates CTEN expression in both CRC cells and normal fibroblasts. Wnt pathway activation drives differentiation of iPSCs into the endoderm that forms colonic tissue and other digestive organs. CTEN is expressed in iPSCs, but CTEN expression and regulation had not been investigated in iPSCs. To explore CTEN expression during Wnt-induced iPSC differentiation, iPSCs were treated with Wnt activators or a Wnt inhibitor. Analysis of CTEN expression showed CTEN is downregulated during Wnt-induced differentiation of iPSCs into endoderm. Thus the relationship between Wnt signalling and CTEN is different in pluripotent cells to that in mature tissue. CTEN is a common feature in the signalling pathway of numerous growth factors and the oncogenic functions of several growth factors may be dependent on CTEN activty. CTEN induces invasion, migration and the EMT in CRC; however, these oncogenic mechanisms are poorly understood. CTEN contains a SH2 domain and can translocate to the nucleus in CRC cells. SH2 domains are important for localisation to focal adhesions in the cytoplasm. To investigate how CTEN functions as an oncogene in CRC, CTEN deletion mutant constructs lacking the SH2 domain and a predicted nuclear localisation signal were created. The ability of CTEN to induce migration and invasion in CRC cells was dependent on the SH2 domain and independent of nuclear localisation of CTEN. Moreover, preliminary results indicated that CTEN may play a role in the hypoxia-induced epithelial- mesenchymal transition. In summary, this thesis highlights number of novel findings regarding the regulation and function of CTEN as an oncogene in CRC and revealed the pattern of CTEN expression during Wnt- induced differentiation of iPSCs into endoderm. This work provides a basis for further investigation of the regulation and biological function of CTEN in CRC, iPSCs and other models

Cten promotes Epithelial–Mesenchymal Transition (EMT) in colorectal cancer through stabilisation of Src

Cten is an oncogene promoting EMT in many signaling pathways, namely through Snail. We investigated whether Cten function could be mediated through Src. Cten levels were modulated by forced expression in HCT116 and gene knockdown in SW620 CRC (colorectal cancer) cell lines. In all cell lines, Cten was a positive regulator of Src expression. The functional importance of Src was tested by simultaneous Cten overexpression and Src knockdown. This resulted in abrogation of Cten motility-inducing activity and reduction of colony formation ability together with failure to induce Cten targets. In SW620ΔCten reduced Src expression increased following restoration of Cten, also leading to increased cell motility and colony formation, which were lost if Src was concomitantly knocked down. By qRT-PCR we showed modulation of Cten had no effect on Src mRNA. However, a CHX pulse chase assay demonstrated stabilization of Src protein by Cten. Finally, expression of Cten and Src was tested in a series of 84 primary CRCs and there was a significant correlation between them (P = 0.001). We conclude that Src is a novel and functionally important target of the Cten signaling pathway and that Cten protein causes post-transcriptional stabilization of Src in promoting EMT and possibly metastasis in CRC

Tensin4 (TNS4) is upregulated by Wnt signalling in adenomas in multiple intestinal neoplasia (Min) mice

ApcMin/+ mice are regarded as a standard animal model of colorectal cancer (CRC). Tensin4 (TNS4 or Cten) is a putative oncogene conferring features of stemness and promoting motility. Our objective was to assess TNS4 expression in intestinal adenomas and determine whether TNS4 is upregulated by Wnt signalling. ApcMin/+ mice (n=11) were sacrificed at approximately 120days old at the onset of anaemia signs. Small intestines were harvested, and Swiss roll preparations were tested for TNS4 expression by immunohistochemistry (IHC). Individual polyps were also separately collected (n=14) and tested for TNS4 mRNA expression and Kras mutation. The relationship between Wnt signalling and TNS4 expression was tested by Western blotting in the human CRC cell line HCT116 after inhibition of β-catenin activity with MSAB or its increase by transfection with a Flag β-catenin expression vector. Overall, 135/148 (91.2%) of the total intestinal polyps were positive for TNS4 expression by IHC, whilst adjacent normal areas were negative. RT-qPCR analysis showed approximately 5-fold upregulation of TNS4 mRNA in the polyps compared to adjacent normal tissue and no Kras mutations were detected. In HCT116, β-catenin inhibition resulted in reduced TNS4 expression, and conversely, β-catenin overexpression resulted in increased TNS4 expression. In conclusion, this is the first report linking aberrant Wnt signalling to upregulation of TNS4 both during initiation of intestinal adenomas in mice and in in vitro models. The exact contribution of TNS4 to adenoma development remains to be investigated, but the ApcMin/+ mouse represents a good model to study this

Evaluation of inhaled nitric oxide (iNO) treatment for moderate-to-severe ARDS in critically ill patients with COVID-19: A multicenter cohort study

Background: Inhaled nitric oxide (iNO) is used as rescue therapy in patients with refractory hypoxemia due to severe COVID-19 acute respiratory distress syndrome (ARDS) despite the recommendation against the use of this treatment. To date, the effect of iNO on the clinical outcomes of critically ill COVID-19 patients with moderate-to-severe ARDS remains arguable. Therefore, this study aimed to evaluate the use of iNO in critically ill COVID-19 patients with moderate-to-severe ARDS. Methods: This multicenter, retrospective cohort study included critically ill adult patients with confirmed COVID-19 treated from March 01, 2020, until July 31, 2021. Eligible patients with moderate-to-severe ARDS were subsequently categorized into two groups based on inhaled nitric oxide (iNO) use throughout their ICU stay. The primary endpoint was the improvement in oxygenation parameters 24 h after iNO use. Other outcomes were considered secondary. Propensity score matching (1:2) was used based on the predefined criteria. Results: A total of 1598 patients were screened, and 815 were included based on the eligibility criteria. Among them, 210 patients were matched based on predefined criteria. Oxygenation parameters (PaO2, FiO2 requirement, P/F ratio, oxygenation index) were significantly improved 24 h after iNO administration within a median of six days of ICU admission. However, the risk of 30-day and in-hospital mortality were found to be similar between the two groups (HR: 1.18; 95% CI: 0.77, 1.82; p = 0.45 and HR: 1.40; 95% CI: 0.94, 2.11; p= 0.10, respectively). On the other hand, ventilator-free days (VFDs) were significantly fewer, and ICU and hospital LOS were significantly longer in the iNO group. In addition, patients who received iNO had higher odds of acute kidney injury (AKI) (OR (95% CI): 2.35 (1.30, 4.26), p value = 0.005) and hospital/ventilator-acquired pneumonia (OR (95% CI): 3.2 (1.76, 5.83), p value = 0.001). Conclusion: In critically ill COVID-19 patients with moderate-to-severe ARDS, iNO rescue therapy is associated with improved oxygenation parameters but no mortality benefits. Moreover, iNO use is associated with higher odds of AKI, pneumonia, longer LOS, and fewer VFDs

Role and regulation of CTEN in colorectal cancer

C-terminal tensin-like (CTEN), also called Tensin4 (TNS4), is the fourth member of the tensin family. The function of CTEN is tissue dependent; it acts as an oncogene in numerous cancers, including colorectal cancer (CRC) but appears to be a tumour suppressor in prostate cancer. While it is barely detectable in normal colorectal tissues, CTEN is upregulated in early-stage colorectal cancer (CRC). CTEN induces invasion and migration in CRC cell lines; however, the mechanisms by which CTEN exerts these effects are poorly understood. This thesis investigated (i) the regulation of CTEN and (ii) the biological function of specific protein domains in CTEN using CRC cell lines and induced pluripotent stem cells (iPSCs). CTEN has been reported to interact with β-catenin in CRC cell lines and form a complex in the nucleus. The oncogenic activity of CTEN and β-catenin, independently, in CRC is well established. However, the biological function of the CTEN/β- interaction is unknown. Firstly, CTEN deletion mapping was used to identify the molecular sites responsible for the CTEN/β- catenin interaction. Co-immunoprecipitation (Co-IP) assays revealed that the N-terminus of CTEN binds β-catenin. Moreover, functional assays demonstrated that the CTEN/β- catenin interaction was not required for CTEN-induced migration and invasion in CRC cells. Upregulation of CTEN and Wnt pathway activation are early events in CRC and induce cancer stemness. β-catenin interacts with CTEN and is a downstream target of the Wnt pathway. To study the involvement of CTEN in the Wnt pathway, the levels of CTEN and β-catenin were modulated in CRC cells and in normal fibroblasts. Western blotting for CTEN and active β- catenin showed that β-catenin positively regulates CTEN expression in both CRC cells and normal fibroblasts. Wnt pathway activation drives differentiation of iPSCs into the endoderm that forms colonic tissue and other digestive organs. CTEN is expressed in iPSCs, but CTEN expression and regulation had not been investigated in iPSCs. To explore CTEN expression during Wnt-induced iPSC differentiation, iPSCs were treated with Wnt activators or a Wnt inhibitor. Analysis of CTEN expression showed CTEN is downregulated during Wnt-induced differentiation of iPSCs into endoderm. Thus the relationship between Wnt signalling and CTEN is different in pluripotent cells to that in mature tissue. CTEN is a common feature in the signalling pathway of numerous growth factors and the oncogenic functions of several growth factors may be dependent on CTEN activty. CTEN induces invasion, migration and the EMT in CRC; however, these oncogenic mechanisms are poorly understood. CTEN contains a SH2 domain and can translocate to the nucleus in CRC cells. SH2 domains are important for localisation to focal adhesions in the cytoplasm. To investigate how CTEN functions as an oncogene in CRC, CTEN deletion mutant constructs lacking the SH2 domain and a predicted nuclear localisation signal were created. The ability of CTEN to induce migration and invasion in CRC cells was dependent on the SH2 domain and independent of nuclear localisation of CTEN. Moreover, preliminary results indicated that CTEN may play a role in the hypoxia-induced epithelial- mesenchymal transition. In summary, this thesis highlights number of novel findings regarding the regulation and function of CTEN as an oncogene in CRC and revealed the pattern of CTEN expression during Wnt- induced differentiation of iPSCs into endoderm. This work provides a basis for further investigation of the regulation and biological function of CTEN in CRC, iPSCs and other models

Alternatively Spliced Isoforms of <i>MUC4</i> and <i>ADAM12</i> as Biomarkers for Colorectal Cancer Metastasis

There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer

Core Analysis of Differentially Expressed Genes using IPA.

<p>Core analysis using IPA was carried out using set of 760 genes that were differentially expressed in tumour samples. Important biological functions (a) pathways (b) and networks (c-e) were revealed by this analysis.</p

Gene Regulatory Network Inference diagram for Tumor and Normal Groups.

<p>A hierarchical network topology is used to visualize the degrees of interaction between transcription factor genes and target genes. (a) The inferred network for tumour group showing RUVBL1 as master regulator. (b) The inferred network for normal group showing TSHZ1as master regulator.</p