One factor that is produced abundantly by stromal cells in the tumor microenvironment is transforming growth factor β (TGFβ). TGFβ is a multifunctional growth factor with a complicated dual role in tumorigenesis (David Padua et al., 2008). TGFβ is induced in response to hypoxia and inflammation and can have a protective effect on tumor cells. However, TGFβ has also been observed to drive an epithelial-to-mesenchymaltransition in cancer cells, which increases their metastatic capability (Welm, 2008). Since its discovery in the early 1980s, TGF signaling has been increasingly recognized as a key driver in cancer (Giannelli, Villa, & Lahn, 2014). Unlike its tumor suppressor function in normal tissue, TGF activation causes tumor promotion in cancer tissue. This switch from tumor suppression to promotion is not well clarified, but several intrinsic and extrinsic factors seem to play important roles (N. Sun, Taguchi, & Hanash, 2016). The loss of cell polarity, acquisition of motile properties, and a mesenchymal phenotype during epithelial-mesenchymaltransition (EMT) are considered crucial intrinsic changes of the tumor cells (Chaffer, San Juan, Lim, & Weinberg, 2016; Wu & Zhou, 2009).
Breast cancer is the leading cause for cancer deaths among females worldwide and its incidence is growing. The high rate of relapse and metastatic spread is attributed to breast cancer stem cells (CSCs) (1). Of note, CSC formation is closely linked to epithelial- mesenchymaltransition (EMT), a process which confers mesenchymal properties on epithelial cells (2). Hence, the pharmacological targeting of EMT may represent a new strategy to overcome tumor progression. Recent reports showed that targeting the endolysosomal machinery is a promising approach to overcome EMT (3). To this end, the vacuolar H + -ATPase (V-ATPase) represents an attractive target. V-ATPases are ubiquitous expressed, multimeric ATP-dependent proton pumps, essential for ligand internalization, endosomal recycling and lysosomal degradation (4). Although V-ATPase has been linked with the recycling of E-cadherin, a crucial player in EMT, detailed information about a potential role of V-ATPase in EMT and CSCs is still missing.
precedes chronic inflammation and eventually leads to conjunctival fibrosis ( Whittum-Hudson et al., 1986; Hu et al., 2013 ). The resulting scarring is the key element in the development of the blinding sequelae leading to inversion of the eyelids and lashes (trichiasis) that wound the ocular surface ( Mohammadpour et al., 2016 ). Chronic keratoconjunctivitis and/or prolonged trichiasis cause corneal opacification and blindness ( Mabey et al., 2003; Brunham and Rey-Ladino, 2005; Wolle et al., 2009 ). Like in any fibrotic process, it is believed that the scar tissue in trachoma originates from activated fibroblasts ( Kechagia et al., 2016 ), and there is supporting evidence that innate and adaptive immunity involved in the resolution of Ct infection may initiate the fibrotic process ( Brunham and Rey-Ladino, 2005 ). Thus, to date, research has been focused on elucidating the fibroblast-stimulating profibrotic mediators. Only recently, an alternative pathway, the process of epithelial- mesenchymaltransition (EMT), was proposed to be involved in the development of end-stage trachoma ( Derrick et al., 2015 ).
hot spot areas with a high tumor density and subjected to Free-D Software 1.09. 18 Boundaries of all tumor cell clus-
ters were followed from every digitalized picture and Our data demonstrate that connected and isolated tumor buds are equal concerning immunohistochemical epithelial– mesenchymaltransition characteristics and histomorphology. Our data also give an insight in the process of tumor budding. While there is a notion that the epithelial–mesenchymaltransition zinc finger E-box-binding homeobox 1–E-Cadherin cascade is initiated by zinc finger E-box-binding homeobox 1, our results are contrary and outline other possible pathways influencing the regulation of E-Cadherin.
Cell-cell adhesive interactions can also influence organotropism during metastasis formation (Chen et al., 2018). During the EMT procedure, cancer cells will frequently decrease the expression of E- cadherin, and in parallel enhance N-cadherin expression (Gloushankova et al., 2017), which is referred to as the “cadherin switch”. E-cadherin plays a key role in tight cell-cell junctions in epithelial cells, while N-cadherin directly mediates homotypic and heterotypic cell-cell adhesion, and is predominantly expressed in neural, endothelial, and stromal cells (Chen et al., 2018; Gloushankova et al., 2017; Mrozik et al., 2018). Recent studies demonstrate that tumor cells can acquire partial or hybrid EMT phenotypes, which means epithelial and mesenchymal markers, including different cadherins, can co- exist in individual cells (Jordan et al., 2011; Pastushenko and Blanpain, 2019; Tan et al., 2014). CTCs/DTCs are considered as the source of metastatic seeds. Theoretically, CTCs/DTCs with a hybrid EMT phenotype may adapt more easily to the distant microenvironment by, for example, transiently changing their expression of cadherins (E- or N- cadherin). Such changes could allow them to tighten or release contacts to cells of various microenvironments, and thus to better cope with changing conditions along the metastatic cascade. In the last decade, many efforts have been made to capture and characterize CTCs and, to lesser extent, DTCs of various tumor types. However, the biological characterization and capture of CTCs and DTCs are still technically challenging (Gabriel et al., 2016). In most CTC isolation techniques, CTCs are captured through the enrichment of rare epithelial cells from blood, by using epithelial markers, e.g. epithelial cell adhesion molecule (EpCAM). Despite being of great value for further prognosis of patients, these techniques might preferentially enrich epithelial cells, thus introducing a bias in the actual nature of CTCs that are enriched. Therefore, EpCAM-dependent CTC enrichment technologies do not allow to study the full spectrum of systemic tumor cells. Hence, the actual contribution of EMT to metastasis formation remains only partially addressed.
Here we used the CRISPR/Cas9 system to knock Tks4 out and investigate the morphologic, phenotypic and transcriptional changes which developed in the lack of the protein. Our results showed that loss of Tks4 initiated EMT or EMT-like phenotypic and molecular changes in this human colorectal carcinoma cell line. The mechanism by which the loss of Tks4 modulates this EMT-like process in tumor cells remains obscure. One possible explanation is that Tks4 is involved in the regulation of cancer cell motility, as indicated by its roles in EGFR signaling [ 24 , 60 ] an EMT-inducing molecular pathway. It has been already shown that the induction of the EGF/EGFR axis can initiate EMT in cholangiocarcinoma (CCA) cell lines and in the human embryonal rhabdomyosarcoma cell line [ 61 ]. In these experiments, increases in the Snail2/Slug and ZEB1 levels were observed upon EGF stimulation. Another group found that EGF upregulates Snail1/2 and ZEB1 expression but does not affect Twist in serous borderline ovarian tumor cells (SBOT) [ 62 ]. Both of these publications also mentioned that EGFR induction decreased E-cadherin levels. We assume that the lack of Tks4 likely alters EGFR signaling in these cells, perhaps resulting in a shift toward a mesenchymal-like motile phenotype. Moreover, due to the loss of Tks4 having similar effects on cancer cells as EGF treatment (EMT induction, reduced E-cadherin expression), it is possible that Tks4 plays a putative negative regulatory role in the EGFR pathway by delaying signal transduction or changing its kinetics.
Downregulation of E-cadherin is a fundamental event in EMT. This calcium-dependent transmembrane glycoprotein is expressed by most epithelial cells and is responsible for the tight junctions connecting adjacent cells (Nieto et al. 2016). EMT-associated transcription factors (ATF) can repress E-cadherin expression by binding and regulating the activity of its promotor, such is the case for Snail, KLF8 and the ZEB family of transcription factors. Others, such as Twist and FoxC2 can indirectly repress E-cadherin transcription. EMT ATFs not only regulate E-cadherin expression but can also initiate the complete EMT program inhibiting and activating several epithelial and mesenchymal genes. Both ZEB and Snail have been associated with activating invasion and metastasis, resisting cell death and enabling replicative immortality, whereas Twist has been linked with induction of angiogenesis, evasion of growth suppressors, and the sustainment of proliferative signaling. Taking together the role of the EMT ATFs, their function has evolved from simple E-cadherin repressors to inducers of most of the traits necessary for tumor development and metastasis (Sanchez-Tillo et al. 2012).
Thomas Seufferlein 1 , Martin Wagner 1,w & Alexander Kleger 1,w
Pancreatic ductal adenocarcinoma (PDAC) is associated with accumulation of particular oncogenic mutations and recent genetic sequencing studies have identiﬁed ataxia telangiectasia-mutated (ATM) mutations in PDAC cohorts. Here we report that conditional deletion of ATM in a mouse model of PDAC induces a greater number of proliferative precursor lesions coupled with a pronounced ﬁbrotic reaction. ATM-targeted mice display altered TGFb-superfamily signalling and enhanced epithelial-to-mesenchymaltransition (EMT) coupled with shortened survival. Notably, our mouse model recapitulates many features of more aggressive human PDAC subtypes. Particularly, we report that low expression of ATM predicts EMT, a gene signature speciﬁc for Bmp4 signalling and poor prognosis in human PDAC. Our data suggest an intimate link between ATM expression and pancreatic cancer progression in mice and men.
heterogeneous phenotypes is one characteristic of CSCs that may help to explain some of the differences which discriminate tumor cells from differentiated somatic cells like immortality, quiescence, invasion, metastasis, and relapse after treatment. Initial studies identified CD44 + HNSCC cells that could generate new tumors in vivo (<5,000 cells injected into mice) 15 , and concluded that CD44 is a CSC marker. Recently, ALDH was demonstrated to be another useful CSC marker to identify CSCs in many epithelial cancers including HNSCC 32,33,41 . In HNSCC, Chen et al. showed in immunocompromised mice that 500 injected ALDH + HNSCC cells resulted in visible tumors in all cases after 6 weeks, while 10 4 ALDH − cells failed to produce tumors 41 . According to our own results and in line with the observations mentioned previously, ALDH + cells showed increased CSCs properties compared to ALDH - cells derived from HNSCC cell lines 26 . We and others could demonstrate that in HNSCC there is a varying overlap of
(Received 5 February 2018; accepted 30 July 2018; published online 22 August 2018)
Aberrant activation of epithelial-mesenchymaltransition (EMT) in carcinoma cells contributes to increased migration and invasion, metastasis, drug resistance, and tumor-initiating capacity. EMT is not always a binary process; rather, cells may exhibit a hybrid epithelial/mesenchymal (E/M) phenotype. ZEB1—a key transcrip- tion factor driving EMT—can both induce and maintain a mesenchymal phenotype. Recent studies have identified two novel autocrine feedback loops utilizing epithe- lial splicing regulatory protein 1 (ESRP1), hyaluronic acid synthase 2 (HAS2), and CD44 which maintain high levels of ZEB1. However, how the crosstalk between these feedback loops alters the dynamics of epithelial-hybrid-mesenchymal transi- tion remains elusive. Here, using an integrated theoretical-experimental framework, we identify that these feedback loops can enable cells to stably maintain a hybrid E/M phenotype. Moreover, computational analysis identifies the regulation of ESRP1 as a crucial node, a prediction that is validated by experiments showing that knockdown of ESRP1 in stable hybrid E/M H1975 cells drives EMT. Finally, in multiple breast cancer datasets, high levels of ESRP1, ESRP1/HAS2, and ESRP1/ ZEB1 correlate with poor prognosis, supporting the relevance of ZEB1/ESRP1 and ZEB1/HAS2 axes in tumor progression. Together, our results unravel how these inter- connected feedback loops act in concert to regulate ZEB1 levels and to drive the dynamics of epithelial-hybrid-mesenchymaltransition. V C 2018 Author(s). All article
Immature truncated O-GalNAc glycosylation is an important feature of the pancreatic ductal adenocarcinomas (PDAC). The expression of truncated O-GalNAc glycans is strongly associated with decreased survival rates and poor prognosis. Aberrant O-GalNAc glycosylation is assumed to influence PDAC signaling and therefore likely to promote oncogenic properties. To address this assumption the ability of aberrant O-GalNAc glycosylation to exercise a triggering and protumorigenic influence over two important PDAC signaling pathways, PI3K/AKT/mTOR and Ras/Raf/MAPK, has been investigated in this study. Different expression levels of proteins associated with PI3K/AKT/mTOR and Ras/Raf/MAPK pathways could be identified in aberrant O-glycosylated PDAC cells by using quantitative proteomics. The proteins that were identified also included the protein kinase AKT, a major key signaling molecule in PDAC. Further investigation of AKT led to the detection of an enhanced S473 phosphorylation and a directly aberrant O-GalNAc-glycosylation. Aberrant O- glycosylated PDAC cells also revealed strong changes in the activity of other signaling molecules belonging to the PI3K/AKT/mTOR signaling pathway. For instance, aberrant O- GalNAc glycosylation was found to stabilize mTOR S2448 phosphorylation. Since numerous cross talks between the PI3K/AKT/mTOR and Ras/Raf/MAPK pathways are known to exist, they were included in the investigation. The analysis identified the Ras/Raf/MAPK pathway as predominantly negatively regulated. Finally, the research result revealed that an aberrant O- GalNAc-glycosylation leads to an enhanced expression of epithelial-mesenchymal-transition markers, known as key features of aggressive PDAC cells.
Epithelial – MesenchymalTransition is a process that is involved in developmental processes and thereby requires several transcriptional regulators. To address, if enhanced Emx2 expression in metastatic melanoma cell lines alters the expression levels of some chosen EMTRs, immunoblotting was performed. Emx2 overexpression does not change protein levels of Snail, Twist, ZEB1 and N-cadherin. Slug however is significantly decreased, revealing reduced protein expression in Emx2 transduced cells up to 50% (figure 10A and B). To determine the distribution of the proteins in both cytoplasm and nucleus, these fractions were analyzed by immunoblotting. Slug has clearly reduced protein levels in the cytoplasm (down to 20%), while the nuclear fraction shows no decrease. ZEB1, which has equal total protein levels, indicates a shift of the protein from the cytoplasm into the nucleus (figure 10C and D).
The epithelial-mesenchymaltransition (EMT), which is a key process during the metastatic cascade in malignant tumors, is operated by the transcription factors Twist, Snail, Slug, ZEB1 and ZEB2. So far the expression of these transcription factors has been only been studied individually. The aim of this study was to investigate and compare the expression of all five transcription factors collectively in a patient cohort of head and neck squamous cell carcinomas (HNSCC). Twist was expressed in the cancer cells in a major proportion of our patients with HNSCC. The immunoreactivity of Snail and Slug in squamous cell carcinoma cells was much less. The expression of ZEB1 and ZEB2 is negligible as they were generally expressed in the stromal cells only and not in the cancer cells themselves. Twist seems to be the most significant transcription factor regulating the EMT in HNSCC. Nonetheless the expression of Twist and Snail as well as Twist and Slug is highly associated and consequently there is a connection between these three transcription factors.
Damage of the endo-epithelial barrier is the major hallmark of acute lung injury upon bacterial infection, associated with oedema formation, alveolar flooding, impaired fluid clearance and gas exchange. Hence, to restore the normal lung function, alveolar repair processes are ultimately initiated (34). Resident alveolar macrophages have been assigned a contributing role in epithelial repair, closely associated with the transition of the pro- inflammatory into the anti-inflammatory macrophage phenotype (62, 94). In the current thesis the potential of early activated, pro-inflammatory resident alveolar macrophages to influence epithelial repair processes was investigated. Moreover, the hypothesis that pro-inflammatory resident alveolar macrophages may contribute to effective epithelial repair after LPS- and K. pneumoniae induced lung injury was tested. Hence, in vitro experiments revealed that alveolar epithelial cells co-cultured with LPS-stimulated resident alveolar macrophages express significantly higher amounts of growth factors, particularly of GM-CSF. Macrophage TNF-α released upon LPS stimulation was identified as a mediator inducing GM-CSF expression in epithelial cells, which in turn elicited autocrine proliferative signalling in type II alveolar epithelial cells. Genetic deletion of GM-CSF resulted in absence of macrophage-induced epithelial cell proliferation. Similarly, in vivo TNF-α neutralization after LPS-induced lung injury impaired epithelial proliferation. Furthermore, GM-CSF-deficient mice displayed reduced AEC II proliferation and sustained alveolar leakage after LPS challenge. Similarly, K. pneumoniae-induced lung injury was associated with early release of TNF-α and GM-CSF, and subsequent TNF-α-dependent AEC II proliferation during the alveolar repair phase. Altogether, these data reveal that alveolar repair processes are initiated early in the inflammatory course of pathogen-induced acute lung injury, and are mediated by macrophage TNF-α and epithelial GM-CSF (Fig. 31).
RESULTS Alterations in abundance and localization of adherens and tight junction proteins in dKO animals might influence and destabilize the maintenance of the epithelial barrier function and thereby the integrity of the entire lung epithelium as well. In order to examine potential epithelial barrier leakage in dKO compared to Ctrl lungs, intra peritoneal (i.p.) injections of 1% Evans Blue Dye (EBD) solution were conducted and leakage of the dye into lung cavity was measured for both groups. After two hours of incubation a lung lavage was performed and bronchio-alveo-lavage fluid (BAL) as well as blood serum were collected to assess the percentage of EBD BAL/Serum ratio by calculating OD 600 of BAL and blood serum. This ratio provides information about the amount of EBD, which passes the epithelial barrier to the lung lumen. The results clearly displayed a more than two-fold increase of EBD BAL/serum ratio for dKOs in comparison to Ctrls (Figure 10D). In fact, this strongly indicated a disruption of the lung epithelial barrier function, which strengthened the hypothesis of a compromised lung epithelial integrity in Numb/Numblike dKOs. The amount of EBD, which accumulated in lung interstitial spaces during incubation time, is displayed by the amount (ng) of extracted EBD per mg lung tissue (Figure 10D; second graph). These results indicated to what extent EBD was absorbed and retained by the interstitium and thereby served as an experimental control. After isolation of lung tissue and extraction of EBD absorbed by the pulmonary interstitium, the amount of EBD/mg lung tissue (ng EBD/mg lung tissue) was determined. Both groups did not reveal any significant differences in the amount of EBD that accumulated in lung interstitial spaces indicating that the absorption of the dye by the interstitium of dKO and Ctrl lungs was equal. Hence, the elevated EBD measured in the lung cavity (BAL) of dKO mice clearly resulted by an enhanced paracellular dye leakage via the epithelium, whereas the content of the dye that remained in lung interstitial spaces of lungs was similar for both groups. In summary, these results suggested that Numb and Numblike mediate lung epithelial cell polarity as well as the maintenance of cell-cell contacts. 3.2 Numb/Numblike in bleomycin induced fibrosis
Als Epithelial-Mesenchymale Transition (EMT) wird eine Vielzahl biochemischer Pro- zesse bezeichnet, die es der ortsständigen und polaren epithelialen Zelle ermöglichen, mesenchymale Eigenschaften und ein migratorisches Potential anzunehmen. Sie spielt eine wichtige Rolle in der Embryogenese und der Organentwicklung. Zunehmende Hinweise legen außerdem eine fundamentale Rolle der EMT in der Tumorbiologie na- he, vor Allem in der Formation von Metastasen, der Tumorprogression sowie der Resis- tenz gegen Chemotherapeutika. Hinweise auf einen Zusammenhang zwischen EMT und Tumorbiologie konnten bereits für verschiedene Tumoren, wie etwa Kolonkarzi- nome gezeigt werden. Um eine mögliche Rolle der EMT in der Biologie humaner Me- ningeome aufzuzeigen, wurde die Expression von E-Cadherin, einem Protein der Adherens Junctions, und E-Cadherin-assoziierter Transkriptionsfaktoren sowie dem Tight Junction assoziierten Protein Zonula-Occludens-1 unter unterschiedlichen expe- rimentellen Bedingungen untersucht. Ferner wurde die Möglichkeit, das EMT- assoziierte Expressionsprofil von Meningeomzellen durch den Wachstumsfaktor Trans- forming Growth Factor-ß (TGF-ß) zu beeinflussen, geprüft. Es konnte gezeigt werden, dass es signifikante Unterschiede zwischen Meningeomen unterschiedlicher WHO- Grade gibt, insbebesondere was die Expression des Glykoproteins E-Cadherin betrifft. Außerdem konnte gezeigt werden, dass sich die Expression der untersuchten EMT- Marker in Meningeomen mit malignen Charakteristika von der Vergleichsgruppe unter- scheidet und, dass diese veränderten Expressionsmuster in wesentlichen Teilen mit ei- ner EMT in Meningeomen vereinbar sind.
This thesis investigates the lamellipodial dynamics of moving cells on 2D surfaces. Cell migration is essential in various biological processes such as embryonic development, im- mune system function, wound healing, synaptic targeting in neural networks and cancer metastasis [ 8 – 10 ]. The movement of cells can be directed by external chemical gradients (chemotaxis) [ 11 ], when cells aim to reach to a specific target (e.g. in wound healing). However, even without external chemical gradients, cells are able to initiate movement in a random direction (e.g in cancer metastasis or even on a substrate in vitro). To initiate the movement, they polarize and form leading edges. On surfaces, cells form sheet-like projections of the cytoskeletal network called lamellipodia or finger-like cytoplasmic pro- jections with aligned filaments called filopodia [ 12 , 13 ]. Mesenchymal motility is defined as a type of cell movement in which cells form actin-rich protrusions such as lamellipodia or filopodia as well as strong adhesion with the substrate, which usually is followed by contractions at the rear of the cell [ 14 ]. Amoeboid motility is another type of motility that includes rapidly moving cells making stable blebs and actin-rich pseudopods (tem- porary cytoplasmic projections) with very low adhesion [ 14 – 16 ]. Cancer cells exhibit both mesenchymal and amoeboid movements. Here, we are interested in understand- ing lamellipodial dynamics in mesenchymal motility and in particular, we compare our findings to the dynamic behavior of B16-F1 melanoma cancer cells.
Today, OC survival has significantly improved and this improvement is in general attributed to optimal surgical treatment (cytoreduction) and to the use of more effective new chemotherapy drugs [51, 52, 53, 54, 55]. Both cytoreductive surgery and chemotherapy require good physiologic capacities and because elderly patients are more likely to have comorbid conditions, they are less likely to receive optimal surgery and chemotherapy . Two retrospective population-based studies have shown that older patients with advanced ovarian cancer were less apt than younger to receive chemotherapy, to be treated by oncology specialists and to undergo adequate primary cytoreductive surgery [92, 93]. Some studies have established a survival advantage for patients who underwent “optimal” vs “suboptimal” primary surgical cytoreduction [94, 95, 96] and recent studies have demonstrated that the intraoperative tumor dissemination pattern and the post-operative residual tumor, therefore, primary radical surgery are decisive for prognosis in epithelial ovarian cancer [91, 97, 98]. Consequently, we can ask if the poorer outcome of the elderly patients is simply due to a less aggressive medical management than what is received by their younger counterparts. The reported prognostic significance of age in woman cancers has been inconsistent, table 5.9.