It has been proposed that epithelial cells can acquire invasive properties through exposure to paracrine signals originated from mesenchymal cells within the tumor microenvironment. Transforming growth factor-? (TGF-?) has been revealed as an active factor that mediates the epithelial-stroma cross-talk that facilitates cell invasion and metastasis. TGF-? signaling is modulated by the coreceptor Endoglin (Eng), which shows a tumor suppressor activity in epithelial cells and regulates the ALK1-Smad1,5,8 as well as the ALK5-Smad2,3 signaling pathways. In the current work, we present evidence showing that cell surface Eng abundance in epithelial MCF-7 breast cancer cells is inversely related with cell motility. Shedding of Eng in MCF-7 cell surface by soluble matrix metalloproteinase-14 (MMP-14) derived from the HS-5 bone-marrow-derived cell line induces a motile epithelial phenotype. On the other hand, restoration of full-length Eng expression blocks the stromal stimulus on migration. Processing of surface Eng by stromal factors was demonstrated by biotin-neutravidin labeling of cell surface proteins and this processing generated a shift in TGF-? signaling through the activation of Smad2,3 pathway. Stromal MMP-14 abundance was stimulated by TGF-? secreted by MCF-7 cells acting in a paracrine manner. In turn, the stromal proteolytic activity of soluble MMP-14, by inducing Eng shedding, promoted malignant progression. From these data, and due to the capacity of TGF-? to regulate malignancy in epithelial cancer, we propose that stromal-dependent epithelial Eng shedding constitutes a putative mechanism that exerts an environmental control of cell malignancy.
Podoplanin (PDPN) is a mucin-like transmembrane glycoprotein that plays an important role in development and cancer. Here, we provide evidence that the intracellular domain (ICD) of podoplanin is released into the cytosol following a sequential proteolytic processing by a metalloprotease and ?-secretase. Western blotting and cell fractionation studies revealed that HEK293T and MDCK cells transfected with an eGFP-tagged podoplanin construct (PDPNeGFP, 50-63kDa) constitutively express two C-terminal fragments (CTFs): a ?33kDa membrane-bound PCTF33, and a ?29kDa cytosolic podoplanin ICD (PICD). While pharmacological inhibition of metalloproteases reduced the expression of PCTF33, treatment of cells with ?-secretase inhibitors resulted in enhanced PCTF33 levels. PCTF33 processing by ?-secretase depends on presenilin-1 (PS1) function: cells expressing a dominant negative form of PS1 (PS1 D385N), and mouse embryonic fibroblasts (MEFs) genetically deficient in PS1, but not in PS2, show higher levels of PCTF33 expression with respect to wild-type MEFs. Furthermore, transfection of PS1 deficient MEFs with wild-type PS1 (PS1 wt) decreased PCTF33 levels. N-terminal amino acid sequencing of the affinity purified PICD revealed that the ?-secretase cleavage site was located between valines 150 and 151, but these residues are not critical for proteolysis. We found that podoplanin CTFs are also generated in cells expressing podoplanin mutants harboring heterologous transmembrane regions. Taken together, these results indicate that podoplanin is a novel substrate for PS1/?-secretase.
Mesenchymal stem cells (MSCs) have been promoted as an attractive option to use as cellular delivery vehicles to carry anti-tumor agents, owing to their ability to home into tumor sites and secrete cytokines. Multiple isolated populations have been described as MSCs, but despite extensive in vitro characterization, little is known about their in vivo behavior.The aim of this study was to investigate the efficacy and efficiency of different MSC lineages derived from five different sources (bone marrow, adipose tissue, epithelial endometrium, stroma endometrium, and amniotic membrane), in order to assess their adequacy for cell-based anti-tumor therapies. Our study shows the crucial importance of understanding the interaction between MSCs and tumor cells, and provides both information and a methodological approach, which could be used to develop safer and more accurate targeted therapeutic applications.
Endoglin (Eng) is a transmembrane glycoprotein that is mainly expressed in endothelial cells, but it is also present in the epidermis and skin appendages. To address the role of Eng in cutaneous wound healing, we compared the kinetics of reepithelialization in Eng heterozygous null (Eng(+/-)) mice and their normal littermates (Eng(+/+)) following skin wounds. The wound area was significantly larger in Eng(+/-) than in Eng(+/+) mice from 2 to 8 days after injury; overall wound closure was delayed by 1 to 2 days. In Eng(+/-) mice, keratinocytes at the wound edges exhibited impaired proliferation but were more migratory, as shown by their elongated morphology and increased keratin 17 expression. Inhibition of nitric oxide (NO) synthesis delayed healing in Eng(+/+) but not in Eng(+/-) mice. Administration of the NO donor LA-803 accelerated wound closure in Eng(+/-) mice, with no effect on normal littermates. The acute stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced Eng expression in mouse epidermal keratinocytes in vivo and in vitro associated with hyperproliferation. Similarly, the skin of Eng(+/-) mice failed to mount a hyperplastic response to acute stimulation with TPA. These results demonstrate an important involvement of Eng in wound healing that is associated with NO bioavailability.
Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression.
The TGF-? (transforming growth factor-?) system signals via protein kinase receptors and Smad mediators to regulate a plethora of biological processes, including morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing and inflammation. In addition, alterations of specific components of the TGF-? signalling pathway may contribute to a broad range of pathologies such as cancer, cardiovascular pathology, fibrosis and congenital diseases. The knowledge about the mechanisms involved in TGF-? signal transduction has allowed a better understanding of the disease pathogenicity as well as the identification of several molecular targets with great potential in therapeutic interventions.
Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.
One of the key problems in cancer gene therapy is the inefficient delivery of therapeutic transgenes to tumour sites, after the systemic injection of the viral vector. Hence, new vector discovery is extremely important for the improvement of gene therapy results. Previously, mammalian cells were proposed as new vector systems; however with recent advances in stem cell research this modality makes them more suitable candidates. Tumours are composed of both malignant and benign cells. As "benign" cell types are able to form blood vessels, and stroma, it has been hypothesised that exogenously administrated cells of a different kind would preferentially engraft at the stromal tumour site and could deliver cancer gene therapy vectors to tumours.
Endoglin (CD105) is an auxiliary membrane receptor of transforming growth factor beta (TGF-?) that interacts with type I and type II TGF-? receptors and modulates TGF-? signaling. Endoglin is overexpressed in the tumor-associated vascular endothelium, where it modulates angiogenesis. This feature makes endoglin a promising target for antiangiogenic cancer therapy. In addition, recent studies on human and experimental models of carcinogenesis point to an important tumor cell-autonomous role of endoglin by regulating proliferation, migration, invasion, and metastasis. These studies suggest that endoglin behaves as a suppressor of malignancy in experimental and human epithelial carcinogenesis, although it can also promote metastasis in other types of cancer. In this review, we evaluate the implication of endoglin in tumor development underlying studies developed in our laboratories in recent years.
Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin-CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.
Endoglin is a coreceptor for transforming growth factor-? (TGF-?) that acts as a suppressor of malignancy during mouse skin carcinogenesis. Because in this model system H-Ras activation drives tumor initiation and progression, we have assessed the effects of endoglin on the expression of H-Ras in transformed keratinocytes. We found that TGF-?1 increases the expression of H-Ras at both messenger RNA and protein levels. The TGF-?1-induced H-Ras promoter transactivation was Smad4 independent but mediated by the activation of the TGF-? type I receptor ALK5 and the Ras-mitogen-activated protein kinase (MAPK) pathway. Endoglin attenuated stimulation by TGF-?1 of both MAPK signaling activity and H-Ras gene expression. Moreover, endoglin inhibited the Ras/MAPK pathway in transformed epidermal cells containing an H-Ras oncogene, as evidenced by the levels of Ras-guanosine triphosphate, phospho-MAPK kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK) as well as the expression of c-fos, a MAPK downstream target gene. Interestingly, in spindle carcinoma cells, that have a hyperactivated Ras/MAPK pathway, endoglin inhibited ERK phosphorylation without affecting MEK or Ras activity. The mechanism for this effect is unknown but strongly depends on the endoglin extracellular domain. Because the MAPK pathway is a downstream mediator of the transforming potential of Ras, the effect of endoglin on the oncogenic function of H-Ras was assessed. Endoglin inhibited the transforming capacity of H-Ras(Q61K) and H-Ras(G12V) oncogenes in a NIH3T3 focus formation assay. The ability to interfere with the expression and oncogenic potential of H-Ras provides a new face of the suppressor role exhibited by endoglin in H-Ras-driven carcinogenesis.
Transforming growth factor-beta1 (TGF-beta1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-beta1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-beta1, while N17TRac1 was inhibitory. TGF-beta1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-beta1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-beta1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.
The purpose of this study was to test the hypothesis that clinically significant depression detected in a population sample increases the risk of diabetes mellitus. The authors examined the effect of characteristics of depression frequently found in the community on the risk of incident diabetes mellitus.
The Na/I symporter (hNIS) promotes concentration of iodine in cells. In cancer gene therapy, this transgene has potential as a reporter gene for molecular imaging of viral biodistribution and as a therapeutic protein promoting (131)I-mediated radiotherapy. Here, we combined the imaging and therapeutic potential of hNIS in an oncolytic adenoviruses targeting colorectal cancer cells.
The transforming growth factor beta (TGF-beta) signaling pathway plays a key role in different physiological processes such as development, cellular proliferation, extracellular matrix synthesis, angiogenesis or immune responses and its deregulation may result in tumor development. The TGF-beta coreceptors endoglin and betaglycan are emerging as modulators of the TGF-beta response with important roles in cancer. Endoglin is highly expressed in the tumor-associated vascular endothelium with prognostic significance in selected neoplasias and with potential to be a prime vascular target for antiangiogenic cancer therapy. On the other hand, the expression of endoglin and betaglycan in tumor cells themselves appears to play an important role in the progression of cancer, influencing cell proliferation, motility, invasiveness and tumorigenicity. In addition, experiments in vitro and in vivo in which endoglin or betaglycan expression is modulated have provided evidence that they act as tumor suppressors. The purpose of this review was to highlight the potential of membrane and soluble forms of the endoglin and betaglycan proteins as molecular targets in cancer diagnosis and therapy.
Podoplanin/PA2.26 antigen is a small transmembrane mucin expressed in different types of cancer where it is associated with increased cell migration, invasiveness and metastasis. Little is known about the mechanisms that control podoplanin expression. Here, we show that podoplanin synthesis can be controlled at different levels. We analyzed podoplanin expression in a wide panel of tumour cell lines. The podoplanin gene (PDPN) is transcribed in cells derived from sarcomas, embryonal carcinomas, squamous cell carcinomas and endometrial tumours, while cell lines derived from colon, pancreatic, ovarian and ductal breast carcinomas do not express PDPN transcripts. PDPN is expressed as two mRNAs of approximately 2.7 and approximately 0.9 kb, both of which contain the coding sequence and arise by alternative polyadenylation. Strikingly, in most of the cell lines where PDPN transcripts were found, no podoplanin or only very low levels of the protein could be detected in Western blot. Treatment of several of these cell lines with the calpain inhibitor calpeptin resulted in podoplanin accumulation, whereas lactacystin, a specific inhibitor of the proteasome, had no effect. In vitro experiments showed that podoplanin is a substrate of calpain-1. These results indicate that at least in some tumour cells absence or reduced podoplanin protein levels are due to post-translational calpain-mediated proteolysis. We also report in this article the identification of a novel podoplanin isoform that originates by alternative splicing and differs from the standard form in lacking two cytoplasmic residues (YS). YS dipeptide is highly conserved across species, suggesting that it might be functionally relevant.
To measure the prevalence of depressive symptoms and its association with a comprehensive set of variables and to study the potential modifying effects of sex and age.
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