Papers by Athina-Myrto Chioni
Proceedings of The Physiological Society, 2005
Pancreatology, 2013
Pancreatic cancer is characterised by desmoplasia, driven by activated pancreatic stellate cells ... more Pancreatic cancer is characterised by desmoplasia, driven by activated pancreatic stellate cells (PSCs). Over-expression of FGFs and their receptors is a feature of pancreatic cancer and correlates with poor prognosis, but whether their expression impacts on PSCs is unclear. At the invasive front of human pancreatic cancer, FGF2 and FGFR1 localise to the nucleus in activated PSCs but not cancer cells. In vitro, inhibiting FGFR1 and FGF2 in PSCs, using RNAi or chemical inhibition, resulted in significantly reduced cell proliferation, which was not seen in cancer cells. In physiomimetic organotypic co-cultures, FGFR inhibition prevented PSC as well as cancer cell invasion. FGFR inhibition resulted in cytoplasmic localisation of FGFR1 and FGF2, in contrast to vehicle-treated conditions where PSCs with nuclear FGFR1 and FGF2 led cancer cells to invade the underlying extra-cellular matrix. Strikingly, abrogation of nuclear FGFR1 and FGF2 in PSCs abolished cancer cell invasion. These findings suggest a novel therapeutic approach, where preventing nuclear FGF/FGFR mediated proliferation and invasion in PSCs leads to disruption of the tumour microenvironment, preventing pancreatic cancer cell invasion.
Fibrogenesis & Tissue Repair, Dec 1, 2008
The advent of co-culture approaches has allowed researchers to more accurately model the behaviou... more The advent of co-culture approaches has allowed researchers to more accurately model the behaviour of epithelial cells in cell culture studies. The initial work on epidermal modelling allowed the development of reconstituted epidermis, growing keratinocytes on top of fibroblasts seeded in a collagen gel at an air-liquid interface to generate terminally differentiated 'skin equivalents'. In addition to developing ex vivo skin sheets for the treatment of burns victims, such cultures have also been used as a means of investigating both the development and repair of the epidermis, in more relevant conditions than simple two-dimensional culture, but without the use of animals. More recently, by varying the cell types used and adjusting the composition of the matrix components, this physiological system can be adapted to allow the study of interactions between tumour cells and their surrounding stroma, particularly with regards to how such interactions regulate invasion. Here we provide a summary of the major themes involved in tumour progression and consider the evolution of the approaches used to study cancer cell behaviour. Finally, we review how organotypic models have facilitated the study of several key pathways in cancer development and invasion, and speculate on the exciting future roles for these models in cancer research.
Springer eBooks, 2015
Fibroblast Growth Factors (FGFs), in a complex with their receptors (FGFRs) and heparan sulphate ... more Fibroblast Growth Factors (FGFs), in a complex with their receptors (FGFRs) and heparan sulphate (HS), impact on a wide range of cellular functions, regulating processes from embryogenesis to metabolism. Upon ligand binding and receptor dimerisation, four key downstream pathways are initiated: MAPK, PI3K/AKT, STAT and PLCγ. Regulation of FGF signalling is critical to ensure a balanced response to receptor stimulation. This occurs through negative feedback mechanisms, including internalisation, cleavage and induction of negative regulators. FGF signalling has been studied in depth by developmental biologists, in a variety of model systems, and plays a critical role in developmental patterning and the establishment of paracrine signalling loops. Both germ line and somatic FGFR mutations are known to play a role in a range of diseases, most notably developmentally regulated diseases such as craniosynostosis dysplasias, dwarfism and hearing loss. Because of the ability of FGFR signalling to induce cell proliferation, migration and survival, FGFRs are readily co-opted by cancer cells. Mutations in, and amplifications of, these receptors are found in a range of cancers. Here, we outline the molecular mechanisms of FGFR signalling and discuss the role of this pathway in development and disease. We also address the rationale for therapeutic intervention and the need for FGFR-targeted therapy to selectively target cancer cells in view of the fundamental roles of FGF signalling in normal physiology.
European Journal of Cell Biology, Jul 1, 2009
FGF-10 plays an important role in development and disease, acting as the key ligand for FGFR2B to... more FGF-10 plays an important role in development and disease, acting as the key ligand for FGFR2B to regulate cell proliferation, migration and differentiation. Aberrant FGF signalling is implicated in tumourigenesis, with several cancer studies reporting FGF-10 or FGFR2B upregulation or identifying activating mutations in Fgfr2. We used 5' RACE to identify a novel transcription start site for murine Fgf-10. Conventional in silico analysis predicted multiple binding sites for the transcription factor PEA3 upstream of this site. Binding was confirmed by chromatin immunopreciptation, and functional significance was studied by both RNAi knockdown and transient over-expression of PEA3. Knockdown of PEA3 message led to increased Fgf-10 expression, whereas overexpression of PEA3 resulted in decreased Fgf-10 expression. Thus, we have identified PEA3 as a negative regulator of Fgf-10 expression in a murine cell line and confirmed that activity also is seen in human breast cancer cell lines (MCF-7 and MDA-MB-231). Furthermore, over-expression of PEA3 in these cells resulted in impaired cell migration, which was rescued by treatment with FGF-10. Thus, PEA3 can regulate the transcription of Fgf-10 and such modulation can control breast cancer cell behaviour.
Breast Cancer Research and Treatment, Jul 13, 2006
Upregulation of functional voltage-gated Na + channels (VGSCs) occurs in metastatic human breast ... more Upregulation of functional voltage-gated Na + channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the 'neonatal' splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by ~90 %) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (~30 %), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33 %, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43 %, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42 %, in a dosedependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.
Cancers, Nov 13, 2021
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
FEBS Journal, Jan 3, 2022
Clinical Science, Apr 10, 2014
FGFR (fibroblast growth factor receptor) signalling plays critical roles in embryogensis, adult p... more FGFR (fibroblast growth factor receptor) signalling plays critical roles in embryogensis, adult physiology, tissue repair and many pathologies. Of particular interest over recent years, it has been implicated in a wide range of cancers, and concerted efforts are underway to target different aspects of FGFR signalling networks. A major focus has been identifying the canonical downstream signalling pathways in cancer cells, and these are now relatively well understood. In the present review, we focus on two distinct but emerging hot topics in FGF biology: its role in stromal cross-talk during cancer progression and the potential roles of FGFR signalling in the nucleus. These neglected areas are proving to be of great interest clinically and are intimately linked, at least in pancreatic cancer. The importance of the stroma in cancer is well accepted, both as a conduit/barrier for treatment and as a target in its own right. Nuclear receptors are less acknowledged as targets, largely due to historical scepticism as to their existence or importance. However, increasing evidence from across the receptor tyrosine kinase field is now strong enough to make the study of nuclear growth factor receptors a major area of interest.
Embo Molecular Medicine, Feb 6, 2014
Pancreatic cancer is characterised by desmoplasia, driven by activated pancreatic stellate cells ... more Pancreatic cancer is characterised by desmoplasia, driven by activated pancreatic stellate cells (PSCs). Over-expression of FGFs and their receptors is a feature of pancreatic cancer and correlates with poor prognosis, but whether their expression impacts on PSCs is unclear. At the invasive front of human pancreatic cancer, FGF2 and FGFR1 localise to the nucleus in activated PSCs but not cancer cells. In vitro, inhibiting FGFR1 and FGF2 in PSCs, using RNAi or chemical inhibition, resulted in significantly reduced cell proliferation, which was not seen in cancer cells. In physiomimetic organotypic co-cultures, FGFR inhibition prevented PSC as well as cancer cell invasion. FGFR inhibition resulted in cytoplasmic localisation of FGFR1 and FGF2, in contrast to vehicle-treated conditions where PSCs with nuclear FGFR1 and FGF2 led cancer cells to invade the underlying extra-cellular matrix. Strikingly, abrogation of nuclear FGFR1 and FGF2 in PSCs abolished cancer cell invasion. These findings suggest a novel therapeutic approach, where preventing nuclear FGF/FGFR mediated proliferation and invasion in PSCs leads to disruption of the tumour microenvironment, preventing pancreatic cancer cell invasion.
Methods in molecular biology, Dec 7, 2016
Organotypic models are 3D in vitro representations of an in vivo environment. Their complexity ca... more Organotypic models are 3D in vitro representations of an in vivo environment. Their complexity can range from an epidermal replica to the establishment of a cancer microenvironment. These models have been used for many years, in an attempt to mimic the structure and function of cells and tissues found inside the body. Methods for developing 3D organotypic models differ according to the tissue of interest and the experimental design. For example, cultures may be grown submerged in culture medium and or at an air-liquid interface. Our group is focusing on an air-liquid interface 3D organotypic model. These cultures are grown on a nylon membrane-covered metal grid with the cells embedded in a Collagen-Matrigel gel. This allows cells to grow in an air-liquid interface to enable diffusion and nourishment from the medium below. Subsequently, the organotypic cultures can be used for immunohistochemical staining of various components of ERK signaling, which is a key player in mediating communication between cells and their microenvironment.
The International Journal of Biochemistry & Cell Biology, Feb 1, 2010
Voltage-gated Na(+) channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles),... more Voltage-gated Na(+) channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles), as well as in some classically &amp;amp;amp;amp;amp;amp;amp;amp;#39;non-excitable&amp;amp;amp;amp;amp;amp;amp;amp;#39; cells (e.g. fibroblasts), and in carcinomas. In general, functional expression of VGSCs in plasma membrane (PM) is hierarchical and dynamic. Previously, we have shown that an activity-dependent positive feedback mechanism involving cAMP-dependent protein kinase A (PKA) plays a significant role in upregulation of VGSCs in strongly metastatic rat prostate cancer Mat-LyLu cells expressing Nav1.7. Here, we investigated the possible role of PKA in VGSC regulation and its functional consequences in strongly metastatic human breast cancer (BCa) MDA-MB-231 cells, where the neonatal splice form of Nav1.5 (nNav1.5) is the predominant VGSC present. Treatment with the PKA activator forskolin for 24h increased mRNA and PM protein levels of nNav1.5, without changing the total VGSC protein level. Opposite effects were obtained by application of the PKA inhibitor KT5720 or the highly specific VGSC blocker tetrodotoxin (TTX), the latter implying activity-dependent upregulation. We tested the possibility, therefore, that the activity dependence of VGSC (nNav1.5) expression involved PKA. Indeed, TTX pretreatment reduced the level of phosphorylated PKA and eliminated basal and PKA-stimulated cellular migration. These data suggested that activity-dependent positive feedback mediated by PKA plays an important role in the functional expression of nNav1.5 in BCa, and in turn, this enhances the cells&amp;amp;amp;amp;amp;amp;amp;amp;#39; metastatic potential.
Tumor suppressor genes (TSGs) play critical roles in preventing tumorigenesis and they are freque... more Tumor suppressor genes (TSGs) play critical roles in preventing tumorigenesis and they are frequently inactivated in tumours. Recently developed high-density microarrays can detect subchromosomal deletions, recurrence of which usually indicates the location of TSGs within the deleted region. We analyzed testicular germ cell tumour (TGCT) clinical samples using SNP arrays and found a frequent small deletion on the region 6q25.3 containing only one known gene, ZDHHC14. While its cellular function is unknown, ZDHHC14 belongs to the recently discovered DHHC family, which are predicted to be involved in protein palmitoylation, a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Consistently, we found a dramatic under-expression of ZDHHC14 mRNA and protein in TGCTs, and this associated with chemoresistance. Oncomine database mining showed that ZDHHC14 is also under-expressed in lymphoma, liposarcoma, brain, kidney, lung and colorectal cancers. Thus, it appears that ZDHHC14 downregulation may be involved in other cancers. We studied ZDHHC14 expression in prostate cancer (PCa), detecting a decrease at mRNA and protein level. We also detected that ZDHHC14 mRNA was downregulated in a pilot study on breast cancer samples. As genomic loss of the ZDHHC14 region was only detected in a small number of PCa samples, we checked whether promoter hypermethylation was the cause for ZDHHC14 downregulation. However, no changes in methylation status were found. We then sequenced the whole genomic region surrounding ZDHHC14 by next generation sequencing in TGCTs and PCa and found several mutations in the promoter, the coding region, as well as in intronic regions. Finally, we tested the function of ZDHHC14 in cell-based studies. We generated a 293 T-REx tetracycline inducible ZDHHC14 overexpressing stable cell line, which showed that ZDHHC14 overexpression decreased cell viability. The induction of apoptosis by ZDHHC14 overexpression was detected both by FACS and caspase 7 and PARP cleavage analyses. This was confirmed by transient ZDHHC14 overexpression in the PCa cell line 22RV1. In vivo we xenografted mice using both tetracycline inducible ZDHHC14 overexpressing 293 T-REx cells and control cells transfected with the empty vector. ZDHHC14 expression was induced by tetracycline at the beginning of inoculation and we detected that ZDHHC14 overexpression blocked tumour initiation completely. In conclusion, these results implicate ZDHHC14 as a tumour suppressor gene commonly inactivated in human cancers, indicating that it might exert its tumor suppressor role through the induction of programmed cell death. This is the first study showing the involvement of ZDHHC14 in a specific pathway, the classic caspase-dependent apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4858. doi:1538-7445.AM2012-4858
Journal of Cell Biology, Jun 4, 2012
Organotypic modelling as a means of investigating epithelial-stromal interactions during tumourig... more Organotypic modelling as a means of investigating epithelial-stromal interactions during tumourigenesis
One of the leading causes of death worldwide, in both man and woman, is cancer. Despite the signi... more One of the leading causes of death worldwide, in both man and woman, is cancer. Despite the significant development in therapeutic strategies, the inevitable emergence of drug resistance limits the success and impedes the curative outcome. Intrinsic and acquired resistance are common mechanisms responsible for cancer relapse. Several factors crucially regulate tumourigenesis and resistance, including physical barriers, tumour microenvironment (TME), heterogeneity, genetic and epigenetic alterations, the immune system, tumour burden, growth kinetics and undruggable targets. Moreover, transforming growth factor-beta (TGF-β), Notch, epidermal growth factor receptor (EGFR), integrin-extracellular matrix (ECM), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), phosphoinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR), wingless-related integration site (Wnt)/β-catenin), Janus kinase/signal transducers and activators of transcription (JAK...
Clinical Cancer Research, Aug 1, 2005
Ion channel activity is involved in several basic cellular behaviors that are integral to metasta... more Ion channel activity is involved in several basic cellular behaviors that are integral to metastasis (e.g., proliferation, motility, secretion, and invasion), although their contribution to cancer progression has largely been ignored. The purpose of this study was to investigate voltagegated Na + channel (VGSC) expression and its possible role in human breast cancer. Experimental Design: Functional VGSC expression was investigated in human breast cancer cell lines by patch clamp recording. The contribution of VGSC activity to directional motility, endocytosis, and invasion was evaluated by in vitro assays. Subsequent identification of the VGSC a-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCa expression and its association with metastasis in vivo. Results:VGSC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGSC activity potentiated cellular directional motility, endocytosis, and invasion. Reverse transcription-PCR revealed that Na v 1.5, in its newly identified ''neonatal'' splice form, was specifically associated with strong metastatic potential in vitro and breast cancer progression in vivo. An antibody specific for this form confirmed up-regulation of neonatal Na v 1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Na v 1.5 expression and clinically assessed lymph node metastasis. Conclusions: Up-regulation of neonatal Na v 1.5 occurs as an integral part of the metastatic process in human breast cancer and could serve both as a novel marker of the metastatic phenotype and a therapeutic target.
Journal of Neuroscience Methods, Sep 1, 2005
Voltage-gated Na(+) channel (VGSC) diversity is achieved through a number of mechanisms: multiple... more Voltage-gated Na(+) channel (VGSC) diversity is achieved through a number of mechanisms: multiple subunits, multiple genes encoding the pore-forming VGSC alpha-subunit and multiple gene isoforms generated by alternative splicing. A major type of VGSCalpha alternative splicing is in D1:S3, which has been proposed to be developmentally regulated. We recently reported a D1:S3 spliced form of Na(v)1.5 in human metastatic breast cancer cells. This novel &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;neonatal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; isoform differs from the counterpart &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;adult&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; form at seven amino acids (in the extracellular loop between S3-S4 of D1). Here, we generated an anti-peptide polyclonal antibody, named NESOpAb, which specifically recognised &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;neonatal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; but not &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;adult&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Na(v)1.5 when tested on cells specifically over-expressing one or other of these Na(v)1.5 spliced forms. The antibody was used to investigate developmental expression of &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;neonatal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; Na(v)1.5 (nNa(v)1.5) in a range of mouse tissues by immunohistochemistry. Overall, the results were consistent with nNa(v)1.5 protein being more abundantly expressed in selected tissues (particularly heart and brain) from neonate as compared to adult animals. Importantly, NESOpAb blocked functional nNa(v)1.5 ion conductance when applied extracellularly at concentrations as low as 0.05 ng/ml. Possible biological and clinical applications of NESOpAb are discussed.
Uploads
Papers by Athina-Myrto Chioni