Occurrence Of Mytilicola-Intestinalis Steuer An Intestinal Copepod Parasite Of Mytilus In Southwest Of England

The occurrence of Mytilicola intestinalis in populations of mussels in south-west England is recorded and compared with previous data. Since 1955 there have been two main changes in the distribution of Mytilicola: (a) it has invaded all the major estuarine mussel populations on the Bristol Channel coast, and (b) many previously uninfested open-coast populations all round the peninsula are now lightly infested. It is suggested that differences in infestation levels between estuarine and open-coast populations of mussels are due primarily to differences in the degree of exposure to wave action although factors such as size, population density and location of the hosts also influence infestation. The chance of the establishment of breeding pairs of Mytilicola depends on the parasite population size and its distribution through the host population

Zinc transporter HKE4 as a new target in antihormone resistance of breast cancer

Background Oestrogen receptor-positive breast cancers develop resistance to anti-oestrogens by utilising alternative growth factor pathways as observed in our tamoxifen-resistant cell line (TAMR). These include EGFR, IGF1-R and Src signalling as well as increased growth and invasion. Zinc is elevated in breast cancer tissue and has been demonstrated to activate certain growth factor signalling pathways. We have tested the expression level of members of the LIV-1 family of zinc influx transporters and discovered that HKE4 (SLC39A7, ZIP7), previously shown by us capable of increasing the intracellular zinc levels, has increased expression in TAMR. We have therefore investigated whether the development of the more aggressive phenotype observed in our TAMR cells, including activation of these signalling pathways as well as increased growth and invasion, is due to an increase of intracellular zinc and as a direct result of increased expression of HKE4. Methods All nine members of the LIV-1 subfamily of ZIP transporters were measured in our model of tamoxifen-resistant breast cancer using Affymetrix arrays. Zinc-induced activation of growth factor signalling pathway components was investigated by western blot and/or fluorescent microscopy. Short-term (15-min) treatments with 20 μM zinc included ionophore, whereas long-term (hours/days) did not. Recombinant LIV-1 family members with a V5 tag were expressed using pcDNA3.1/V5-His-TOPO vector, and siRNA (Dharmacon smartpools with relevant controls) was used to reduce endogenous expression. Results HKE4 (SLC39A7), a ZIP transporter from the LIV-1 subfamily, was discovered to be elevated in TAMR cells by Affymetrix analysis and confirmed by PCR and western blot. We have observed that our TAMR cells have a twofold increase in intracellular zinc compared with wild-type cells, using the zinc-specific fluorescent dye Newport Green. Short-term zinc treatment of TAMR cells activates the signalling pathways implicated in antihormone-resistant proliferation and is reduced by both the zinc chelator TPEN and the Src kinase inhibitor SU6556. The same effects are observed after longer term (6 days) zinc treatment with additional increases in cell growth and invasion through Matrigel. Since we have previously demonstrated that HKE4 is capable of increasing intracellular zinc in cells and, more recently, that these TAMR have elevated intracellular zinc levels, we have tested the hypothesis that elevated HKE4 expression is directly responsible for the aggressive phenotype observed in our TAMR cells. Reducing HKE4 levels by siRNA demonstrated a role for this molecule in driving the zinc-induced activation of multiple signalling pathways. In the presence of siRNA for HKE4, the previously observed zinc-induced activation of EGFR, Src, and IGF1-R was eradicated and the EGF-stimulated activation was also decreased. Additionally, we have demonstrated the converse by transfecting recombinant HKE4 into wild-type cells and/or treating them with zinc to observe the activation of these signalling pathways and increases in invasive capability. Interestingly, we have observed a similar role of HKE4 in our model of faslodex-resistant breast cancer. Conclusion The presented results propose that HKE4, a member of the LIV-1 subfamily of ZIP transporters, is directly involved in the activation of the aggressive phenotype observed with the development of antihormone resistance, and as such is a potential new target for the prevention of resistance to antihormones in breast cancer progression

Recruitment of insulin receptor substrate-1 by erbB3 impacts on IGF-IR signalling in oestrogen receptor-positive breast cancer cells

Insulin-like growth factor receptor (IGF-IR) signalling classically involves phosphorylation of insulin receptor substrate-1 (IRS-1) to recruit key down-stream signalling pathways effecting breast cancer cell proliferation and survival. Recently, we have shown a further capacity for IRS-1 to associate with the epidermal growth factor receptor (EGFR/erbB1), with activation of EGFR promoting recruitment and phosphorylation of IRS-1 in an oestrogen receptor (ER)-positive tamoxifen-resistant breast cancer cell line. In this study, we examined recruitment of IRS-1 by another member of the erbB receptor family, erbB3, in three ER-positive breast cancer cell lines. Our studies revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT474 cells with HRGβ1 treatment significantly enhancing this recruitment and promoting IRS-1 phosphorylation at tyrosine (Y) 612, a specific phosphoinositide 3-kinase (PI3K) binding site. IRS-1 appears to play a key role in erbB3 signalling in MCF-7 and T47D cells as its knockdown using siRNA greatly impaired HRGβ1 signalling via PI3K/AKT in these cell lines. This novel interaction may have clinical relevance as immunohistochemical analysis of ER-positive breast cancer patient samples revealed IRS-1 Y612 expression positively correlated with total erbB3, p-AKT and Ki67 expression. Importantly, we found that recruitment of IRS-1 by erbB3 impaired IRS-1 recruitment by IGF-IR in both MCF-7 and T47D cells, whilst blockade of IGF-1R enhanced erbB3/IRS-1 interaction and sensitised both cell lines to HRGβ1. Consequently, blockade of erbB3 signalling enhanced the effects of IGF-IR inhibition in these cells. In conclusion, these and previous findings suggest that IRS-1 can be recruited to IGF-1R, EGFR and erbB3 in ER-positive breast cancer cells and this may provide an adaptive resistance mechanism when these receptors are targeted individually. Consequently co-targeting of IGF-IR and erbB receptors may prove to be a more effective strategy for the treatment of ER-positive breast cancer

Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin β1

Introduction We have previously reported that induction of epidermal growth factor receptor and ErbB2 in response to antihormonal agents may provide an early mechanism to allow breast cancer cells to evade the growth-inhibitory action of such therapies and ultimately drive resistant cell growth. More recently, the other two members of the ErbB receptor family, ErbB3 and ErbB4, have been implicated in antihormone resistance in breast cancer. In the present study, we have investigated whether induction of ErbB3 and/or ErbB4 may provide an alternative resistance mechanism to antihormonal action in a panel of four oestrogen receptor (ER)-positive breast cancer cell lines. Methods MCF-7, T47D, BT474 and MDAMB361 cell lines were exposed to fulvestrant (100 nM) for seven days, and effects on ErbB3/4 expression and signalling, as well as on cell growth, were assessed. Effects of heregulin β1 (HRGβ1) were also examined in the absence and presence of fulvestrant to determine the impact of ER blockade on the capacity of this ErbB3/4 ligand to promote signalling and cell proliferation. Results Fulvestrant potently reduced ER expression and transcriptional activity and significantly inhibited growth in MCF-7, T47D, BT474 and MDAMB361 cells. However, alongside this inhibitory activity, fulvestrant also consistently induced protein expression and activity of ErbB3 in MCF-7 and T47D cells and ErbB4 in BT474 and MDAMB361 cell lines. Consequently, fulvestrant treatment sensitised all cell lines to the actions of the ErbB3/4 ligand HRGβ1 with enhanced ErbB3/4-driven signalling activity, reexpression of cyclin D1 and significant increases in cell proliferation being observed when compared to untreated cells. Indeed, in T47D and MDAMB361 HRGβ1 was converted from a ligand having negligible or suppressive growth activity into one that potently promoted cell proliferation. Consequently, fulvestrant-mediated growth inhibition was completely overridden by HRGβ1 in all four cell lines. Conclusions These findings suggest that although antihormones such as fulvestrant may have potent acute growth-inhibitory activity in ER-positive breast cancer cells, their ability to induce and sensitise cells to growth factors may serve to reduce and ultimately limit their inhibitory activity

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells

Introduction Anti-oestrogens have been the mainstay of therapy in patients with oestrogen-receptor (ER) positive breast cancer and have provided significant improvements in survival. However, their benefits are limited by tumour recurrence in a significant proportion of initially drug-responsive breast cancer patients because of acquired anti-oestrogen resistance. Relapse on such therapies clinically presents as local and/or regional recurrences, frequently with distant metastases, and the prognosis for these patients is poor. The selective ER modulator, tamoxifen, classically exerts gene inhibitory effects during the drug-responsive phase in ER-positive breast cancer cells. Paradoxically, this drug is also able to induce the expression of genes, which in the appropriate cell context may contribute to an adverse cell phenotype. Here we have investigated the effects of tamoxifen and fulvestrant treatment on invasive signalling and compared this with the direct effects of oestrogen withdrawal to mimic the action of aromatase inhibitors. Methods The effect of oestrogen and 4-hydroxy-tamoxifen on the invasive capacity of endocrine-sensitive MCF-7 cells, in the presence or absence of functional E-cadherin, was determined by Matrigel invasion assays. Studies also monitored the impact of oestrogen withdrawal or treatment with fulvestrant on cell invasion. Western blotting using phospho-specific antibodies was performed to ascertain changes in invasive signalling in response to the two anti-oestrogens versus both oestradiol treatment and withdrawal. Results To the best of our knowledge, we report for the first time that tamoxifen can promote an invasive phenotype in ER-positive breast cancer cells under conditions of poor cell-cell contact and suggest a role for Src kinase and associated pro-invasive genes in this process. Our studies revealed that although this adverse effect is also apparent for further classes of anti-oestrogens, exemplified by the steroidal agent fulvestrant, it is absent during oestrogen withdrawal. Conclusions These data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate

Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

Introduction Resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies is an emerging clinical problem. The efficacy of anti-EGFR therapies can be influenced by the presence of heregulins (HRGs), which can bind erbB3/4 receptors and can activate alternative signalling pathways. In the present study we have examined whether HRG signalling can circumvent EGFR blockade in an EGFR-positive tamoxifen-resistant MCF-7 (Tam-R) breast cancer cell line. Methods Tam-R cells, incubated with the selective EGFR tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839), were exposed to HRGβ1 and the effects on erbB receptor dimerization profiles and on activation of associated downstream signalling components were assessed by immunoprecipitation, western blotting and immunocytochemistry. The effects of HRGβ1 on gefitinib-treated Tam-R cell growth and invasion were also examined, and HRGβ1 expression levels were assessed in breast cancer tissue by immunohistochemistry to address the potential clinical relevance of such a resistance mechanism. Results In Tam-R cells, HRGβ1 promoted erbB3/erbB2 and erbB3/EGFR heterodimerization, promoted ERK1/2 and AKT pathway activation and increased cell proliferation and invasion. Gefitinib prevented HRGβ1-driven erbB3/EGFR heterodimerization, ERK1/2 activation and Tam-R cell proliferation, but HRGβ1-driven erbB3/erbB2 heterodimerization, AKT activation and Tam-R cell invasion were maintained. A combination of gefitinib and the phosphatidylinositol 3-kinase inhibitor LY294002 effectively blocked HRGβ1-mediated intracellular signalling activity, growth and invasion in Tam-R cells. Similarly, targeting erbB2 with trastuzumab in combination with gefitinib in Tam-R cells reduced HRGβ1-induced erbB2 and ERK1/2 activity; however, HRGβ1-driven AKT activity and cell growth were maintained while cell invasion was significantly enhanced with this combination. In clinical tissue all samples demonstrated cytoplasmic tumour epithelial HRGβ1 protein staining, with expression correlating with EGFR positivity and activation of both AKT and ERK1/2. Conclusion HRGβ1 can overcome the inhibitory effects of gefitinib on cell growth and invasion in Tam-R cells through promotion of erbB3/erbB2 heterodimerization and activation of the phosphatidylinositol 3-kinase/AKT signalling pathway. This may have implications for the effectiveness of anti-EGFR therapies in breast cancer as HRGβ1 is enriched in many EGFR-positive breast tumours

Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation-sensitivity in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Activator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.</p> <p>Methods</p> <p>We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.</p> <p>Results</p> <p>We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed <it>Cell Death </it>and <it>Cell Adhesion and Migration </it>as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. <it>Ctgf, Nrp1</it>, <it>Tnfaip3, Gsta3</it>) and AP-2 and other main apoptosis players and to create a unique network.</p> <p>Conclusions</p> <p>Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.</p

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

INTRODUCTION Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years

Recognising facial expressions in video sequences

We introduce a system that processes a sequence of images of a front-facing human face and recognises a set of facial expressions. We use an efficient appearance-based face tracker to locate the face in the image sequence and estimate the deformation of its non-rigid components. The tracker works in real-time. It is robust to strong illumination changes and factors out changes in appearance caused by illumination from changes due to face deformation. We adopt a model-based approach for facial expression recognition. In our model, an image of a face is represented by a point in a deformation space. The variability of the classes of images associated to facial expressions are represented by a set of samples which model a low-dimensional manifold in the space of deformations. We introduce a probabilistic procedure based on a nearest-neighbour approach to combine the information provided by the incoming image sequence with the prior information stored in the expression manifold in order to compute a posterior probability associated to a facial expression. In the experiments conducted we show that this system is able to work in an unconstrained environment with strong changes in illumination and face location. It achieves an 89\% recognition rate in a set of 333 sequences from the Cohn-Kanade data base

Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

<p>Abstract</p> <p>Background</p> <p>Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) promotes neural progenitor proliferation <it>in vitro </it>in cultures of rodent hippocampal and human cortical neural progenitors, and <it>in vivo </it>in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation.</p> <p>Methods</p> <p>In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging.</p> <p>Results</p> <p>Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC₅₀of 110 ± 15 nM and a maximal response occurring at three days <it>in vitro</it>. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La³⁺, or the L-type calcium channel blocker nifedipine. Furthermore, the GABA_Areceptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise.</p> <p>Conclusion</p> <p>Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABA_Areceptor and L-type calcium channel. These data suggest that APα-induced intracellular calcium concentration increase serves as the initiation mechanism whereby APα promotes neurogenesis.</p