SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian))

Review on SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), with data on DNA, on the protein encoded, and where the gene is implicated

The Role of BCA2 in the Endocytic Trafficking of EGFR and Significance as a Prognostic Biomarker in Cancer

Breast Cancer Associated gene 2 (BCA2) is an E3 ubiquitin ligase that is over-expressed in >50% of primary breast cancers, and has been shown to increase in vitro cell proliferation and invasion. The protein has been linked to alterations in EGFR degradation, however there is some dispute as to its role and influence on the biology of this receptor. Our work aimed to ascertain the role of BCA2 in EGFR endocytosis and down-regulation and to examine its links with breast cancer outcome. Data generated with the online expression analysis tool KM-Plotter showed that high BCA2 levels are associated with poor prognosis in ovarian, gastric and breast cancer, particularly HER2 over-expressing breast cancers. Experimentally, we demonstrate that over-expression of BCA2 induced a reduction in total EGFR levels. BCA2 over-expressing cells stimulated with EGF exhibited reduced lysosomal degradation of both this ligand and its receptor. Signalling downstream of EGFR in BCA2 over-expressing cells was characterized by a lower magnitude but increased duration. Our findings support a role for BCA2 in receptor endocytosis. Consistent with this we show that BCA2 over-expression reduces the level of vesicle-associated Rab7, a regulator of late endocytosis and documented interaction partner of BCA2. Levels of transferrin receptor and the uptake of transferrin were unaltered by over-expression of BCA2 indicating that trafficking changes may be limited to late endocytic sorting events. This report offers a thorough exploration of BCA2 biology and suggests a context-dependent role for the protein in the endocytic regulation of EGFR and as a prognostic biomarker in cancer

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

Overexpression of CD44 in acquired tamoxifen-resistant breast cancer cells augments their migratory response to heregulin beta 1

Background Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling [1] and a highly migratory cell phenotype [2]. Interestingly, in tamoxifen-resistant (TamR) MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein known to interact with, and modulate the function of, growth factor receptors [3]. Here we have explored the role of CD44 as a modulator of heregulin beta-1-induced migratory signalling in TamR cells. Methods Expression of CD44 (standard and v3 isoforms) were confirmed by RT-PCR and western blotting and their association with erbB family members determined by both immunofluorescence microscopy and immunoprecipitation. Activation of intracellular signalling following heregulin beta 1 treatment (10 ng/ml) in the presence or absence of CD44 (using siRNA-mediated inhibition) was determined by western blotting using phosphospecific antibodies. Cellular migration was determined by seeding cells (control and CD44 siRNA-treated) into fibronectin-coated transwell chambers (8.0 μm pore size) in the presence or absence of heregulin beta 1. After 24 hours, migratory cells were fixed, stained with crystal violet and counted. Results Both standard and v3 isoforms of CD44 were overexpressed in TamR cells at both gene and protein levels (mean fold increase in CD44s protein (TamR versus MCF7): 4.26 ± 1.2, P < 0.05). Moreover, CD44s and v3 colocalised with Her2 and Her3 receptors at the cell surface and were also detectable in Her2/Her3 cellular immunoprecipitates. Treatment of TamR cells with heregulin resulted in phosphorylation of erbB receptors together with a number of downstream signalling intermediates, including Akt, Src and FAK, and resulted in enhanced cellular migration. Significantly, heregulin-induced intracellular signalling was dramatically reduced in cells in which the expression of CD44 was suppressed (via siRNA), with a corresponding loss of heregulin-induced migratory behaviour (mean fold change in cell migration versus untreated control: 6.7 ± 1.1, P < 0.05 (heregulin beta 1); 1.8 ± 0.9 (CD44 siRNA); 1.47 ± 0.6, P < 0.05 (heregulin beta 1 + CD44 siRNA)). Conclusion These data demonstrate a role for CD44 as a modulator of erbB receptor function in endocrine-resistant breast cancer cells, where it augments heregulin beta 1 migratory signalling

The effects of n-6 polyunsaturated fatty acids on the expression of nm-23 in human cancer cells.

This study examined the effect of n-6 polyunsaturated fatty acids (PUFAs) on the expression of nm-23, a metastasis-suppressor gene, in two highly invasive human cancer cell lines, HT115 and MDA MB 231. A range of n-6 and n-3 PUFAs were tested. We report that while linoleic acid and arachidonic acid reduced the expression of nm-23-H1, gamma linolenic acid (GLA) and its soluble lithium salt markedly increased the expression of the molecules. The stimulation of the expression of nm-23 by GLA was seen at both protein and mRNA levels. Up-regulation of nm-23 was also associated with a reduction of the in vitro invasiveness of these cells. It is concluded that gamma linolenic acid (GLA) enhances the expression of nm-23. This contributes to the inhibition of the in vitro invasion of tumour cells

Inhibition of hepatocyte growth factor-induced motility and in vitro invasion of human colon cancer cells by gamma-linolenic acid.

In this study we have determined the effects of the n-6 essential fatty acid gamma-linolenic acid (GLA) on the motility and invasive/metastatic nature of the human colon cancer cell lines HT115, HT29 and HRT18. Cell motility was induced by hepatocyte growth factor/scatter factor (HGF/SF) and measured by both colony scattering and dissociation from carrier beads. Invasiveness was measured in vitro by cellular invasion into extracellular matrix. At concentrations up to 100 microM (which had no effect on cell growth over the duration of the experiments) both cell motility and invasion induced by HGF/SF were markedly reduced by GLA and its lithium salt. The attachment of these cells to the extracellular matrix components (Matrigel and fibronectin) was also inhibited. There were also changes in the cell-surface E-cadherin, but not fibronectin receptor at similar concentrations. It is concluded that n-6 essential fatty acids have the ability to inhibit both motility and invasiveness of human colon cancer cells, perhaps by modifying cell-surface adhesion molecules

Deep splicing plasticity of the human adenovirus type 5 transcriptome drives virus evolution

Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic. Adenovirus transcription and splicing is especially complex. We used long-read direct RNA sequencing to study adenovirus transcription and splicing during infection. This revealed a previously unappreciated complexity of alternative splicing and potential for secondary initiating codon usage. Moreover, we find that most viral transcripts tend to shorten polyadenylation lengths as infection progresses. Development of an open reading frame centric bioinformatics analysis pipeline provided a deeper quantitative and qualitative understanding of adenovirus’s genetic potential. Across the viral genome adenovirus makes multiple distinctly spliced transcripts that code for the same protein. Over 11,000 different splicing patterns were recorded across the viral genome, most occurring at low levels. This low-level use of alternative splicing patterns potentially enables the virus to maximise its coding potential over evolutionary timescales

Phosphorylated c-Src in the nucleus is associated with improved patient outcome in ER-positive breast cancer

Elevated c-Src protein expression has been shown in breast cancer and &lt;i&gt;in vitro&lt;/i&gt; evidence suggests a role in endocrine resistance. To investigate whether c-Src is involved in endocrine resistance, we examined the expression of both total and activated c-Src in human breast cancer specimens from a cohort of oestrogen receptor (ER)-positive tamoxifen-treated breast cancer patients. Tissue microarray technology was employed to analyse 262 tumour specimens taken before tamoxifen treatment. Immunohistochemistry using total c-Src and activated c-Src antibodies was performed. Kaplan–Meier survival curves were constructed and log-rank test were performed. High level of nuclear activated Src was significantly associated with improved overall survival (&lt;i&gt;P&lt;/i&gt;=0.047) and lower recurrence rates on tamoxifen (&lt;i&gt;P&lt;/i&gt;=0.02). Improved patient outcome was only seen with activated Src in the nucleus. Nuclear activated Src expression was significantly associated with node-negative disease and a lower NPI (&lt;i&gt;P&lt;/i&gt;&#60;0.05). On subgroup analysis, only ER-positive/progesterone receptor (PgR)-positive tumours were associated with improved survival (&lt;i&gt;P&lt;/i&gt;=0.004). This shows that c-Src activity is increased in breast cancer and that activated Src within the nucleus of ER-positive tumours predicts an improved outcome. In ER/PgR-positive disease, activated Src kinase does not appear to be involved in &lt;i&gt;de novo&lt;/i&gt; endocrine resistance. Further study is required in ER-negative breast cancer as this may represent a cohort in which it is associated with poor outcome

Evaluating putative repellent 'push' and attractive 'pull' components for manipulating the odour orientation of host-seeking malaria vectors in the peri-domestic space

BACKGROUND: Novel malaria vector control approaches aim to combine tools for maximum protection. This study aimed to evaluate novel and re-evaluate existing putative repellent 'push' and attractive 'pull' components for manipulating the odour orientation of malaria vectors in the peri-domestic space. METHODS: Anopheles arabiensis outdoor human landing catches and trap comparisons were implemented in large semi-field systems to (i) test the efficacy of Citriodiol((R)) or transfluthrin-treated fabric strips positioned in house eave gaps as push components for preventing bites; (ii) understand the efficacy of MB5-baited Suna-traps in attracting vectors in the presence of a human being; (iii) assess 2-butanone as a CO2 replacement for trapping; (iv) determine the protection provided by a full push-pull set up. The air concentrations of the chemical constituents of the push-pull set-up were quantified. RESULTS: Microencapsulated Citriodiol((R)) eave strips did not provide outdoor protection against host-seeking An. arabiensis. Transfluthrin-treated strips reduced the odds of a mosquito landing on the human volunteer (OR 0.17; 95% CI 0.12-0.23). This impact was lower (OR 0.59; 95% CI 0.52-0.66) during the push-pull experiment, which was associated with low nighttime temperatures likely affecting the transfluthrin vaporisation. The MB5-baited Suna trap supplemented with CO2 attracted only a third of the released mosquitoes in the absence of a human being; however, with a human volunteer in the same system, the trap caught < 1% of all released mosquitoes. The volunteer consistently attracted over two-thirds of all mosquitoes released. This was the case in the absence ('pull' only) and in the presence of a spatial repellent ('push-pull'), indicating that in its current configuration the tested 'pull' does not provide a valuable addition to a spatial repellent. The chemical 2-butanone was ineffective in replacing CO2. Transfluthrin was detectable in the air space but with a strong linear reduction in concentrations over 5 m from release. The MB5 constituent chemicals were only irregularly detected, potentially suggesting insufficient release and concentration in the air for attraction. CONCLUSION: This step-by-step evaluation of the selected 'push' and 'pull' components led to a better understanding of their ability to affect host-seeking behaviours of the malaria vector An. arabiensis in the peri-domestic space and helps to gauge the impact such tools would have when used in the field for monitoring or control

Heat Shock Protein 70 family members interact with Crimean-Congo hemorrhagic fever virus and Hazara virus nucleocapsid proteins and perform a functional role in the nairovirus replication cycle

The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast HAZV is non-pathogenic to humans, and thus represents an excellent model to study aspects of CCHFV biology under more accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N, and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells using small molecule inhibitors significantly reduced HAZV titres, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease