erbB3 recruitment of insulin receptor substrate 1 modulates insulin-like growth factor receptor signalling in oestrogen receptor-positive breast cancer cell lines

Introduction Recently we reported that insulin receptor substrate 1 (IRS-1), classically an adaptor protein for the insulin-like growth factor type I receptor (IGF-IR), associates with the epidermal growth factor receptor in oestrogen receptor (ER)-positive (ER+) tamoxifen-resistant breast cancer cells. In this study, we examined whether IRS-1 also associates with another erbB receptor family member, erbB3, and what impact this might have on IGF-IR signalling in three ER+ breast cancer cell lines. Methods Immunoprecipitation and Western blot analysis were utilised to examine the potential association between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells in the absence and presence of the erbB3/4 ligand heregulin β1 (HRGβ1). Subsequently, the impact of a selective IGF-IR/IR inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-(N, N-dimethylamino)propoxy)anilino]pyrimidine on this association and HRGβ1 signalling was assessed in these cell lines. Immunohistochemical analysis of a small cohort of ER+ breast cancer patient samples was also performed to determine the potential clinical relevance of this novel interaction. Results Immunoprecipitation and Western blot analysis revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells, with HRGβ1 significantly enhancing this recruitment and promoting IRS-1 phosphorylation at Y612. IRS-1 participates in erbB3 signalling in MCF-7 and T47D cells as IRS-1 knockdown impaired HRGβ1 signalling. Importantly, recruitment of IRS-1 by erbB3 reduced IRS-1 association with IGF-IR in MCF-7 and T47D cells, whilst blockade of IGF-IR-enhanced erbB3-IRS-1 interaction and sensitised both cell lines to HRGβ1, allowing HRGβ1 to override IGF-IR blockade. Consequently, suppression of IRS-1 signalling enhanced the effects of IGF-IR inhibition in these cells. This novel interaction may have clinical relevance, as immunohistochemical analysis of a small ER+ breast tumour series revealed significant positive correlations between phosphorylated IRS-1 Y612 expression and total erbB3, phosphorylated Akt and Ki-67 expression. Conclusions IRS-1 can be recruited to IGF-IR and erbB3 in ER+ breast cancer cells, and this provides an adaptive resistance mechanism when these receptors are targeted individually. Consequently, cotargeting IGF-IR and either erbB3 or IRS-1 should prove to be a more effective strategy for the treatment of ER+ breast cancer

A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry : application to the characterisation of metabolites in rat urine

A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.</p

Increased Ret signalling and impact of vandetanib in acquired tamoxifen resistant breast cancer

Deregulation of the tyrosine kinase Ret and its coreceptors (GFRα) has been implicated in neoplasia, and Ret is of interest as a therapeutic target in endocrine-treated breast cancer. This study evaluated in vitro impact of vandetanib, a tyrosine kinase inhibitor able to target Ret in addition to EGFR and VEGFR2, in ER+ breast cancer cells that have acquired resistance to tamoxifen treatment. Tamoxifen resistant TAMR and endocrine responsive MCF7 cells were grown in vitro for 7 days +/- vandetanib (0.5-5μM) +/- exogenous growth factors (10-50ng/ml), and also in continuous culture with vandetanib (1μM), to monitor growth impact and emergence of vandetanib resistance. For Western blotting or immunoprecipitation, log phase cells were transferred for 24hr to serum-free medium and pre-treated for 1hr +/- vandetanib followed by Ret ligands GDNF or artemin for 5mins. Immunohistochemistry for Ret activity was performed on an ER+ tamoxifen-treated clinical breast cancer TMA sample series using Y1062 Ret phospho-antibody with HScore staining assessment. Growth of TAMR cells was substantially inhibited by vandetanib (p<0.001, IC50 0.6μM) with complete cell loss by 17weeks, contrasting rapid emergence of resistance in endocrine responsive MCF7 cells. TAMR cells were more sensitive to vandetanib vs. gefitinib (p<0.001; 1μM each agent), indicating mitogenic signalling in addition to EGFR contributed to TAMR growth. TAMR cells had elevated basal Ret expression, activity and interaction with elevated GFRα3 coreceptor expression; mature VEGFR2 was not detected in TAMR cells. Exogenous GFRα3 ligands artemin or GDNF modestly stimulated TAMR cell growth and hyperactivated Ret, downstream kinases (including MAPK, AKT) and ER Ser167, confirming functional GFRα/Ret signalling and its cross-talk with ER. Increased phospho-Ret immunostaining was also associated with shortened DFI (p=0.036) and survival (p=0.011) in tamoxifen-treated clinical ER+ breast cancers. Vandetanib (0.5-1μM) depleted GFRα3/Ret activity and decreased phospho-EGFR in TAMR cells under basal and Ret ligand-stimulated conditions, inhibited MAPK, p70S6K and S6RP phosphorylation, and partially-reduced levels of phospho-AKT and phospho-ER. However, vandetanib failed to consistently impact on HER2, 3 or 4 activity; moreover, hyperactivation of all erbB receptors by exogenous heregulin B1 (10ng/ml) recovered AKT, MAPK, p70S6K and ER AF-1 phosphorylation in the presence of vandetanib and was able to overcome the basal growth-inhibitory impact of this agent in TAMR cells. These findings demonstrate a central importance for increased Ret signalling and its cross-talk with ER in tamoxifen resistant breast cancer that can be targeted in vitro by vandetanib. Further studies are required to determine optimal combination treatments with vandetanib to circumvent potential intrinsic resistance in clinical breast cancers that exhibit heregulin B1 enrichment

Biological effects of fulvestrant on estrogen receptor positive human breast cancer: Short, medium and long-term effects based on sequential biopsies.

We report the first study of the biological effect of fulvestrant on ER positive clinical breast cancer using sequential biopsies through to progression. Thirty-two locally/systemically advanced breast cancers treated with first-line fulvestrant (250 mg/month) were biopsied at therapy initiation, 6 weeks, 6 months and progression and immunohistochemically-analyzed for Ki67, ER, EGFR and HER2 expression/signaling activity. This series showed good fulvestrant responses (duration of response [DoR] = 25.8 months; clinical benefit = 81%). Ki67 fell (p < 0.001) in 79% of tumours by 6 months and lower Ki67 at all preprogression time-points predicted for longer DoR. ER and PR significantly decreased in all tumours by 6 months (p < 0.001), with some declines in ER (serine 118) phosphorylation and Bcl-2 (p = 0.007). There were modest HER2 increases (p = 0.034, 29% tumours) and loss of any detectable EGFR phosphorylation (p = 0.024, 50% tumours) and MAP kinase (ERK1/2) phosphorylation (p = 0.019, 65% tumours) by 6 months. While ER remained low, there was some recovery of Ki67, Bcl-2 and (weakly) EGFR/MAPK activity in 45–67% patients at progression. Fulvestrant's anti-proliferative impact is related to DoR, but while commonly downregulating ER and indicators of its signaling and depleting EGFR/MAPK signaling in some patients, additional elements must determine response duration. Residual ER at fulvestrant relapse explains reported sensitivity to further endocrine therapies. Occasional modest treatment-induced HER2 and weakly detectable EGFR/HER2/MAPK signaling at relapse suggests targeting of such activity might have value alongside fulvestrant in some patients. However, unknown pathways must drive relapse in most. Ki67 has biomarker potential to predict fulvestrant outcome and as a quantitative measure of response

Clinical characteristics, multiorgan dysfunction and outcomes of patients with COVID-19: a prospective case series

BACKGROUND: Characterizing the multiorgan manifestations and outcomes of patients hospitalized with COVID-19 will inform resource requirements to address the long-term burden of this disease. We conducted a descriptive analysis using prospectively collected data to describe the clinical characteristics and spectrum of organ dysfunction, and in-hospital and longer-term clinical outcomes of patients hospitalized with COVID-19 during the first wave of the pandemic at a Canadian centre. METHODS: We conducted a prospective case series involving adult patients (aged ≥ 18 yr) with COVID-19 admitted to 1 of 2 hospitals in London, Ontario, from Mar. 17 to June 18, 2020, during the first wave of the pandemic. We recorded patients\u27 baseline characteristics, physiologic parameters, measures of organ function and therapies administered during hospitalization among patients in the intensive care unit (ICU) and in non-ICU settings, and compared the characteristics of hospital survivors and nonsurvivors. Finally, we recorded follow-up thoracic computed tomography (CT) and echocardiographic findings after hospital discharge. RESULTS: We enrolled 100 consecutive patients (47 women) hospitalized with COVID-19, including 32 patients who received ICU care and 68 who received treatment in non-ICU settings. Respiratory sequelae were common: 23.0% received high-flow oxygen by nasal cannula, 9.0% received noninvasive ventilation, 24.0% received invasive mechanical ventilation and 2.0% received venovenous extracorporeal membrane oxygenation. Overall, 9.0% of patients had cerebrovascular events (3.0% ischemic stroke, 6.0% intracranial hemorrhage), and 6.0% had pulmonary embolism. After discharge, 11 of 19 patients had persistent abnormalities on CT thorax, and 6 of 15 had persistent cardiac dysfunction on echocardiography. INTERPRETATION: This study provides further evidence that COVID-19 is a multisystem disease involving neurologic, cardiac and thrombotic dysfunction, without evidence of hepatic dysfunction. Patients have persistent organ dysfunction after hospital discharge, underscoring the need for research on long-term outcomes of COVID-19 survivors

Correction to: Heregulin ?1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

© 2018 The Author(s). After the publication of this work [1], an error was noticed in Fig. 2b and Fig. 4b as well as Fig. 4b. and Fig. 5d. Images of the ERK1/2 blots were accidentally duplicated. In Fig. 5a. and Fig. 5c., the last lane for p-ERK1/2 was mistakenly cropped out of the final image. The original blot for Fig. 4b., "total EGFR" (or lane 2) is shown below to avoid any misunderstanding of the data. We apologize for this error, which did not affect any of the interpretations or conclusions of the article

Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

Background: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. Methods: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. Results: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. Conclusions: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance

Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells

Background: Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved. Methods: The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference. Results: Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells. Conclusion: The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance

Crop Updates 2001 - Grower Booklet

1. Strategies for leaf disease management in wheat, Jatinderpal Bhathal1, Cameron Weeks2, Kith Jayasena1 and Robert Loughman1, 1Agriculture Western Australia. 2Mingenew-Irwin Group Inc. 2. Burn stubble windrows: to diagnose soil fertility problems, Bill Bowden, Chris Gazey and Ross Brennan, Agriculture Western Australia 3. Rainfall – what happened in 2000 and the prospects for 2001, Ian Foster, Agriculture Western Australia 4. Strategies for leaf disease management in malting barley, K. Jayasena1, Q. Knight2 and R. Loughman1, 1Agriculture Western Australia, 2IAMA Agribusiness 5. Planning your cropping program in season 2001, Dr Ross Kingwell, Agriculture Western Australia and University of Western Australia 6. Rotational crops and varieties for management of root lesion nematodes in Western Australia, S.B. Sharma, S. Kelly and R. Loughman, Crop Improvement Institute, Agriculture Western Australia 7. When and where to grow oats, Glenn McDonald, Agriculture Western Australia 8. Managing Gairdner barley for quality, Kevin Young and Blakely Paynter, Agriculture Western Australia FARMING SYSTEMS, PASTURES AND WEEDS 9.Evaluation of pasture species for phase pasture systems, Keith Devenish, Agriculture Western Australia 10. Competitiveness of wild radish in a wheat – lupin rotation, Abul Hashem, Nerys Wilkins, and Terry Piper, Agriculture Western Australia 11. Can we eradicate barley grass? Sally Peltzer, Agriculture Western Australia 12. Short term pasture phase for weed control, Clinton Revell and Candy Hudson, Agriculture Western Australia 13. Herbicide tolerance of some annual pasture legumes adapted to coarse textured sandy soils, Clinton Revell and Ian Rose, Agriculture Western Australia 14. Integrated weed management: Cadoux, Alexandra Wallace, Agriculture Western Australia LUPINS 15. Inter-row knockdowns for profitable lupins, Paul Blackwell, Agriculture Western Australia and Miles Obst, farmer, Mingenew 16.. Wild radish – the implications for our rotations, Dr David Bowran, Centre for Cropping Systems 17. Lupin variety performance: Are you making the most of it? Bevan J. Buirchell, Senior Plant Breeder, Agriculture Western Australia 18. Anthracnose in lupins – understanding the risk, Moin Salam, Art Diggle, Geoff Thomas, Mark Sweetingham and Bill O’Neill, Agriculture Western Australia OILSEEDS 19. Effect of stubble, seeding technique and seed size on crop establishment and yield of canola, Rafiul Alam, Glen Riethmuller and Greg Hamilton, Agriculture Western Australia 20. Canola – More responses to lime, Chris Gazey and Paul Carmody,Agriculture Western Australia 22. Performance of new canola varieties in AGWEST variety trials in 2000, G. Walton, Crop Improvement Institute, Agriculture Western Australia PULSES 23. The ascochyta management package for 2001, B. MacLeod, Agriculture Western Australia 24. Herbicide tolerance of new field pea varieties and lines, M. Seymour, H. Dhammu, T. Piper, D. Nicholson, M. D\u27Antuono, Agriculture Western Australi