Loss of oestrogen receptor alpha in long-term antioestrogen-resistant cells: reversal by a c-src inhibitor

Background Tamoxifen still remains the most frequently used antioestrogen for the treatment of breast cancer. However, its efficacy is often limited by the emergence of acquired resistance and it has been suggested that, in some instances, this may involve oestrogen receptor (ER) loss. This study addresses this issue by examining long-term tamoxifen treatment of breast cancer cells and identifies that progressive ER loss does occur, leading to greatly increased aggressive tumour cell behaviour. Encouragingly, even after 30 months treatment, ER loss is reversible by a c-src inhibitor. Our data therefore provide a new model to study the cellular mechanisms associated with antihormone promoted ER loss and its possible prevention/reversal by signal transduction inhibitors. Methods Using quantitative PCR based on SYBR Green fluorescence, the expression of total ERα mRNA and its constituent mRNA variants were quantified in MCF7 cells and in our in vitro developed tamoxifen-resistant breast cancer cells (TamR), which have been cultured in the presence of tamoxifen for 30 months. Specific PCR amplification of all ERα mRNA variants was possible using forward primers designed to bind specifically to the 5' untranslated regions of ERα mRNA and used separately with a common reverse primer that anneals to the 5' end of the protein encoding region of exon 1 of ERα cDNA. Expression of ERα protein was assessed by western blot and immunohistochemistry. Results In MCF7 cells, the ERα mRNA isoforms A, B and C were detected as the most predominant variants, with C ERα mRNA showing the highest expression level. In TamR cells, about a 40% fall in total ERα mRNA was observed in comparison with MCF7 cells and was most apparent for the C variant. Extension of the tamoxifen treatment period to 30 months produced a further dramatic decrease in ERα mRNA (all variants) and protein levels, resulting in ER negativity being recorded in >90% of the cells by immunohistochemistry. These cells show increased levels of phosphorylated Erk 1&2, AKT, PKCα and src, and are highly aggressive in their growth behaviour, with increased cell motility and invasiveness. Treatment of the cells with the demethylating agent 5-azacytidine did not restore ERα expression, suggesting that epigenetic alterations are unlikely to be responsible for the reduced ER levels. However, Affymetrix data in the TamR cells showed that some positive regulators of ER expression, such as p53 and Foxo3A, are downregulated during the development of the resistant phenotype and their continued absence may contribute to the progressive ER loss. Significantly, pathway inhibitor studies revealed c-src to be an important regulator of ER loss, since its inhibition rapidly restored ER levels. Conclusion Our data indicate that considerable ER loss can occur during antihormonal treatment of breast cancer cells and that this can lead to a more aggressive phenotype. Encouragingly, however, even after 30 months exposure to tamoxifen, the process is reversible by inhibition of c-src. These data suggest that combinations of antihormones with signal transduction inhibitors could retain ER functions in treated cells and prevent a drift towards more aggressive cancer cell behaviour

Effects of Longwall Mining on Hydrology, Leslie County, Kentucky Part 2: During-Mining Conditions

The effects of longwall coal mining on hydrology in the Eastern Kentucky Coal Field are being investigated. The study area is in the Edd Fork watershed in southern Leslie County, over Shamrock Coal Company\u27s Beech Fork Mine. Longwall panels approximately 700 ft wide are separated by three-entry gateways that are approximately 200 ft wide. The mine is operated in the Fire Clay (Hazard No. 4) coal; overburden thickness ranges from 300 to 800 ft. Mining began in panel 1 in September 1991 and concluded with panel 8 in September 1994. Long-term monitoring consisting of a network of piezometers and time-domain reflectometry (TDR) cables previously installed over panel 7, in conjunction with a continuously recording rain gage and flume, is continuing after the completion of mining. Mining in panel 5 affected water levels in three of 24 piezometers installed over panel 7; the level went down in one piezometer and rose in two. Mining in panel 6 affected 16 of 24 piezometers; the level went down in 11 piezometers and rose in five. Mining in panel 7 affected water levels in 20 of 24 piezometers. Different water-level responses were recorded as the mine approached and passed by the instrumental sites. Thirteen piezometers failed as a result of undermining. These piezometers penetrated the zone of deep fracturing that extends upward approximately 450 ft (or 60 times greater than the mined thickness) above the mine. Only one piezometer showed a net increase in water level as a result of mining. Mining-induced surface fractures, observed along roads in the watershed, were generally parallel to the slope of the land surface or mining direction and probably contributed to ground-water recharge. The surface stream was unaffected until it was undermined by panel 8; then the stream went dry. TDR cables in the Hazard coal zone were deformed as mining passed by on the adjacent panel. Water levels in piezometers in the Hazard coal zone declined at the same time. TDR cables broke completely twice. The deepest complete break was in the Hazard coal zone and occurred when the active mine face was approaching, but still approximately 1,000 ft away from, the affected cable in panel 7. This corresponds to an angle of influence of 60 to 70°. Rock broke in the shallow subsurface (less than 50 ft deep) when the cable was directly undermined. Water-level responses in piezometers adjacent to mining are related to the complex flow system, rather than a defined angle of hydrologic influence. Coal beds and other conductive strata transmit water-level responses as far away as 1,450 ft, whereas nonconductive strata transmit little water-level change at closer distances. The water-level responses observed in this study support existing subsidence models. Piezometers in the zone of intensive fracturing failed as a result of rock breakage. An aquiclude zone developed in the ridge. The integrity of strata and piezometers was generally maintained. The most variable effects were observed in the zone of surface fracturing, within 50 ft of the surface

Effects of Longwall Mining on Hydrogeology, Leslie County, Kentucky Part 3: Post-Mining Conditions

The effects of longwall coal mining on hydrology in the Eastern Kentucky Coal Field have been investigated since 1991. The study area is in the Edd Fork watershed in southern Leslie County, over Shamrock Coal Company\u27s Beech Fork Mine. Longwall panels approximately 700 ft wide are separated by three-entry gateways that are approximately 200 ft wide. The mine is operated in the Fire Clay (Hazard No. 4) coal; overburden thickness ranges from 300 to 800 ft. Mining began in panel 1 in September 1991 and concluded with panel 8 in September 1994. Long-term monitoring consisting of a network of piezometers and time-domain reflectometry (TDR) cables previously installed over panel 7, in conjunction with a continuously recording rain gage and flume, began after the completion of mining. Two new core holes were drilled over panel 7 approximately 1 year after mining ceased in panel 8 to determine depth of collapse and hydraulic conductivity of strata. Water levels were measured in two new monitoring wells installed after mining to complement the 11 piezometers installed prior to mining that were still functioning. Precipitation was measured through July 1996, and streamflow was measured in Edd Fork on a monthly basis using a cross-section gaging method. Physical failure of piezometers, core drilling, and the movement of air into deeper piezometers after mining indicate that extensive fracturing occurred to a height of 450 ft above the mine, which is approximately 60 times the extracted coal-seam thickness. Hydraulic conductivity values determined from pressure-injection tests were 10 to 100 times greater after mining than before mining; many values were in the range of 10-2 to 10-4 ft/min for all lithologies. At a minimum, a zone of rock approximately 200 ft above the mined coal was dewatered beneath Edd Fork. Ground-water levels in ridgetop piezometers fluctuated slightly more after mining than they did before, which indicates that the upper part of the ridge is more hydraulically connected to surface recharge from precipitation since mining took place. The existence of ground water in the shallow ridgetop piezometers suggests that an underlying aquitard zone developed during mine collapse, which retards the downward movement of shallow ground water to the mined-out area. Water level declined in a sandstone unit approximately 300 ft above the mine after mining, but recovered within a year. This indicates that the underlying regional aquitard still retards downward ground-water movement, despite the hydraulic conductivity of the unit increasing 100 times after mining. Edd Fork, approximately 375 ft above the mine in panel 7, resumed surface flow 2 months after completion of mining; however, flow diminishes downstream at about the centerline of panel 8. Mining is still active in other areas of the mine, and mechanical dewatering activities will most likely keep water levels in the deep zones artificially depressed in the study area until mining is completed and dewatering activities cease

Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro

Introduction: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells. Methods: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. Results: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. Conclusions: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance

The articulation of enkinaesthetic entanglement

In this article I present an argument for the necessary co-articulation of meaning within our felt enkinaesthetic engagement with our world. The argument will be developed through a series of stages, the first of which will be an elaboration of the notion of articulation of and through the body. This will be followed by an examination of enkinaesthetic experiential entanglement and the role it plays in rendering our world meaningful and our actions values-realising. At this stage I will begin to extend Husserl’s notion of intentional transgression to the enkinaesthetic sphere of lived experience, and in support of this claim I will examine the theoretical and practical work of osteopathic manual listening [Gens &amp; Roche 2014] and the ‘felt sense’ in focusing [Gendlin] which makes possible a shift from a somatic articulation to a semantic, and potentially conceptual, one. Throughout, my position will be compatible with Merleau-Ponty’s claim that “Whenever I try to understand myself, the whole fabric of the perceptible world comes too, and with it comes the others who are caught in it.” [Merleau-Ponty 1964a, p.15]

Overexpression of Mcl-1 exacerbates lymphocyte accumulation and autoimmune kidney disease in lpr mice

Cell death by apoptosis has a critical role during embryonic development and in maintaining tissue homeostasis. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas/APO-1; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by prosurvival and pro-apoptotic members of the Bcl-2 family of proteins. Pro-survival Mcl-1, which can block activation of the proapoptotic proteins, Bax and Bak, appears critical for the survival and maintenance of multiple haemopoietic cell types. To investigate the impact on haemopoiesis of simultaneously inhibiting both apoptosis pathways, we introduced the vavP-Mcl-1 transgene, which causes overexpression of Mcl-1 protein in all haemopoietic lineages, into Faslpr/lpr mice, which lack functional Fas and are prone to autoimmunity. The combined mutations had a modest impact on myelopoiesis, primarily an increase in the macrophage/monocyte population in Mcl-1tg/lpr mice compared with lpr or Mcl-1tg mice. The impact on lymphopoiesis was striking, with a marked elevation in all major lymphoid subsets, including the non-conventional double-negative (DN) T cells (TCRβ+ CD4– CD8– B220+ ) characteristic of Faslpr/lpr mice. Of note, the onset of autoimmunity was markedly accelerated in Mcl-1tg/lpr mice compared with lpr mice, and this was preceded by an increase in immunoglobulin (Ig)-producing cells and circulating autoantibodies. This degree of impact was surprising, given the relatively mild phenotype conferred by the vavP-Mcl-1 transgene by itself: a two- to threefold elevation of peripheral B and T cells, no significant increase in the non-conventional DN T-cell population and no autoimmune disease. Comparison of the phenotype with that of other susceptible mice suggests that the development of autoimmune disease in Mcl-1tg/lpr mice may be influenced not only by Ig-producing cells but also other haemopoietic cell types

The impact of induction and/or concurrent chemoradiotherapy on acute and late patient-reported symptoms in oropharyngeal cancer:Application of a mixed-model analysis of a prospective observational cohort registry

BACKGROUND The goal of this study was to comprehensively investigate the association of chemotherapy with trajectories of acute symptom development and late symptom recovery in patients with oropharyngeal cancer (OPC) by comparing symptom burden between induction chemotherapy followed by concurrent chemoradiotherapy (ICRT), concurrent chemo-radiotherapy (CRT), or radiotherapy (RT) alone.METHODS Among a registry of 717 patients with OPC, the 28-item patient-reported MD Anderson Symptom Inventory-Head and Neck Module (MDASI-HN) symptoms were collected prospectively at baseline, weekly during RT, and 1.5, 3 to 6, 12, and 18 to 24 months after RT. The effect of the treatment regimen (ICRT, CRT, and RT alone) was examined with mixed-model analyses for the acute and late period. In the CRT cohort, the chemotherapy agent relationship with symptoms was investigated.RESULTS Chemoradiation (ICRT/CRT) compared with RT alone resulted in significantly higher acute symptom scores in the majority of MDASI-HN symptoms (ie, 21 out of 28). No late symptom differences between treatment with or without chemotherapy were observed that were not attributable to ICRT. Nausea was lower for CRT with carboplatin than for CRT with cisplatin; cetuximab was associated with particularly higher scores for acute and late skin, mucositis, and 6 other symptoms. The addition of ICRT compared with CRT or RT alone was associated with a significant increase in numbness and shortness of breath.CONCLUSION The addition of chemotherapy to definitive RT for OPC patients was associated with significantly worse acute symptom outcomes compared with RT alone, which seems to attenuate in the late posttreatment period. Moreover, induction chemotherapy was specifically associated with worse numbness and shortness of breath during and after treatment.LAY SUMMARYChemotherapy is frequently used in addition to radiotherapy cancer treatment, yet the (added) effect on treatment-induced over time is not comprehensively investigatedThis study shows that chemotherapy adds to the symptom severity reported by patients, especially during treatment</p

Patterns of Failure after IMRT in Head and Neck Squamous Cell Carcinoma of Unknown Primary: Implication of Elective Nodal and Mucosal Dose Coverage

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