RESUMEN
BACKGROUND: Invasive lobular breast cancer (ILC) is the second most common type of breast cancer after invasive breast cancer of no special type (NST), representing up to 15% of all breast cancers. DESIGN: Latest data on ILC are presented, focusing on diagnosis, molecular make-up according to the European Society for Medical Oncology Scale for Clinical Actionability of molecular Targets (ESCAT) guidelines, treatment in the early and metastatic setting and ILC-focused clinical trials. RESULTS: At the imaging level, magnetic resonance imaging-based and novel positron emission tomography/computed tomography-based techniques can overcome the limitations of currently used imaging techniques for diagnosing ILC. At the pathology level, E-cadherin immunohistochemistry could help improving inter-pathologist agreement. The majority of patients with ILC do not seem to benefit as much from (neo-)adjuvant chemotherapy as patients with NST, although chemotherapy might be required in a subset of high-risk patients. No differences in treatment efficacy are seen for anti-human epidermal growth factor receptor 2 (HER2) therapies in the adjuvant setting and cyclin-dependent kinases 4 and 6 inhibitors in the metastatic setting. The clinical utility of the commercially available prognostic gene expression-based tests is unclear for patients with ILC. Several ESCAT alterations differ in frequency between ILC and NST. Germline BRCA1 and PALB2 alterations are less frequent in patients with ILC, while germline CDH1 (gene coding for E-cadherin) alterations are more frequent in patients with ILC. Somatic HER2 mutations are more frequent in ILC, especially in metastases (15% ILC versus 5% NST). A high tumour mutational burden, relevant for immune checkpoint inhibition, is more frequent in ILC metastases (16%) than in NST metastases (5%). Tumours with somatic inactivating CDH1 mutations may be vulnerable for treatment with ROS1 inhibitors, a concept currently investigated in early and metastatic ILC. CONCLUSION: ILC is a unique malignancy based on its pathological and biological features leading to differences in diagnosis as well as in treatment response, resistance and targets as compared to NST.
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Neoplasias de la Mama , Carcinoma Ductal de Mama , Carcinoma Lobular , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Cadherinas/uso terapéutico , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/diagnóstico , Carcinoma Lobular/genética , Carcinoma Lobular/terapia , Femenino , Humanos , Pronóstico , Proteínas Proto-OncogénicasRESUMEN
The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.
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Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Humanos , Pirrolidinonas/farmacología , Quinolonas/farmacología , Raltegravir PotásicoRESUMEN
Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.
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Aequorina/biosíntesis , Aequorina/genética , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Aequorina/metabolismo , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Aparato de Golgi/efectos de los fármacos , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/farmacologíaRESUMEN
Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.
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Señalización del Calcio , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Queratinocitos/metabolismo , Animales , Células COS , ATPasas Transportadoras de Calcio/genética , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , TransfecciónRESUMEN
Non-mitochondrial intracellular Ca2+ stores contain both thapsigargin-sensitive sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and thapsigargin-insensitive secretory-pathway Ca2+-ATPases (SPCA1). We now have studied the Ca2+-release properties of the compartments associated with these pumps in intact, i.e. non-permeabilized, cells of different origin (HeLa, keratinocytes, 16HBE14o-, COS-1, A7r5) and with different approaches (45Ca2+ fluxes, Ca2+ imaging and measurements of the free luminal [Ca2+] in the endoplasmic-reticulum and the Golgi apparatus using targeted aequorin). Application of an extracellular agonist in the absence of thapsigargin induced in all cells a Ca2+ release from both the endoplasmic-reticulum and the Golgi apparatus. The agonists were not able to release Ca2+ in the presence of 10 microM thapsigargin, except in COS-1 cells overexpressing SPCA1, where this pump not only appeared in the Golgi compartment but also overflowed into the agonist-sensitive part of the endoplasmic-reticulum. We conclude that the subcompartments of the endoplasmic-reticulum and of the Golgi complex that endogenously express SPCA1 are insensitive to agonist stimulation.
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ATPasas Transportadoras de Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Inositol 1,4,5-Trifosfato/agonistas , Inositol 1,4,5-Trifosfato/biosíntesis , Tapsigargina/farmacología , Animales , Células COS , Calcio/metabolismo , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Células HeLa , Humanos , Masculino , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.
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ATPasas Transportadoras de Calcio/metabolismo , Músculo Esquelético/enzimología , Adaptación Fisiológica , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/genética , Frío , Cartilla de ADN , ADN Complementario , Hidrólisis , Cinética , Datos de Secuencia Molecular , Músculo Esquelético/fisiología , Ranidae , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Homología de Secuencia de AminoácidoRESUMEN
In recent years, it has been well established that the Ca(2+) concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca(2+) and Mn(2+) concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I. We found evidence for alternative splicing in the 5'-untranslated region, but no indication for the generation of different protein isoforms. C. elegans PMR1 overexpressed in COS-1 cells transports Ca(2+) and Mn(2+) with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca(2+) can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca(2+) store.
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Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Aparato de Golgi/metabolismo , Magnesio/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Clonación Molecular , Datos de Secuencia Molecular , Mutación , Alineación de SecuenciaRESUMEN
The sarco-endoplasmic reticulum Ca(2+)-transport ATPase (SERCA) loads intracellular releasable Ca(2+) stores by transporting cytosolic Ca(2+) into the endoplasmic (ER) or sarcoplasmic reticulum (SR). We characterized the only SERCA homologue of the nematode Caenorhabditis elegans, which is encoded by the sca-1 gene. The sca-1 transcript is alternatively spliced in a similar mode as the vertebrate SERCA2 transcript, giving rise to two protein variants: CeSERCAa and CeSERCAb. These proteins showed structural and functional conservation to the vertebrate SERCA2a/b proteins. The CeSERCAs were primarily expressed in contractile tissues. Loss of CeSERCA through gene ablation or RNA interference resulted in contractile dysfunctioning and in early larval or embryonic lethality, respectively. Similar defects could be induced pharmacologically using the SERCA-specific inhibitor thapsigargin, which bound CeSERCA at a conserved site. The conservation of SERCA2 homologues in C. elegans will allow genetic and chemical suppressor analyses to identify promising drug targets and lead molecules for treatment of SERCA-related diseases such as heart disease.
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Caenorhabditis elegans/enzimología , ATPasas Transportadoras de Calcio/metabolismo , Músculos/fisiología , Animales , Secuencia de Bases , Células COS , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/fisiología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Larva/crecimiento & desarrollo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tapsigargina/farmacologíaRESUMEN
Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.