Role of the NF-kB pathway and nitric oxide in mammary gland involution after weaning. Implications in breast cancer

INTRODUCCIÓN La glándula mamaria es un órgano dinámico que alcanza su máximo desarrollo funcional con la lactancia, momento en el cual, a través de la producción de leche, aporta nutrientes y protección inmunológica a las crías de los mamíferos. Cuando la lactancia finaliza con el destete, el exceso de tejido glandular debe desaparecer y la glándula sufre una extensa remodelación hasta alcanzar nuevamente un estado pre-gestacional, preparada para un nuevo ciclo. La involución del tejido mamario es un proceso complejo durante el cual se suceden de forma coordinada una serie de eventos como la muerte de las células epiteliales secretoras, la proliferación de tejido adiposo circundante y la remodelación de la estructura glandular. El proceso involutivo puede clasificarse en dos etapas, siendo la primera un proceso reversible en el que predomina la apoptosis, y la segunda fase un fenómeno irreversible, en el que se da la remodelación del epitelio ductal y en el que participan proteasas como las metaloproteasas (MMPs) y las catepsinas. Cabe señalar que las vías de señalización que coordinan este proceso también están implicadas en el desarrollo de cáncer de mama. Es más, si bien desde una perspectiva epidemiológica, el embarazo y la lactancia protegen del desarrollo de neoplasias mamarias a largo plazo, se ha demostrado que los principales mecanismos que controlan la involución también controlan procesos de carcinogénesis. Las cuatro vías principales de señalización que regulan la involución post-lactancia son STAT5/STAT3, NF-κB, TGF-β y PI3K/AKT. Los factores de transcripción STAT3 y NF-κB regulan la expresión de genes de respuesta inflamatoria. Nuestro grupo ha demostrado en trabajos previos que uno de los genes inducidos por NF-κB es la Óxido Nítrico Sintasa Inducible (NOS2). Este enzima es responsable del aumento de los niveles de óxido nítrico (NO) en la glándula mamaria durante el destete. OBJETIVOS: Los objetivos de el presente trabajo son: i) estudiar el papel del NO en la regulación de dos de las principales vías del proceso involutivo, STAT3 y NF-κB; ii) estudiar la existencia de modificaciones postraduccionales inducidas por el NO en proteínas, centrándonos en la nitración de resíduos de tirosina. Posteriormente se trata de esclarecer el efecto de estas modificaciones postraduccionales; iii) estudiar qué mecanismos fisiológicos regulados por NO durante el destete también son activados en células de cáncer de mama que son expuestas a altas concentraciones de NO. RESULTADOS: Para estudiar el papel del NO en la involución de la glándula mamaria tras la lactancia utilizamos ratones knockout (KO) para el gen NOS2 (NOS2-KO). Si bien no se apreciaron diferencias entre los ratones NOS2-KO y los wild-type (WT) durante el embarazo se objetivó que los ratones KO tenían una mayor producción de leche y sus crías una mayor ganancia ponderal durante la lactancia. A su vez, existía un retraso evidente en el inicio de la involución en estos ratones con respecto a los WT. Los animales NOS2-KO presentaban un aumento en la fosforilación de STAT5 durante el destete, con un aumento de los niveles de expresión de β-caseina con respecto al tejido glandular de los WT. Ambos parámetros son características distintivas del período de lactancia. A su vez, si bien en ambos tipos de ratones se objetivó una activación de STAT3, que alcanzó su pico a las 24h tras el destete, esta activación estaba claramente reducida en los ratones NOS2-KO. De hecho, se vio tanto una activación de STAT3 y NF-κB como un aumento de la apoptosis en el tejido glandular de los ratones WT tras el destete; sin embargo, durante el mismo periodo de tiempo, se observó una menor apoptosis (medida como actividad de caspasa 3) así como un retraso en la activación de NF-κB en los ratones KO, en comparación con los WT. Estos resultados sugieren que el NO tiene un papel en la regulación del proceso involutivo post-lactancia, dado que, en ausencia de NOS2, la activación de las cascadas de señalización que rigen la involución se encuentra retrasada, lo cual dilata el inicio de la fase de apoptosis y consecuentemente, de la fase de remodelación tisular. Por otra parte, NO actúa como una especie reactiva e induce modificaciones nitrosativas en lípidos y proteínas, que pueden conllevar activación o inhibición de actividad enzimática. En proteínas estas modificaciones suelen ser s-nitrosilación de tioles de cisteína y nitración de residuos tirosina. Realizamos estudios de nitroproteómica en homogenados de glándula mamaria de rata lactante y tras 72h de destete en los que se demostró un aumento del patrón de nitración durante el destete. Objetivamos que la catepsina D (CD) se nitra durante el destete y que esta modificación postraduccional suponía un aumento en su actividad del 50% en estudios in vitro. El papel de la nitración en la actividad de la CD se confirmó en estudios in vivo mediante los cuales objetivamos que no había diferencias en el procesamiento de la proteasa, pero su actividad proteolítica se encontraba significativamente reducida durante el destete en los ratones KO. Finalmente, mediante un estudio de espectrometría de masas, se determinó que uno de los residuos tirosina nitrados se encontraba en la posición 168 de la cadena pesada. Finalmente, realizamos estudios preliminares para intentar dilucidar qué vías de señalización reguladas por NO durante el destete también se veían afectadas en células de carcinoma de mama. Para ello se utilizó un modelo in vitro de células MCF-7 tratadas con altas concentraciones de SIN-1, un donante de NO. De acuerdo con la literatura, la exposición a concentraciones crecientes de NO inducía disminución de la viabilidad celular, sin embargo, también se objetivó la activación de vías de señalización pro-supervivencia. Concretamente comprobamos que a concentraciones mayores de NO existía un aumento de los niveles de AKT fosforilado y un descenso en IκBα, un inhibidor de NF-κB, el cual se acompañaba de un aumento en los niveles de mRNA de MMP-9, una diana transcripcional de NF-κB. Estos datos preliminares apuntan a la posibilidad de que en células de cáncer de mama, tras estímulo con NO, se activen las mismas vías reguladas por esta molécula durante la involución post-lactancia. CONCLUSIONES: La ausencia de NOS2 implica una disminución en los niveles de NO durante la involución de la glándula mamaria tras el destete, lo cual retrasa pero no impide este proceso fisiológico. Las diferencias más importantes objetivadas han sido a nivel de la activación de las vías STAT3 y NF-κB, lo cual retrasa el inicio de la primera fase del proceso, en la cual la apoptosis predomina. Este retraso también tiene un impacto en la segunda fase de la involución en la que se da la remodelación de la matriz extracelular. Además, el NO induce modificaciones postraduccionales específicas que modulan la función enzimática, por ejemplo la nitración de residuos de tirosina. En este trabajo describimos la nitración de CD que induce su activación. En un modelo de cáncer de mama luminal, la exposición a altas dosis de NO parece inducir señales contradictorias dado que por una parte reduce la viabilidad celular y por otra, active vías anti-apoptóticas como AKT y NF-κB, lo que apunta al papel importante de esta molécula en la regulación de múltiples procesos celulares.INTRODUCTION: The mammary gland is a highly dynamic tissue that reaches its maximal functional differentiation during lactation, when, by means of milk production, provides nutrition and immunological protection to the mammalian offspring. When lactation ends and weaning of the pups takes place, the excess tissue that was developed during pregnancy and lactation is no longer needed and mammary gland has to regress to a pre-pregnant state. Involution of mammary gland is an intricate process during which the events that take place are programmed cell death of secretory epithelial cells, differentiation of adipose tissue, and remodelling of the glandular structure. These coordinated events can be classified in two phases, the first being reversible and consisting mainly of apoptosis, and a second irreversible phase where the remodelling of the ductal epithelium takes place and many proteases are involved, such as metalloproteases (MMPs) and cathepsins. Of note, many of the pathways involved in the post-lactation regression process have also been implicated in breast cancer development, and, although epidemiologically speaking, pregnancy and lactation confer women protection from breast cancer, it has been shown that the main hubs that control mammary gland involution are also involved in tumour development and progression. There are many signalling molecules that play a role in involution, being the main four pathways that orchestrate this process STAT5/STAT3, NF-κB, TGF-β and PI3K/AKT. Among these, STAT3 and NF-κB regulate the expression of genes involved in pro-inflammatory response. Previous work from our group demonstrated that one of the pro-inflammatory genes induced during mammary gland involution is inducible nitric oxide synthase (NOS2). This gene is regulated by NF-κB and it is responsible in part for the increase of nitric oxide (NO) levels found in mammary gland upon weaning. AIMS: The aims of this work were: i) to explore the role of NO as a signalling molecule in the regulation of two of the main pathways implicated in the involution process, STAT3 and NF-κB; ii) to study if there are specific nitrosative modifications induced by NO in proteins that are implicated in mammary gland involution, and how these post-translational modifications affect their function; and finally, iii) to find which physiological mechanisms regulated by NO during weaning are also recruited by neoplastic breast cells when exposed to high NO concentrations. RESULTS: In order to study the role of NO in the involution of mammary tissue after lactation, NOS2-KO (knockout) mice were used. No apparent differences were observed between NOS2- KO and WT (wild-type) animals during pregnancy. However, during lactation KO mice produced more milk and their pups gained more weight. Upon cessation of lactation, a notable delay in involution was observed, compared with WT mice. NOS2-KO mice showed increased phosphorylation of STAT (signal transducer and activator of transcription) 5 during weaning, concomitant with increased β-casein mRNA levels when compared with weaned WT glands, both hallmarks of the lactating period. In contrast, activation of STAT3, although maximal at 24h after weaning, was significantly reduced in NOS2-KO mice. Indeed, activation of both STAT3 and NF-κB was observed in WT mice during weaning, concomitant with an increased apoptotic rate. During the same period, less apoptosis, in terms of caspase 3 activity, was found in NOS2-KO mice and NF-κB activity was significantly delayed when compared with WT mice. These results emphasize the role of NO in the fine regulation of the weaning process, since, in the absence of NOS2, the switching on of the cascades that trigger involution is hindered for a time, retarding apoptosis of the epithelial cells and extracellular matrix remodelling. On the other hand, it is well known that NO can act as a reactive species and induce nitrosative modifications in lipids and proteins, leading to gain or loss of function. In proteins, these modifications manifest in two main forms, s-nitrosylation of cysteine thiols and nitration of tyrosine residues. For the second aim of our work we performed proteomic studies in the mammary gland of control lactating and 72h weaned rats that showed that there is an increased pattern of nitrated proteins during weaning when compared with controls. We established the novel finding that cathepsin D (CD) is nitrated during weaning. The expression and protein levels of this enzyme were increased after 8 h of litter removal and this up-regulation declined 5days after weaning. However, there was a marked delay in CD activity since it did not increase until 2 days post-weaning and remained high thereafter. This delay between expression and activity pointed to the possibility of a post-translational modification that allowed for the activation of the protease. In order to find out whether nitration of CD regulates its activity, the previously mentioned NOS2-KO model was used. The expression and protein levels of this enzyme were similar to WT animals, but the proteolytic activity was significantly reduced during weaning in KO compared to WT mice. In vitro treatment of recombinant human CD or lactating mammary gland homogenates with relatively low concentrations of peroxynitrite enhanced the nitration as well as specific activity of this enzyme. Using mass spectrometry, we showed that the residue Tyr168 is nitrated. All of these results point that NO plays a role in the regulation of the pathways involved in mammary gland remodelling after weaning. Lastly, we performed some preliminary work in order to figure out which of the pathways we found to be regulated by NO during the physiological process of involution, were also affected in a luminal breast cancer model. For this, we performed in vitro experiments in which we exposed breast cancer MCF-7 cell line to high concentrations of a NO donor (SIN-1). We saw that even if exposure of cells to high concentrations of NO decreased cell viability, there was also recruitment of pro-survival mechanisms such as AKT and NF-κB. Indeed, we confirmed an increase in levels of phosphorylated AKT as well as a decrease in the NF-κB negative regulator IκBα, which was accompanied with an increase of MMP-9 mRNA levels, a gene known to be regulated at a transcriptional level by NF-κB. This preliminary data point to the possibility that breast tumour cells, upon NO stimulation, activate the same mechanisms observed during weaning. CONCLUSIONS: The absence of NOS2 results in lower NO levels during post-lactational mammary gland involution, which in turn delays this process. The most remarkable differences have been observed specifically in the activation of the Stat5/Stat3 switch and the activation of NF-κB affecting the first phase of the process, where apoptosis is mainly involved, this delay has further impact on the extracellular matrix (ECM) remodelling that takes place during the second phase of involution. Also, NO induces specific post-translational modifications that modulate protein function such as tyrosine-nitration of CD which results in its activation. Finally, in a luminal breast cancer model, in vitro exposure to high concentrations of NO seems to induce contradictory signals since it leads to cell death but the remaining cells present AKT phosphorylation and NF-κB activation, which are pro-survival signals, underlining the important role of this molecule in the fine-tuning of many cellular processes

Selective inhibition of HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.Research in the E.D.A. lab is supported by Asociación Española Contra el Cáncer (AECC), the Ministry of Science of Spain-FEDER (CIBERONC, PI1700464, PI2000003, RD06/0020/0059)S. D.G.D. and L.H.P. are supported by CIBERONC (CB16/12/00361). D.G.D., M.J.R. and L.H.P. are PhD researchers funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016, ECAI F2-0012-2018 and PI-0013-2018, respectively).Peer reviewe

Manual de señalética

Con el objetivo de garantizar unas indicaciones claras y efectivas que ayuden a orientarse en las instalaciones de la Universidad de Zaragoza, se crea este Manual de Señalética. Además, este documento trata de unificar y consolidad la imagen gráfica presente en los espacios de la Universidad de Zaragoza, basada en la actual imagen corporativa. Por tanto, el punto de partida de este manual lo constituye el Manual de Identidad Corporativa aprobado por el Consejo de Gobierno de la Universidad de Zaragoza el 6 de julio de 2010. El presente manual cumple con la normativa actual vigente UNE 170002 Requisitos de accesibilidad para la rotulación. Durante el proceso de redacción, el manual ha sido consultado por la Oficina Universitaria de Atención a la Diversidad para ofrecer un mayor grado de accesibilidad en los diseños propuestos

Process evaluation of the Bridging the Age Gap in Breast Cancer decision support intervention cluster randomized trial [abstract only]

Aims/Objectives: Shared decision making on the choice of treatment for older women with breast cancer involves many factors. Comprehensive geriatric assessment (CGA) is recognised to have a role in older patients with cancer, but how this should be utilised is still debatable. A pilot study involving older women newly diagnosed with early operable primary breast cancer was conducted aiming to explore the potential value of CGA. Methods: Decision of primary treatment followed consultation with the clinical team and was not guided by any aspect of this study. CGA, using a validated cancer-specific tool from our collaborator, A Hurria, was conducted within 6 weeks after diagnosis, regardless of date of surgery/first treatment. A total of 178 female patients aged ≥70 years with a new diagnosis of early (stage 1 or 2; cT0-2, N0-1, M0) operable primary breast cancer proven histologically, were thus far recruited from three UK centres. Results: Among these 178 patients, 149 underwent primary surgery and 29 received non-surgical treatment (primary endocrine therapy (N=28) or radiotherapy (N=1)). CGA determined that increasing age (p=0.006), reduced independence with activities of daily living (ADLs) (p=0.001) and independent activities of daily living (IADLS) (p=0.001), increased number and severity of comorbidity (p=0.043), reduced Karnofsky performance status when rated both by the patient (p=0.001) and physician (p=0.003), were significantly related to non-surgical treatment within 6 weeks after diagnosis. Other CGA parameters measured which were not significant include number of daily medications, level of social support, level of social activity, cognition, number of falls, 'Timed up and go' score. Conclusions: The pilot study has confirmed that CGA may have value in assessing this cohort of patients. Generally, it appears that patients receiving non-surgical treatment are more frail than their counterparts undergoing surgery. The study is ongoing and has expanded to include an international centre

A polymorphism at the 3'-UTR region of the aromatase gene defines a subgroup of postmenopausal breast cancer patients with poor response to neoadjuvant letrozole

<p>Abstract</p> <p>Background</p> <p>Aromatase (<it>CYP19A1</it>) regulates estrogen biosynthesis. Polymorphisms in <it>CYP19A1 </it>have been related to the pathogenesis of breast cancer (BC). Inhibition of aromatase with letrozole constitutes the best option for treating estrogen-dependent BC in postmenopausal women. We evaluate a series of polymorphisms of <it>CYP19A1 </it>and their effect on response to neoadjuvant letrozole in early BC.</p> <p>Methods</p> <p>We analyzed 95 consecutive postmenopausal women with stage II-III ER/PgR [+] BC treated with neoadjuvant letrozole. Response to treatment was measured by radiology at 4^thmonth by World Health Organization (WHO) criteria. Three polymorphisms of <it>CYP19A1</it>, one in exon 7 (rs700519) and two in the 3'-UTR region (rs10046 and rs4646) were evaluated on DNA obtained from peripheral blood.</p> <p>Results</p> <p>Thirty-five women (36.8%) achieved a radiological response to letrozole. The histopathological and immunohistochemical parameters, including hormonal receptor status, were not associated with the response to letrozole. Only the genetic variants (AC/AA) of the rs4646 polymorphism were associated with poor response to letrozole (p = 0.03). Eighteen patients (18.9%) reported a progression of the disease. Those patients carrying the genetic variants (AC/AA) of rs4646 presented a lower progression-free survival than the patients homozygous for the reference variant (p = 0.0686). This effect was especially significant in the group of elderly patients not operated after letrozole induction (p = 0.009).</p> <p>Conclusions</p> <p>Our study reveals that the rs4646 polymorphism identifies a subgroup of stage II-III ER/PgR [+] BC patients with poor response to neoadjuvant letrozole and poor prognosis. Testing for the rs4646 polymorphism could be a useful tool in order to orientate the treatment in elderly BC patients.</p

Commentaries on viewpoint : physiology and fast marathons

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A global experiment on motivating social distancing during the COVID-19 pandemic

Finding communication strategies that effectively motivate social distancing continues to be a global public health priority during the COVID-19 pandemic. This cross-country, preregistered experiment (n = 25,718 from 89 countries) tested hypotheses concerning generalizable positive and negative outcomes of social distancing messages that promoted personal agency and reflective choices (i.e., an autonomy-supportive message) or were restrictive and shaming (i.e., a controlling message) compared with no message at all. Results partially supported experimental hypotheses in that the controlling message increased controlled motivation (a poorly internalized form of motivation relying on shame, guilt, and fear of social consequences) relative to no message. On the other hand, the autonomy-supportive message lowered feelings of defiance compared with the controlling message, but the controlling message did not differ from receiving no message at all. Unexpectedly, messages did not influence autonomous motivation (a highly internalized form of motivation relying on one’s core values) or behavioral intentions. Results supported hypothesized associations between people’s existing autonomous and controlled motivations and self-reported behavioral intentions to engage in social distancing. Controlled motivation was associated with more defiance and less long-term behavioral intention to engage in social distancing, whereas autonomous motivation was associated with less defiance and more short- and long-term intentions to social distance. Overall, this work highlights the potential harm of using shaming and pressuring language in public health communication, with implications for the current and future global health challenges

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

High expression of cholesterol biosynthesis genes is associated with resistance to statin treatment and inferior survival in breast cancer

There is sufficient evidence that statins have a protective role against breast cancer proliferation and recurrence, but treatment predictive biomarkers are lacking. Breast cancer cell lines displaying diverse sensitivity to atorvastatin were subjected to global transcriptional profiling and genes significantly altered by statin treatment were identified. Atorvastatin treatment strongly inhibited proliferation in estrogen receptor (ER) negative cell lines and a commensurate response was also evident on the genome-wide transcriptional scale, with ER negative cells displaying a robust deregulation of genes involved in the regulation of cell cycle progression and apoptosis. Interestingly, atorvastatin upregulated genes involved in the cholesterol biosynthesis pathway in all cell lines, irrespective of sensitivity to statin treatment. However, the level of pathway induction; measured as the fold change in transcript levels, was inversely correlated to the effect of statin treatment on cell growth. High expression of cholesterol biosynthesis genes before treatment was associated with resistance to statin therapy in cell lines and clinical biopsies. Furthermore, high expression of cholesterol biosynthesis genes was independently prognostic for a shorter recurrence-free and overall survival, especially among ER positive tumors. Dysregulation of cholesterol biosynthesis is therefore predictive for both sensitivity to anti-cancer statin therapy and prognosis following primary breast cancer diagnosis