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[EN] Deep knowledge of the regeneration processes after a forest fire is key to addressing their adverse environmental impacts, these are especially evident in the vegetation. In the post-fire environment context, the fire severity constitutes a critical variable that affects the ecosystem response in terms of vegetation recovery and hydrogeomorphological dynamics after the fire. Therefore, the severity accurate assessment is essential for the burned areas management because of it allows the identification of priority areas and, therefore, it helps to carry out recovery strategies and measures. The area of interest is located in the natural place of Las Peñuelas (Huelva), where a large fire took place on June 24, 2017 that affected almost 10 000 ha. The methodology was based on the calculation of the RBR (Relativized Burn Ratio) spectral index to estimate the severity of the fire, and the NDVI (Normalized Difference Vegetation Index) index to evaluate the recovery of vegetal vigor. For the work, images from the Sentinel-2 and Pleiades satellites, images acquired by UAV (Unmanned Aerial Vehicle) and field samplings were used. The result was a cartography showing the levels of recovery or degradation of the affected vegetation., [ES] Conocer en profundidad los procesos de regeneración después de un incendio forestal es fundamental para afrontar las consecuencias adversas de éstos sobre el medio natural. En este contexto, la severidad del fuego constituye una variable crítica porque condiciona la respuesta del ecosistema en términos de regeneración vegetal y dinámica hidrogeomorfológica tras el incendio. Por tanto, su correcta evaluación es fundamental para la gestión de las zonas quemadas ya que permite priorizar las áreas de actuación y, por tanto, ayuda en la adopción de estrategias y medidas de recuperación. El área de trabajo está localizada en el paraje de Las Peñuelas (Huelva), donde tuvo lugar un gran incendio el 24 de junio de 2017 que afectó a unas 10 000 ha. La metodología se basó en el cálculo del índice espectral RBR (Relativized Burn Ratio) para la estimación de la severidad, y el índice NDVI (Normalized Difference Vegetation Index) para la evaluación de la recuperación del vigor vegetal. Para el trabajo se utilizaron imágenes procedentes de los satélite Sentinel-2 y Pléiades, fotografías adquiridas mediante UAV (Unmanned Aerial Vehicle), y muestreos de campo. El resultado fue una cartografía donde se muestran los niveles de recuperación o retroceso de la vegetación afectada., En el marco de los trabajos del Grupo de Trabajo Científico-Técnico encargado de la restauración del incendio de “Las Peñuelas” 2017, este artículo ha sido posible gracias a la colaboración del Espacio Natural de Doñana, por su contribución en la recogida de datos en las distintas campañas de campo y sus aportaciones técnicas en relación con el conocimiento exhaustivo del ámbito afectado. Por otro lado, agradecemos a AMAYA y La Caixa la aportación de información procedente de la campaña de campo con dron. También queremos agradecer a AIRBUS Defence and Space su colaboración en la optimización de procesos operativos y administrativos para la adquisición de imágenes Pléiades por parte de Rediam.

When reinforced concrete elements are subjected to high temperatures, such as in a fire, they are susceptible to physical and chemical changes that cause spalling, thereby undermining their performance under such conditions. It is known that the age and the internal moisture content of concrete are factors that contribute to this event, but the intensity of spalling is not yet a consensus. This study aimed to assess the influence of age and internal moisture on the performance of concrete walls at high temperatures. Therefore, 6 real-scale walls were built with dimensions of 3.15 × 3.00 m, with the same composition of concrete, for tests in a vertical furnace under the ISO 834 curve, for ages of 7, 14, 28, 56, 84 and 830 days. Moisture was measured as per the electrical resistivity of concrete. It was noted that walls with ages above 84 days showed no spalling whatsoever, due to the internal moisture of concrete. The most severe spalling took place at 14 days, thus evidencing that pore interconnectivity and hydrated cement crystallization can contribute as well. Keywords: Reinforced concrete, Spalling, Fire resistance, Non-load bearing wall system

Ahead of the incorporation of residues in concrete composition, there is a decline in the environmental impact of buildings. One of the goals of today’s development is the employment of low-impact energy sources, such as thermoelectric. Thermoelectric industries display a high rate of residues, among which is pointed out coal bottom ash (CBA) (15% total residues), which have density superior to fly ash and accumulates in silos. Considering this scenario, this paper assessed the incorporation feasibility of CBA in the composition of cellular concrete, replacing silica fume. This study comprehended CBA characterization and application. For such, it was performed a scanning electron microscope (SEM) associated with Energy dispersive spectroscopy (EDS) analysis, laser granulometry, X-ray diffraction (XRD), X-ray efflorescence and density. Following the incorporation of the residues in cellular concrete in place of silica fume, it was performed compression strength analysis and SEM anew, evaluating the impact of CBA’s insertion in concrete’s microstructure. The XRD results are complementary to the other analyses. With SEM technique, it was observed the predominance of spherical-shape particles. The compressive strength of CBA concretes was superior to the reference concrete from 0,13 to 0,74MPa. RESUMO Diante da incorporação de resíduos no concreto, ocorre uma diminuição do impacto ambiental das edificações. Um dos objetivos do desenvolvimento atual é o emprego de fontes de energia de baixo impacto, como a cinzas pesadas de carvão (coal bottom ash, CBA) (15% resíduos totais), que possuem densidade superior às cinzas volantes e se acumulam nos silos. Diante desse cenário, o presente trabalho avaliou a viabilidade de incorporação da CBA na composição do concreto celular, em substituição à sílica ativa. Este estudo compreendeu a caracterização e aplicação da CBA. Para tal, foi realizada microscopia eletrônico de varredura (MEV) associado à análise de energia dispersiva (EDS), granulometria a laser, difração de raios X (DRX), eflorescência de raios X e densidade. Após a incorporação dos resíduos no concreto celular substituindo a sílica ativa, foi realizada análise de resistência à compressão e novamente MEV, avaliando o impacto da inserção da CBA na microestrutura do concreto. Os resultados de XRD são complementares às outras análises. Com a técnica SEM, observou-se a predominância de partículas com formato esférico. A resistência à compressão dos concretos CBA foi superior ao concreto de referência variando de 0,13 a 0,74MPa.

Accountability, involvement, empowerment of the patients and families/caregiver, as well as the incentive for health behaviors based on physical exercise and healthy living habits with the support of health professionals, are fundamental in meeting the demands of the current society. The main objective of this report is to describe the process of developing skills in rehabilitation nursing that was possible in the context of the final stage in which an intervention program in the area of cardiac rehabilitation was designed, implemented and evaluated. In this quantitative study with exploratory characteristics, it was used the collection of observable and quantifiable data, from patients submitted to coronary artery bypass grafting. During the study, the Kolmogorov-Smirnov test was applied-being verified the normal distribution-and Student t-test, which aimed at comparing outcomes at the beginning and end of the program. When 6-min walk test was applied, there was an optimization of functional capacity (P=0.05), can be proved by the fact that all the patients had statistically significant improvements in the time-walk relationship. Furthermore, the quality of life in those who participated in the program was moderate in the preoperative period with 53.09%, and at the end a significant improvement was observed with 75.02%. Cardiac rehabilitation is fundamental in the patient submitted to coronary artery bypass grafting, taking into account the educational, physical exercise, and quality of life dimensions. Given the prevalence of risk factors associated with cardiovascular disease, differentiated intervention in rehabilitation nursing becomes essential in the training of patients for self-care.

Simple Summary Therapeutic options for the treatment of men with metastatic castration-resistant prostate cancer are limited. Docetaxel—a taxane-based chemotherapeutic agent—was the first treatment to demonstrate significant efficacy in the treatment of this disease. However, responses to docetaxel are frequently curtailed by development of drug resistance, and patients eventually succumb to disease progression due to acquisition of drug resistance. In this study, we established drug-resistant prostate cancer cell lines and identified several mechanisms that may be associated with the development of drug resistance in prostate cancer. Actioning these mechanisms could provide a potential approach to re-sensitize drug-resistant cancer cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with metastatic castration-resistant prostate cancer. Abstract Docetaxel—a taxane-based chemotherapeutic agent—was the first treatment to demonstrate significant improvements in overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, the response to docetaxel is generally short-lived, and relapse eventually occurs due to the development of resistance. To explore the mechanisms of acquired docetaxel resistance in prostate cancer (PCa) and set these in the context of androgen deprivation therapy, we established docetaxel-resistant PCa cell lines, derived from the androgen-dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line. We generated two docetaxel-resistant LNCaPR and C4-2BR sub-lines, with IC50 values 77- and 50-fold higher than those of the LNCaP and C4-2B parental cells, respectively. We performed gene expression analysis of the matched sub-lines and found several alterations that may confer docetaxel resistance. In addition to increased expression of ABCB1, an ATP-binding cassette (ABC) transporter, and a well-known gene associated with development of docetaxel resistance, we identified genes associated with androgen signaling, cell survival, and overexpression of ncRNAs. In conclusion, we identified multiple mechanisms that may be associated with the development of taxane drug resistance in PCa. Actioning these mechanisms could provide a potential approach to re-sensitization of docetaxel-resistant PCa cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with mCRPC.

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. (c) 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

This study aimed to investigate if combined analysis of pro-Neuropeptide Y (NPY) and ERG expression in tumor tissue are associated with biochemical failure (BF), castration-based treatment, castration-resistant prostate cancer (CRPC), and prostate cancer (PCa)-specific death for men undergoing radical prostatectomy (RP) for PCa. This study included 315 patients, who underwent RP from 2002 to 2005. Both pro-NPY and ERG expression were analyzed using immunohistochemistry and were scored as low or high and negative or positive, respectively. Risk of BF, castration-based treatment, CRPC, and PCa-specific death were analyzed with multiple cause-specific Cox regression analyses and stratified cumulative incidences using competing risk assessment. Median follow-up was 13.0 years (95% CI: 12.7-13.2). In total, 85.7% were pro-NPY high and 14.3% were pro-NPY low. The combined analyses of pro-NPY and ERG expression was not associated with risk of BF (p = 0.7), castration-based treatment (p = 0.8), CRPC (p = 0.4) or PCa-specific death (p = 0.5). In the multiple cause-specific Cox regression analysis, pro-NPY high and ERG positivity was not associated with BF (HR: 1.02; 95% CI 0.6-1.7; p = 0.94). In conclusion the combination of pro-NPY and ERG expression did not show association with risk of BF, castration-based treatment, CRPC, and PCa-specific death following RP.

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2 , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+ /H+ exchanger NHE1, the Na+ - HCO3- cotransporter NBCn1, and the lactate-H+ cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA-mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line significantly reduced steady-state intracellular pH (pHi ) and capacity for pHi recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA-MB-231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor-free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that comprises various disease entities, all of which share a set of common features: a lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, respectively. Because of their receptor status, conventional chemotherapy remains the main therapeutic option for TNBC patients. We employed a reverse phase protein array approach (RPPA), complemented by immunohistochemistry, to quantitatively profile the activation state of 84 actionable key signaling intermediates and phosphoproteins in a set of 44 TNBC samples. We performed supervised and unsupervised approaches to proteomic data analysis to identify groups of samples sharing common characteristics that could be amenable to existing therapies. We found the heterogenous activation of multiple pathways, with PI3 K/AKT/mTOR signaling being the most common event. Some specific individualized therapeutic possibilities include the expression of oncogenic KIT in association with cytokeratin 15 and Erk1/2 positive tumors, both of which may have clinical value.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide causing an estimated 700 000 deaths annually. Different types of treatment are available for patients with advanced metastatic colorectal cancer, including targeted biological agents, such as cetuximab, a monoclonal antibody that targets EGFR. We have previously reported a study indicating multiple levels of interaction between metallopeptidase inhibitor 1 (TIMP‐1) and the epidermal growth factor (EGF) signaling axis, which could explain how TIMP‐1 levels can affect the antitumor effects of EGFR inhibitors. We also reported an association between TIMP‐1‐mediated cell invasive behavior and KRAS status. To gain insight into the molecular mechanisms underlying the effects of TIMP‐1 in CRC, we examined by transcriptomics, proteomics, and kinase activity profiling a matched pair of isogenic human CRC isogenic DLD‐1 CRC cell clones, bearing either an hemizygous KRAS wild‐type allele or KRAS G13D mutant allele, exposed, or not, to TIMP‐1. Omics analysis of the two cell lines identified the receptor tyrosine kinase c‐Kit, a proto‐oncogene that can modulate cell proliferation and invasion in CRC, as a target for TIMP‐1. We found that exposure of DLD‐1 CRC cells to exogenously added TIMP‐1 promoted phosphorylation of c‐Kit, indicative of a stimulatory effect of TIMP‐1 on the c‐Kit signaling axis. In addition, TIMP‐1 inhibited c‐Kit shedding in CRC cells grown in the presence of exogenous TIMP‐1. Given the regulatory roles that c‐Kit plays in cell proliferation and migration, and the realization that c‐Kit is an important oncogene in CRC, it is likely that some of the biological effects of TIMP‐1 overexpression in CRC may be exerted through its effect on c‐Kit signaling., We identified a proto‐oncogene, c‐Kit, as a potential mediator of metallopeptidase inhibitor 1 (TIMP‐1) function in colorectal cancer (CRC) cells. Given the role that c‐Kit plays in regulation of cell proliferation and migration, it is likely that some of the effects of TIMP‐1 overexpression in CRC may be exerted through its effect on c‐Kit signaling.

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Here, we describe an MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from pathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including less than 10,000 cancer cells isolated by laser-capture microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total work up took less than a day. We observed a moderate trend towards lower protein identifications in long-term stored samples (>15 years) but clustering into distinct proteomic subtypes was independent of archival time. Our results underline the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and prove the feasibility of analyzing large tissue cohorts in a robust, timely and streamlined manner.

HER2/ErbB2 activation turns on transcriptional processes that induce local invasion and lead to systemic metastasis. The early transcriptional changes needed for ErbB2-induced invasion are poorly understood. Here, we link ErbB2 activation to invasion via ErbB2-induced, SUMO-directed phosphorylation of a single serine residue, S27, of the transcription factor myeloid zinc finger-1 (MZF1). Utilizing an antibody against MZF1-pS27, we show that the phosphorylation of S27 correlates significantly (p

The AMP (adenosine 5′-monophosphate)-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis that co-ordinates metabolic processes to ensure energy supply meets demand. At the cellular level, AMPK is activated by metabolic stresses that increase AMP or adenosine 5′-diphosphate (ADP) coupled with falling adenosine 5′-triphosphate (ATP) and acts to restore energy balance by choreographing a shift in metabolism in favour of energy-producing catabolic pathways while inhibiting non-essential anabolic processes. AMPK also regulates systemic energy balance and is activated by hormones and nutritional signals in the hypothalamus to control appetite and body weight. Failure to maintain energy balance plays an important role in chronic diseases such as obesity, type 2 diabetes and inflammatory disorders, which has prompted a major drive to develop pharmacological activators of AMPK. An array of small-molecule allosteric activators has now been developed, several of which can activate AMPK by direct allosteric activation, independently of Thr172 phosphorylation, which was previously regarded as indispensable for AMPK activity. In this review, we summarise the state-of-the-art regarding our understanding of the molecular mechanisms that govern direct allosteric activation of AMPK by adenylate nucleotides and small-molecule drugs.

Background We have previously identified tissue inhibitor of metalloproteinases-1 (TIMP-1) as a prognostic marker in glioblastomas. TIMP-1 has been associated with chemotherapy resistance, and CD63, a known TIMP-1-binding protein, has been suggested to be responsible for this effect. The aim of this study was to assess CD63 expression in astrocytomas focusing on the prognostic potential of CD63 alone and in combination with TIMP-1. Methods CD63 expression was investigated immunohistochemically in a cohort of 111 astrocytomas and correlated to tumor grade and overall survival by semi-quantitative scoring. CD63 expression in tumor-associated microglia/macrophages was examined by double-immunofluorescence with ionized calcium-binding adapter molecule 1 (Iba1). The association between CD63 and TIMP-1 was investigated using previously obtained TIMP-1 data from our astrocytoma cohort. Cellular co-expression of TIMP-1 and CD63 as well as TIMP-1 and the tumor stem cell-related markers CD133 and Sox2 was investigated with immunofluorescence. TIMP-1 and CD63 protein interaction was detected by an oligonucleotide-based proximity ligation assay and verified using co-immunoprecipitation. Results The expression of CD63 was widely distributed in astrocytomas with a significantly increased level in glioblastomas. CD63 levels did not significantly correlate with patient survival at a protein level, and CD63 did not augment the prognostic significance of TIMP-1. Up to 38% of the CD63+ cells expressed Iba1; however, Iba1 did not appear to impact the prognostic value of CD63. A significant correlation was found between TIMP-1 and CD63, and the TIMP-1 and CD63 proteins were co-expressed at the cellular level and located in close molecular proximity, suggesting that TIMP-1 and CD63 could be co-players in glioblastomas. Some TIMP-1+ cells expressed CD133 and Sox2. Conclusion The present study suggests that CD63 is highly expressed in glioblastomas and that TIMP-1 and CD63 interact. CD63 does not add to the prognostic value of TIMP-1. Co-expression of TIMP-1 and stem cell markers as well as the wide expression of CD63 might suggest a role for TIMP-1 and CD63 in glioblastoma stemness. Electronic supplementary material The online version of this article (10.1186/s12885-018-4179-y) contains supplementary material, which is available to authorized users.

Purpose: An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood. Experimental Design: We applied mass spectrometry–based proteomics to a unique set of 17 prostate cancer patient–derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation. Results: An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy. Conclusions: This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.

Textile wastewaters are rated among the most polluting in all industrial sectors, including high organic loads and dyes that are generally resistant to aerobic biotreatment. Alternative, staged anaerobic/aerobic regimes have been tested, with clear success in the biodecolorization of the widely used azo dyes. The novel aerobic granular sludge (AGS) technology has excellent potential in this context, due to the anoxic/anaerobic zones within granules and their increased tolerance to toxicity. In this study the performance of anaerobic/aerobic, flocculent and AGS sequencing batch reactors (SBR) was compared in the treatment of a simulated textile wastewater. Similar color removal yields (75–80%) of an azo dye were attained in both systems but with higher anaerobic and overall COD removal yields in the AGS SBR, thus demonstrating the latter's good potential. Comparison of the toxicity of samples collected from both SBR using yeast-based assays highlighted the better performance of the AGS SBR as compared with the flocculent SBR, with respect to detoxification potential.

Topoisomerase-1 (Top1) targeting drugs have shown promising efficacy in patients with metastatic breast cancer (BC). However, these drugs are rather toxic calling for development and validation of predictive biomarkers to increase the therapeutic index. As these drugs are targeting the Top1 protein and since no validated anti-Top1 antibodies for immunohistochemistry have been reported, we raised the hypothesis that TOP1 gene amplifications may serve as a proxy for the Top1 protein and thereby a biomarker of response to treatment with Top1 inhibitors in BC. The aim was to determine the prevalence of TOP1 gene copy gain in BC. The prevalence of TOP1 gene copy gain was investigated by fluorescence in situ hybridization with a TOP1/CEN-20 probemix in normal breast tissue (N = 100) and in tissue from patients with metastatic BC in a discovery (N = 100) and a validation cohort (N = 205). As amplification of 20q including CEN-20 is common in BC a TOP1/CEN-2 probemix was applied to the validation cohort. More than 30% of the patients had gene copy numbers of ≥ 4 and ∼20% of the patients had TOP1/CEN-20 ratios ≥ 1.5. The CEN-2 probe did not add any information. Gain of the TOP1 gene appears to be common in BC making the gene a potential biomarker for response to treatment with Top1 inhibitors. As 20q amplification is a common finding in BC and as no other suitable reference gene has yet been identified, TOP1 copy number may be a more valid method of detecting gain than using a gene/centromere ratio.

Circulating forms of the urokinase plasminogen activator receptor (uPAR) are associated with prognosis in patients with colorectal cancer. Preclinical studies have shown that uPAR can influence the state of phosphorylation and signalling activity of the epidermal growth factor receptor (EGFR) in a ligand-independent manner. The purpose of the study was to evaluate whether plasma soluble intact and cleaved uPAR(I-III)+(II-III) levels could identify a subpopulation of patients with metastatic colorectal cancer (mCRC) where treatment with cetuximab would have a beneficial effect. Plasma samples were available from 453 patients treated in the NORDIC VII study. Patients were randomized between FLOX and FLOX + cetuximab. The levels of uPAR(I-III)+(II-III) were determined by time-resolved fluorescence immunoassay. We demonstrated that higher baseline plasma uPAR(I-III)+(II-III) levels were significantly associated with shorter progression-free survival (PFS) (HR = 1.30, 1.14-1.48, p = 0.0001) and overall survival (OS) (HR = 1.75, 1.52-2.02, p

Cancer cells utilize lysosomes for invasion and metastasis. Myeloid Zinc Finger1 (MZF1) is an ErbB2-responsive transcription factor that promotes invasion of breast cancer cells via upregulation of lysosomal cathepsins B and L. Here we identify let-7 microRNA, a well-known tumor suppressor in breast cancer, as a direct negative regulator of MZF1. Analysis of primary breast cancer tissues reveals a gradual upregulation of MZF1 from normal breast epithelium to invasive ductal carcinoma and a negative correlation between several let-7 family members and MZF1 mRNA, suggesting that the inverse regulatory relationship between let-7 and MZF1 may play a role in the development of invasive breast cancer. Furthermore, we show that MZF1 regulates lysosome trafficking in ErbB2-positive breast cancer cells. In line with this, MZF1 depletion or let-7 expression inhibits invasion-promoting anterograde trafficking of lysosomes and invasion of ErbB2-expressing MCF7 spheres. The results presented here link MZF1 and let-7 to lysosomal processes in ErbB2-positive breast cancer cells that in non-cancerous cells have primarily been connected to the transcription factor EB. Identifying MZF1 and let-7 as regulators of lysosome distribution in invasive breast cancer cells, uncouples cancer-associated, invasion-promoting lysosomal alterations from normal lysosomal functions and thus opens up new possibilities for the therapeutic targeting of cancer lysosomes.

Objectives Several polyphenols from renewable sources were surveyed for dentin biomodification. However, phenols from cashew nut shell liquid (CNSL, Anacardium occidentale) and from Aroeira ( Myracrodruon urundeuva ) extract have never been evaluated. The present investigation aimed to compare the dentin collagen crosslinking (biomodification) effectiveness of polyphenols from Aroeira stem bark extract, proanthocyanidins (PACs) from grape-seed extract ( Vitis vinifera ), cardol and cardanol from CNSL after clinically relevant treatment for one minute. Methods Three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams before and after biomodification, whilst color change and mass variation were evaluated after four weeks water biodegradation. Color aspect was assessed by optical images after biodegradation whereas collagen cross-linking was investigated by micro-Raman spectroscopy. Statistical analysis was performed with repeated-measures two way ANOVA and Tukey’s test (p Results The increase in elastic modulus after biomodification was in the order cardol > cardanol > aroeira = PACs with cardol solution achieving mean 338.2% increase. The mass increase after biomodification followed the same order aforementioned. Nevertheless, after four weeks aging, more hydrophobic agent (cardanol) induced the highest resistance against water biodegradation. Aroeira and cardol attained intermediate outcomes whereas PACs provided the lower resistance. Tannin-based agents (Aroeira and PACs) stained the specimens in dark brown color. No color alteration was observed with cardol and cardanol treatments. All four agents achieved crosslinking in micro-Raman after one minute application. Significance In conclusion, major components of CNSL yield overall best dentin biomodification outcomes when applied for one minute without staining the dentin collagen.

The burden of colorectal cancer (CRC) is considerable&mdash, approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.

Tissue inhibitor of metalloproteinases 1 (Timp-1), is one of the four known endogenous inhibitors of matrix metalloproteinases (MMPs); in recent years, Timp-1 has become increasingly recognized as a multifunctional protein that, independently of its MMP inhibitory activity, is able to regulate core cellular processes such as cell proliferation and apoptosis. Consistent with this pro-survival function, Timp-1 expression was shown to be able to protect cancer cells from epirubicin or paclitaxel-mediated cytotoxicity. Consistent with this effect, clinical studies have shown high Timp-1 tumor levels to be predictive of resistance to adjuvant anthracycline-based chemotherapy in metastatic breast cancer patients. In spite of abundant evidence directly involving Timp-1 in regulation of cell growth and apoptosis, the downstream mechanisms of Timp-1–mediated cell signaling underlying these effects, and its biological consequences, have remained unclear. In order to address this issue, we aimed to identify cellular binding partners for Timp-1, which may be able to induce signaling. Therefore, we performed yeast two hybrid screening using a mammary gland cDNA library. We report here the identification of a novel Timp-1 interactor, CD74. CD74, also known as MHC class II invariant chain (li), was mainly thought to function as an MCH class II chaperone promoting the exit of MHC class II molecules from the endoplasmic reticulum (ER). However, a fraction of cellular CD74 has been found to traffic to the plasma membrane where it functions as an accessory-signaling molecule, being quickly recycled back into the endosomal pathway. The interaction between TIMP-1 and CD74 was confirmed by co-immunoprecipitation studies in the triple negative breast cancer cell line MDA-MB-231, and we showed that CD74 is necessary for Timp-1 cellular internalization and Timp-1-mediated activation of Akt signaling. To determine the applicability of our findings to a broader context, we analyzed a breast cancer patient cohort for expression of CD74, CD63 and Timp-1 by immunohistochemistry (IHC) and in situ hybridization (ISH). We found that cancer cells which were negative for TIMP-1 mRNA but positive for Timp-1 protein, indicating an active transport of Timp-1 into the cells. These results raise the possibility that CD74 may be a useful target for effecting Timp-1 mediated chemoresistance. Citation Format: José M Moreira, Mikkel Høeberg, Ulrik Ulrik Lademann, Birgitte Viuff, Lena V Jensen, Jan Stenvang, Sune B Nygård, Maj S Ørum-Madsen, Mette V Vistesen, Anja T Fuglsang, Siqi Liu, Nils Brünner. Identification and characterization of a new TIMP-1 binding protein [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-08.

As part of a regulatory loop linking cell metabolism, growth, and proliferation, CIP2A promotes mTORC1-mediated cell growth and autophagy inhibition but is itself down-regulated by autophagy., mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation.

Tumor interstitial fluid (TIF) is a proximal fluid that, in addition to the set of blood soluble phase-borne proteins, holds a subset of aberrantly externalized components, mainly proteins, released by tumor cells and tumor microenvironment through various mechanisms, which include classical secretion, non-classical secretion, secretion via exosomes and membrane protein shedding. Consequently, the interstitial aqueous phase of solid tumors is a highly promising resource for the discovery of molecules associated with pathological changes in tissues. Firstly, it allows one to delve deeper into the regulatory mechanisms and functions of secretion-related processes in tumor development. Secondly, the anomalous secretion of molecules that is innate to tumors and the tumor microenvironment, being associated with cancer progression, offers a valuable source for biomarker discovery and possible targets for therapeutic intervention. Here we provide an overview of the features of tumor-associated interstitial fluids, based on recent and updated information obtained mainly from our studies of breast cancer. Data from the study of interstitial fluids recovered from several other types of cancer are also discussed. This article is a part of a Special Issue entitled: The Updated Secretome.

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)(-) and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.

A limited number of cancer cells within a tumor are thought to have self-renewing and tumor-initiating capabilities that produce the remaining cancer cells in a heterogeneous tumor mass. Elucidation of central pathways preferentially used by tumor-initiating cells/cancer stem cells (CSCs) may allow their exploitation as potential cancer therapy targets. We used single cell cloning to isolate and characterize four isogenic cell clones from a triple-negative breast cancer cell line; two exhibited mesenchymal-like and two epithelial-like characteristics. Within these pairs, one, but not the other, resulted in tumors in immunodeficient NOD/Shi-scid/IL-2 Rγ null mice and efficiently formed mammospheres. Quantitative proteomics and phosphoproteomics were used to map signaling pathways associated with the tumor-initiating ability. Signaling associated with apoptosis was suppressed in tumor-initiating versus nontumorigenic counterparts with pro-apoptotic proteins, such as Bcl2-associated agonist of cell death (BAD), FAS-associated death domain protein (FADD), and myeloid differentiation primary response protein (MYD88), downregulated in tumor-initiating epithelial-like cells. Functional studies confirmed significantly lower apoptosis in tumor-initiating versus nontumorigenic cells. Moreover, central pathways, including β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signaling, exhibited increased activation in the tumor-initiating cells. To evaluate the CSC model as a tool for drug screening, we assessed the effect of separately blocking NF-κB and Wnt/β-catenin signaling and found markedly reduced mammosphere formation, particularly for tumor-initiating cells. Similar reduction was also observed using patient-derived primary cancer cells. Furthermore, blocking NF-κB signaling in mice transplanted with tumor-initiating cells significantly reduced tumor outgrowth. Our study demonstrates that suppressed apoptosis, activation of pathways associated with cell viability, and CSCs are the major differences between tumor-initiating and nontumorigenic cells independent of their epithelial-like/mesenchymal-like phenotype. These altered pathways may provide targets for future drug development to eliminate CSCs, and the cell model may be a useful tool in such drug screenings.

O intenso crescimento populacional acompanhado de tratamentos inadequados de residuos, tem resultado na destruicao de ambientes dulciaquicolas em ecossistemas urbanos. O rio Sapato constitui o principal efluente na divisa dos municipios de Lauro de Freitas e Salvador, no estado da Bahia. Nao obstante o crescente fluxo de despejo domestico ao qual esta submetido, inexistem dados ecotoxicologicos sobre este recurso hidrico. O objetivo do presente estudo foi avaliar a citogenotoxidade da agua do rio Sapato, em pontos de proximidade de esgotamento sanitario, utilizando o bioensaio de micronucleos (MN) e alteracoes nucleares (AN) em tilapia nilotica ( Oreochromis niloticus ). A analise foi feita em regiao sob emissao de esgoto sanitario e nascente, nas estacoes seca (novembro e dezembro/2006) e chuvosa (marco e abril/2007), sendo observadas 3000 celulas por animal (n=10). Os resultados foram analisados com o teste Kruskal Wallis e indicaram diferencas sazonais e uma frequencia maior de MN e AN no ponto submetido a esgotamento sanitario, em comparacao aos valores do sitio referencia. Conclui-se que as areas impactadas ja apresentam danos genocitotoxicos. Palavras-chave : anormalidades nucleares, esgotamento urbano, genotoxicidade, micronucleo.

Genome-wide association studies recently linked the locus for Na(+),HCO(3)(-)-cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO(3)(-) into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na(+),HCO(3)(-)-cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20-30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na(+)/H(+)-exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH-sensitive fluorophores, we showed that Na(+),HCO(3)(-)-cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in human primary breast carcinomas. At intracellular pH (pH(i) ) levels6.6, CO(2)/HCO(3)(-)-dependent mechanisms accounted for90% of total net acid extrusion. Na(+)/H(+)-exchange activity was prominent only at lower pH(i) -values. Furthermore, steady-state pH(i) was 0.35 ± 0.06 units lower in the absence than in the presence of CO(2)/HCO(3)(-). In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na(+),HCO(3)(-)-cotransport is a major determinant of pH(i) in breast cancer and the modest DIDS-sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.

Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified signaling network activates the transcription of cathepsin B gene (CTSB) via myeloid zinc finger-1 transcription factor that binds to an ErbB2-responsive enhancer element in the first intron of CTSB. This work provides a model system for ErbB2-induced breast cancer cell invasiveness, reveals a signaling network that is crucial for invasion in vitro, and defines a specific role and targets for the identified serine-threonine kinases.

Introduction The major histocompatibility complex (MHC) class II invariant chain (CD74) is a protein which functions as a chaperone for MHC class II in antigen-presenting cells. Furthermore, it acts as a receptor for the cytokine macrophage migration inhibitory factor (MIF), mediating downstream signalling. Many clinical studies have determined that CD74 is overexpressed at the protein level in a subset of human breast tumours. However, there are discrepant findings regarding its correlation to clinical parameters. In some reports, CD74 has been correlated to triple-negative (TN) status and increased presence of metastases, but in separate studies it correlated with better overall survival. In vitro studies of CD74’s molecular functions in cancer cells have generally supported that it has a cancer-promoting effect through stimulating proliferation, survival and invasion. Material and methods Human breast cancer cell lines MDA-MB-231 and M4A4 were transfected with siRNA targeting CD74 or a negative control. Cells were then subjected to in vitro analysis of proliferation or invasion through matrigel. Lysates of transfected cells were analysed by immunoblot for protein levels of cell survival and autophagy markers. A tumour microarray (TMA) containing 651 human breast tumour cores was stained by immunohistochemistry (IHC) for CD74 protein expression. Staining intensity was manually scored for each sample in a blinded fashion. Statistical analysis was performed in R. Results and discussions siRNA knockdown of CD74 in MDA-MB-231 and M4A4 resulted in reduced proliferation and invasion. Furthermore, survival signalling through Akt was decreased. These findings replicate previously published in vitro studies of CD74’s functions in cancer cells. Furthermore, a novel finding was that CD74 knockdown resulted in reduced levels of markers of autophagy. The correlations between CD74 IHC staining of TMA samples and TN status, lymph node status and overall survival were examined. In contrast to previous studies we observed no correlation between expression of CD74 and TN status or cancer spread to the lymph nodes. Instead, survival analysis revealed increased overall survival for cancers with moderate or high CD74 intensity. Conclusion Our results from cell lines support that CD74, when studied in vitro, has functions that stimulate cancer cells to proliferate and invade. However, results from clinical samples show a correlation of CD74 expression with increased survival.

Udgivelsesdato: 2010 Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up-regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up-regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of calreticulin, cellular retinoic acid-binding protein II, chloride intracellular channel protein 1, EF-1-beta, galectin 1, peroxiredoxin-2, platelet-derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl-terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.

The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 β, ɛ, σ, τ, and ζ protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.

We have applied protein expression profiling technologies in combination with immunohistochemistry, using fresh tissue and urine samples, to assess bladder cancer heterogeneity and prognosis as well as to generate protein markers for tumor progression and early diagnosis of the disease. Here, we review some selected lines of investigation and approaches undertaken by our laboratory, drawing on more than 15 years of experience in bladder cancer proteomics, to highlight a number of issues that may be useful for researchers entering the field. In particular, we address the identification of markers for bladder cancer progression and exemplify the potential of gel-based proteomic profiling of urine samples for the early detection of urothelial carcinomas. In addition, we provide a brief description of a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the tumor microenvironment.

Cork oak (Quercus suber) is an evergreen tree characterized by a thick bark, which grows in Mediterranean schlerophyllous forests. It is most prevalent in the southwestern Iberian Peninsula. Despite the potential of the province of Huelva (southwestern Spain) to maintain mature forests of cork oak, the tree has been severely depleted and most forests have either disappeared or are seriously threatened. This paper presents a spatial distribution model of cork oak for the province of Huelva with a view to determining the optimal areas for reforestation. The model draws on all available digital cartographic information with respect to cork oak distribution: topographic data (altitude, slope and orientation) were obtained from a Digital Terrain Model (20 m scale); rainfall, temperature and PET models were based on data collected from a network of meteorological stations; litologic data derive from the litologic map of Huelva (1:100,000). The result of this work is a mesh of points at a resolution of 100 m, sufficient to meet the needs of any kind of reforestation or management programmes in the area studied. Each point of this mesh contains the corresponding values for bioclimatic, topographic and litologic variables in a georeferenced data matrix. The independent variables responsible for cork oak distribution (binary dependent variables) were then identified by means of binary logistic regression analysis. North-facing slopes, abundant annual rainfall and litology were the main explaining variables. The spatial distribution model was produced by applying the formula obtained to spatial analysis software. This model is proposed as a basis for future reforestation plans, especially in those areas most affected by forest fires.

Background Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30 % response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. Methods We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. Results We found that the resistant cell lines showed 7–100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. Conclusions Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2071-1) contains supplementary material, which is available to authorized users.

// Line S. Tarpgaard 1, * , Maj Sofie Orum-Madsen 2, * , Ib J. Christensen 3 , Cathrine Nordgaard 2 , Julie Noer 2 , Tormod K. Guren 4 , Bengt Glimelius 5 , Halfdan Sorbye 6, 7 , Tone Ikdahl 8 , Elin H. Kure 9 , Kjell M. Tveit 8 , Hans J. Nielsen 10 , Per Pfeiffer 1, # , Nils Brunner 2, # , Jose M. A. Moreira 2, # 1 Department of Oncology, Odense University Hospital, Odense, Denmark and University of Southern Denmark, Odense, Denmark 2 Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 3 The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark and Biotech Research and Innovation Center (BRIC), University of Copenhagen, Copenhagen, Denmark 4 Department of Oncology and K. G. Jebsen Centre for Colorectal Cancer Research, Oslo University Hospital Oslo, Norway 5 Departments of Radiology, Oncology and Radiation Science, Uppsala University, Uppsala and Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden 6 Department of Oncology, Haukeland University Hospital, Bergen, Norway 7 Department of Clinical Science, University of Bergen, Bergen, Norway 8 Department of Oncology, Oslo University Hospital, Oslo, Norway 9 Department of Genetics, Oslo University Hospital, Oslo, Norway 10 Department of Surgical Gastroenterology, Copenhagen University Hospital, Hvidovre, Denmark * These authors contributed equally to this work # These authors share senior authorship Correspondence to: Jose M. A. Moreira, email: jomo@sund.ku.dk Keywords: metastatic colorectal cancer, plasma TIMP-1, KRAS mutations, prognosis, prediction Received: April 14, 2016 Accepted: June 17, 2016 Published: August 08, 2016 ABSTRACT It is now widely accepted that therapeutic antibodies targeting epidermal growth factor receptor (EGFR) can have efficacy in KRAS wild-type advanced colorectal cancer (CRC) patients. What remains to be ascertained is whether a subgroup of KRAS -mutated CRC patients might not also derive benefit from EGFR inhibitors. Metalloproteinase inhibitor 1 (TIMP-1) is a pleiotropic factor predictive of survival outcome of CRC patients. Levels of TIMP-1 were measured in pre-treatment plasma samples ( n = 426) of metastatic CRC patients randomized to Nordic FLOX (5-fluorouracil and oxaliplatin) +/− cetuximab (NORDIC VII study). Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 protein levels, KRAS status and treatment with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (> 3rd quartile) showing a significantly longer overall survival if treated with cetuximab (HR, 0.48; 95% CI, 0.25 to 0.93). To gain mechanistic insights into this association we analyzed a set of five different CRC cell lines. We show here that EGFR signaling induces TIMP-1 expression in CRC cells, and that TIMP-1 promotes a more aggressive behavior, specifically in KRAS mutated cells. The two sets of data, clinical and in vitro , are complementary and support each other, lending strength to our contention that TIMP-1 plasma levels can identify a subset of patients with KRAS -mutated metastatic CRC that will have benefit from EGFR-inhibition therapy.

Recently, we presented evidence – based on the analysis of benign hyperproliferative lesions of the breast – for the presence of cells that express the stem cell marker cytokeratin (CK) 15 in combination with CK19, a protein widely expressed by mammary epithelial cells. Here we report the finding of a subset of breast carcinomas characterized by expression of CK15. CK15 expressing tumors constituted 5% (6 out of 120; 4 of ductal type and 2 of lobular type) of the high‐risk breast carcinomas examined by gel‐based proteomics and immunohistochemistry. Five out of the six CK15+ carcinomas were CK15+/CK19−. The remaining tumor was mainly composed of cells expressing both CK15 and CK19 (CK15+/CK19+), but it also contained invasive areas with cells expressing only one of these makers (CK15+/CK19− and CK15−/CK19+ cells). To address the relationship between putative luminal progenitor/amplified CK15+ cells and malignant disease, and to determine whether cells/lesions lose expression of CK15 as a result of tumour initiation and/or progression, we searched among our sample set for carcinomas in which invasive tumor areas co‐existed with non‐malignant cells and hyperproliferative and known pre‐malignant lesions. Only one such tumour was found (T71), a CK15−/CK19+/p53+ carcinoma that contained p53 negative non‐malignant epithelial cells exhibiting a variety of, CK15/CK19 cellular phenotypes (CK15+/CK19+; CK15+/CK19−; CK15−/CK19+; CK15−/CK19−), often associated with simple columnar cells. Single layers of epithelial cells exhibiting all four CK15/CK19 phenotypes were observed contiguous to areas of atypical ductal hyperplasia that contained p53 positive cells that lost CK15 expression (CK15−/CK19+) and had a very similar phenotype to those of the neighboring ductal carcinoma in situ (DCIS) and invasive cells. The undifferentiated CK15+/CK19+ cells, which had the phenotype CK15+/CK19+/CK14+/CK8+ and −/ER−/PgR−/AR−/CD44+ (weak)/CK17−/p63−/vimentin+/Ki67−/Bcl‐2+ (weak)/GATA‐3−/p53−, most likely correspond to lineage‐restricted luminal progenitor cells able to generate the other more differentiated CK15/CK19 cellular phenotypes, thus giving rise to the daunting intratumour heterogeneity displayed by carcinoma T71. Cells with a very similar phenotype to the CK15+/CK19+ progenitor cells were observed in a juvenile fibroadenoma as well as in the large collecting ducts of the breast. The latter, however, expressed in addition CK14 and had a phenotype (CK15+/CK19+/CK14+/CK8+ (weak)/ER−/PgR−/AR−/CD44+ (weak)/CK17−/p63−/vimentin−/Ki67−/Bcl‐2+/GATA‐3−/p53−) that resembled that of the putative normal adult breast stem cells as inferred from published data. Further molecular characterization of these progenitor cells as well as unraveling of the signaling pathways that regulate their growth and differentiation may prove invaluable for developing novel therapeutic strategies that target cancer at an early stage.

Despite the advances that have taken place in the past decade, including the development of novel molecular targeted agents, cytotoxic chemotherapy remains the mainstay of cancer treatment. In breast cancer, anthracyclines and taxanes are the two main chemotherapeutic options used on a routine basis. Although effective, their usefulness is limited by the inevitable development of resistance, a lack of response to drug-induced cancer cell death. A large body of research has resulted in the characterization of a plethora of mechanisms involved in resistance; ATP-binding cassette transporter proteins, through their function in xenobiotic clearance, play an important role in resistance. We review here the current evidence for drug transporters as biomarkers and the benefit of adding drug transporter modulators to conventional chemotherapy.

Up to one-third of women aged 30–50 years have cysts in their breasts and are presumed to be at increased risk of developing breast cancer. Here we present an extensive proteomic and immunohistochemistry (IHC) study of breast apocrine cystic lesions aimed at generating specific biomarkers and elucidating the relationship, if existent, of apocrine cysts with cancer phenotype. To this end we compared the expression profiles of apocrine macrocysts obtained from mastectomies from high risk cancer patients with those of cancerous and non-malignant mammary tissue biopsies collected from the same patients. We identified two biomarkers, 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase, that were expressed specifically by apocrine type I cysts as well as by apocrine metaplastic cells in type II microcysts, terminal ducts, and intraductal papillary lesions. No expression of these markers was observed in non-malignant terminal ductal lobular units, type II flat cysts, stroma cells, or fat tissue as judged by IHC analysis of matched non-malignant tissue samples collected from 93 high risk patients enrolled in our cancer program. IHC analysis of the corresponding 93 primary tumors indicated that most apocrine changes have little intrinsic malignant potential, although some may progress to invasive apocrine cancer. None of the apocrine lesions examined, however, seemed to be a precursor of invasive ductal carcinomas, which accounted for 81% of the tumors analyzed. Our studies also provided some insight into the origin, development, and enlargement of apocrine cysts in mammary tissue. The successful identification of differentially expressed proteins that characterize specific steps in the progression from early benign lesions to apocrine cancer opens a window of opportunity for designing and testing new approaches for pharmacological intervention, not only in a therapeutic setting but also for chemoprevention, to inhibit cyst development as both 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase are currently being targeted for chemoprevention strategies in various malignancies.

14-3-3 proteins comprise a family of highly conserved and broadly expressed multifunctional regulatory proteins that are involved in various cellular processes such as cell cycle progression, cell growth, differentiation, and apoptosis. Transcriptional expression of the sigma isoform of 14-3-3 is frequently impaired in human cancers, including carcinomas of the breast, which has led to the suggestion that this protein might be involved in the neoplastic transformation of breast epithelial cells. Here we report on the analysis of 14-3-3sigma expression in primary breast tumors using a proteomic approach complemented by immunohistochemical analysis by means of specific antibodies against this isoform. We show that the levels of expression of 14-3-3sigma were similar in non-malignant breast epithelial tissue and matched malignant tissue with only sporadic loss of expression observed in 3 of the 68 tumors examined. Moreover we show that 14-3-3sigma immunoreactivity was restricted to epithelial cells and significantly stronger in the myoepithelial cells that line the mammary ducts and lobules. The lack of expression of 14-3-3sigma in the three breast carcinomas was not associated with high levels of expression of the dominant-negative transcriptional regulator DeltaNp63 or with increased expression of estrogen-responsive finger protein, a ubiquitin-protein ligase (E3) that targets 14-3-3sigma for proteolysis. Validation of the results was performed retrospectively on an independent clinical tumor sample set using a tissue microarray containing 65 primary tumors. Our data suggest that, contrary to what was previously thought, loss of expression of 14-3-3sigma protein is not a frequent event in breast tumorigenesis.

It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease.

Evaluation of: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print).Techniques for in situ detection and quantification of proteins in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer patients. Several techniques are currently available for detection of single proteins and post-translational modifications, but very few are suitable for detection of protein complexes. Methods that enable simultaneous detection and quantification of protein complexes provide novel possibilities for understanding the biological role(s) of protein complexes and may open new opportunities to improve clinical biomarker research...

Discovery-driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform ‘omic’ data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge-based complementary procedures into a systems biology approach to fight breast cancer.