Improved process to obtain nanofibrillated cellulose (CNF) reinforced starch films with upgraded mechanical properties and barrier character

Nowadays, the interest on nanofibrillated cellulose (CNF) has increased owing to its sustainability and its capacity to improve mechanical and barrier properties of polymeric films. Moreover, this filler shows some drawbacks related with its high capacity to form aggregates, hindering its dispersion in the matrix. In this work, an improved procedure to optimize the dispersability of CNF in a thermoplastic starch was put forward. On the one hand, CNF needs a hydrophilic dispersant to be included in the matrix, and on the other, starch needs a hydrophilic plasticizer to obtain a thermoformable material. Glycerol was used to fulfil both targets at once. CNF was predispersed in the plasticizer before nanofibrillation and later on was included into starch, obtaining thin films. The tensile strength of these CNF–starch composite films was 60% higher than the plain thermoplastic starch at a very low 0.36% w/w percentage of CNF. The films showed a noticeable correlation between water uptake, and temperature and humidity. Regarding permeability, a ca. 55% oxygen and water vapor permeability drop was found by nanofiller loading. The hydrolytic susceptibility of the composite was confirmed, being similar to that of the thermoplastic starch.Peer ReviewedPostprint (published version

Loxl3 Promotes Melanoma Progression and Dissemination Influencing Cell Plasticity and Survival

Malignant melanoma is the most lethal skin cancer due to its aggressive clinical behavior and therapeutic resistance. A comprehensive knowledge of the molecular mechanisms underlying melanoma progression is urgently needed to improve the survival of melanoma patients. Phenotypic plasticity of melanoma cells has emerged as a key process in melanomagenesis and therapy resistance. This phenotypic plasticity is sustained by an epithelial-to-mesenchymal (EMT)-like program that favors multiple intermediate states and allows adaptation to changing microenvironments along melanoma progression. Given the essential role of lysyl oxidase-like 3 (LOXL3) in human melanoma cell survival and its contribution to EMT, we generated mice with conditional melanocyte-specific targeting of Loxl3, concomitant to Braf activation and Pten deletion. Our results supported a key role of Loxl3 for melanoma progression, metastatic dissemination, and genomic stability, and supported its contribution to melanoma phenotypic plasticity by modulating the expression of several EMT transcription factors (EMT-TFs). Malignant melanoma is a highly aggressive tumor causing most skin cancer-related deaths. Understanding the fundamental mechanisms responsible for melanoma progression and therapeutic evasion is still an unmet need for melanoma patients. Progression of skin melanoma and its dissemination to local or distant organs relies on phenotypic plasticity of melanoma cells, orchestrated by EMT-TFs and microphthalmia-associated TF (MITF). Recently, melanoma phenotypic switching has been proposed to uphold context-dependent intermediate cell states benefitting malignancy. LOXL3 (lysyl oxidase-like 3) promotes EMT and has a key role in human melanoma cell survival and maintenance of genomic integrity. To further understand the role of Loxl3 in melanoma, we generated a conditional Loxl3-knockout (KO) melanoma mouse model in the context of BrafV600E-activating mutation and Pten loss. Melanocyte-Loxl3 deletion increased melanoma latency, decreased tumor growth, and reduced lymph node metastatic dissemination. Complementary in vitro and in vivo studies in mouse melanoma cells confirmed Loxl3's contribution to melanoma progression and metastasis, in part by modulating phenotypic switching through Snail1 and Prrx1 EMT-TFs. Importantly, a novel LOXL3-SNAIL1-PRRX1 axis was identified in human melanoma, plausibly relevant to melanoma cellular plasticity. These data reinforced the value of LOXL3 as a therapeutic target in melanoma

miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP, CK2-α, CCNJ, and MEGF9

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3'-UTRs of ENPEP, CK2-alpha, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-alpha, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40-56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-alpha was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-alpha, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-alpha, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer

Capturing the biological impact of CDKN2A and MC1R genes as an early predisposing event in melanoma and non melanoma skin cancer

Germline mutations in CDKN2A and/or red hair color variants in MC1R genes are associated with an increased susceptibility to develop cutaneous melanoma or non melanoma skin cancer. We studied the impact of the CDKN2A germinal mutation p.G101W and MC1R variants on gene expression and transcription profiles associated with skin cancer. To this end we set-up primary skin cell co-cultures from siblings of melanoma prone-families that were later analyzed using the expression array approach. As a result, we found that 1535 transcripts were deregulated in CDKN2A mutated cells, with over-expression of immunity-related genes (HLA-DPB1, CLEC2B, IFI44, IFI44L, IFI27, IFIT1, IFIT2, SP110 and IFNK) and down-regulation of genes playing a role in the Notch signaling pathway. 3570 transcripts were deregulated in MC1R variant carriers. In particular, genes related to oxidative stress and DNA damage pathways were up-regulated as well as genes associated with neurodegenerative diseases such as Parkinson's, Alzheimer and Huntington. Finally, we observed that the expression signatures indentified in phenotypically normal cells carrying CDKN2A mutations or MC1R variants are maintained in skin cancer tumors (melanoma and squamous cell carcinoma). These results indicate that transcriptome deregulation represents an early event critical for skin cancer development

T-type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E)

Endoglin overexpression modulates cellular morphology, migration, and adhesion of mouse fibroblasts

10 p.-9 fig.-1 tab.Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Endoglin glycoprotein is a component of the transforming growth factor type ß (TGF-ß) receptor system which is highly expressed by endothelial cells, and at lower levels on fibroblasts and smooth muscle cells, suggesting the involvement of these lineages in the HHT1 vascular dysplasia. Overexpression of endoglin in mouse NCTC929 fibroblasts led to decreased migration in chemotactic and wound healing assays, as well as changes in the cellular morphology. When plated on uncoated surfaces, endoglin transfectants formed intercellular clusters, endoglin being not specifically localized to the cell-cell junctions, but homogenously distributed on the cellular surface. Although the expression of α5ß1 integrin and of an activation epitope of ß1 integrin were unchanged, a polyclonal antibody to α5ß1 integrin was able to inhibit cluster formation, suggesting the involvement of integrin ligand/s. In fact, coating with fibronectin, laminin, or an RGD-containing 80 kDa fragment of fibronectin were able to prevent the cellular clustering. Furthermore, synthesis of plasminogen activator inhibitor 1 (PAI-1), and to a weak extent that of fibronectin, were inhibited in endoglin transfectants. Thus, the presence of endoglin in mouse NCTC929 fibroblasts is associated with reduced production of certain extracellular matrix (ECM) components, which might explain their altered morphology, migration and intercellular cluster formation.This work has been supported by grants from Camisión Interministerial de Ciencia yTecnología (CICYT-SAF97-0034 to C. Bernabéu, and CICYT-SAF97-0064-C03-02 to A. García-Pardo), Comunidad Autónoma de Madrid (CAM) and Biomed Program of the European Community (BMH4-CT95-0995 to C. Bernabéu).Peer reviewe

PTEN mediates Notch-dependent stalk cell arrest in angiogenesis.

Coordinated activity of VEGF and Notch signals guides the endothelial cell (EC) specification into tip and stalk cells during angiogenesis. Notch activation in stalk cells leads to proliferation arrest via an unknown mechanism. By using gain- and loss-of-function gene-targeting approaches, here we show that PTEN is crucial for blocking stalk cell proliferation downstream of Notch, and this is critical for mouse vessel development. Endothelial deletion of PTEN results in vascular hyperplasia due to a failure to mediate Notch-induced proliferation arrest. Conversely, overexpression of PTEN reduces vascular density and abrogates the increase in EC proliferation induced by Notch blockade. PTEN is a lipid/protein phosphatase that also has nuclear phosphatase-independent functions. We show that both the catalytic and non-catalytic APC/C-Fzr1/Cdh1-mediated activities of PTEN are required for stalk cells' proliferative arrest. These findings define a Notch-PTEN signalling axis as an orchestrator of vessel density and implicate the PTEN-APC/C-Fzr1/Cdh1 hub in angiogenesis

Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor

et al.Overexpression of the transcription factor Snail in epithelial MDCK cells promotes the epithelial-mesenchymal transition (EMT) and the acquisition of an invasive phenotype. We report here that the expression of Snail is associated with an increase in the promoter activity and expression of the matrix metalloproteinase MMP-9. The effect of Snail silencing on MMP-9 expression corroborates this finding. Induced transcription of MMP-9 by Snail is driven by a mechanism dependent on the MAPK and phosphoinositide 3-kinase (PI3K) signalling pathways. Although other regions of the promoter were required for a complete stimulation by Snail, a minimal fragment (nucleotides -97 to +114) produces a response following an increased phosphorylation of Sp-1 and either Sp-1 or Ets-1 binding to the GC-box elements contained in this region. The expression of a dominant negative form of MEK decreased these complexes. A moderate increase in the binding of the nuclear factor κB (NF κB) to the upstream region (nucleotide -562) of the MMP-9 promoter was also observed in Snail-expressing cells. Interestingly, oncogenic H-Ras (Ras V12) synergistically co-operates with Snail in the induction of MMP-9 transcription and expression. Altogether, these results indicate that MMP-9 transcription is activated in response to Snail expression and that it might explain, at least in part, the invasive properties of the Snail-expressing cells.This work was supported by the Spanish Ministry of Science and Technology (SAF 2002-817 to A.F. and SAF 2004-00361 to A.C.) and the Instituto de Salud Carlos III, RTICCC, FIS 03-C03/10.Peer Reviewe

Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors

Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors. © 2007 Elsevier Inc. All rights reserved.This work was supported by the Spanish Ministry of Science and Technology (SAF 2002-00817 and SAF 2005-03259 to AF; SAF 2004-00361 and NAN 2004-09230 C04-02 to AC); the Instituto de Salud Carlos III, RTICCC, FIS 03-C03/10 and EU (MRTN-CT-2004-005428).Peer Reviewe

Snail silencing effectively suppresses tumour growth and invasiveness

The transcription factor Snail has been recently proposed as an important mediator of tumour invasion because of its role in downregulation of E-cadherin and induction of epithelial-mesenchymal transitions (EMT). This behaviour has led to the consideration of Snail as a potential therapeutic target to block tumour progression. In this report, we provide evidence for this hypothesis. We show that silencing of Snail by stable RNA interference in MDCK-Snail cells induces a complete mesenchymal to epithelial transition (MET), associated to the upregulation of E-cadherin, downregulation of mesenchymal markers and inhibition of invasion. More importantly, stable interference of endogenous Snail in two independent carcinoma cell lines leads to a dramatic reduction of in vivo tumour growth, accompanied by increased tumour differentiation and a significant decrease in the expression of MMP-9 and angiogenic markers and invasiveness. These results indicate that use of RNA interference can be an effective tool for blocking Snail function, opening the way for its application in new antiinvasive therapies. © 2007 Nature Publishing Group All rights reserved.This work was supported by grants from the biotech company Advanced in vitro Cell Technologies SL (Advancell) and from the Spanish Ministry of Science and Technology (SAF2004-00361; SAF2005-03259; NAN2004-09230-C04-02) and Instituto de Salud Carlos III (RTICCC, FIS03/C03/10; PI050656).Peer Reviewe