Exogenous melatonin ameliorates embryo-maternal cross-talk in early pregnancy in sheep.

Early pregnancy losses cause 25% of pregnancy failures in small ruminants because of an asynchrony between conceptus and uterine signals. In that context, melatonin plays a crucial role in sheep reproductive dynamics, but little is known about its effects in the peri-implantation period. We hypothesized that melatonin supports embryo implantation by modulating the uterine microenvironment. This study aimed to assess the effects of exogenous melatonin on the endometrium and early placenta rearrangement. Ten multiparous ewes either did (MEL, n=5) or did not (CTR, n=5) receive a subcutaneous melatonin implant (18 mg) 50 d before a synchronized mating. On Day 21 of pregnancy, sheep were euthanized. MEL ewes exhibited a higher prolificity rate (2.8 vs. 2.0 embryos/ewe) and plasma progesterone levels (3.84 vs. 2.96 ng/mL, P<0.05) than did CTR ewes. Groups did not differ significantly in embryo crown-rump length. MEL placenta had significantly (P<0.001) more binucleated trophoblast cells in the chorion region and ovine placental lactogen expression was significantly (P<0.05) more strongly upregulated than it was in CTR. Exogenous melatonin increased significantly (P<0.05) gene expression of angiogenic factors (VEGFA, VEGFR1, IGF1R), IFNAR2, and PR in the caruncular endometrium. Expression of the MT2 receptor in the endometrium and placenta was significantly (P<0.05) higher in MEL group. Results indicate that melatonin implants acted differentially on the uterus and placenta rearrangement. Melatonin increases differentiation in the placenta and induces changes that could promote vessel maturation in the endometrium, which suggests that it enhances the uterine microenvironment in the early stage of pregnancy in sheep.

A Deep Learning Approach for Accurate Path Loss Prediction in LoRaWAN Livestock Monitoring.

The agricultural sector is amidst an industrial revolution driven by the integration of sensing, communication, and artificial intelligence (AI). Within this context, the internet of things (IoT) takes center stage, particularly in facilitating remote livestock monitoring. Challenges persist, particularly in effective field communication, adequate coverage, and long-range data transmission. This study focuses on employing LoRa communication for livestock monitoring in mountainous pastures in the north-western Alps in Italy. The empirical assessment tackles the complexity of predicting LoRa path loss attributed to diverse land-cover types, highlighting the subtle difficulty of gateway deployment to ensure reliable coverage in real-world scenarios. Moreover, the high expense of densely deploying end devices makes it difficult to fully analyze LoRa link behavior, hindering a complete understanding of networking coverage in mountainous environments. This study aims to elucidate the stability of LoRa link performance in spatial dimensions and ascertain the extent of reliable communication coverage achievable by gateways in mountainous environments. Additionally, an innovative deep learning approach was proposed to accurately estimate path loss across challenging terrains. Remote sensing contributes to land-cover recognition, while Bidirectional Long Short-Term Memory (Bi-LSTM) enhances the path loss model's precision. Through rigorous implementation and comprehensive evaluation using collected experimental data, this deep learning approach significantly curtails estimation errors, outperforming established models. Our results demonstrate that our prediction model outperforms established models with a reduction in estimation error to less than 5 dB, marking a 2X improvement over state-of-the-art models. Overall, this study signifies a substantial advancement in IoT-driven livestock monitoring, presenting robust communication and precise path loss prediction in rugged landscapes.

CD49f+ mammary epithelial cells decrease in milk from dairy cows stressed by overstocking during the dry period.

The work reported in this Research Communication describes the modification in epithelial cell populations during the first and the last month of milking in Holstein Friesian cows that have undergone different management during the dry period, and we report the differential expression of CD49f+ and cytokeratin18+ cell subpopulations. Twenty six cows were randomly divided into 2 balanced groups that were housed at stocking density of either 11 m2 (CTR) or 5 m2 from 21 ± 3 d before the expected calving until calving. Cells collected from milk samples taken in early lactation and late lactation were directly analysed for CD45, CD49f, cytokeratin 14, cytokeratin 18 and cell viability. We observed a differential expression with a significant reduction in CD49f+ (P < 0·01) and cytokeratin 18+ (P < 0·05) cells in early lactation. Differences were still evident in late lactation but were not significant. These observations suggest that mammary epithelial cell immunophenotypes could be associated with different animal management in the dry period and we hypothesise they may have a role as biomarkers for mammary gland function in dairy cows.

Clonogenic assay allows for selection of a primitive mammary epithelial cell population in bovine.

Adult mammary stem cells have been identified in several species including the bovine. They are responsible for the development of the gland and for cyclic remodeling during estrous cycles and pregnancy. Epithelial cell subpopulations exist within the mammary gland. We and others showed previously that the Colony Forming Cell (CFC) assay can be used to detect lineage-restricted mammary progenitors. We carried out CFCs with bovine mammary cells and manually separated colonies with specific morphologies associated with either a luminal or a myoepithelial phenotype. Expression of specific markers was assessed by immunocytochemistry or by flow cytometry to confirm that the manual separation resulted in isolation of phenotipically different cells. When transplanted in recipient immunodeficient mice, we found that only myoepithelial-like colonies gave rise to outgrowths that resembled bovine mammary alveoli, thus proving that adult stem cells were maintained during culture and segregated with myoepithelial cells. After recovery of the cells from the transplanted mice and subsequent progenitor content analysis, we found a tendency to detect a higher progenitor frequency when myoepithelial-like colonies were transplanted. We here demonstrate that bovine adult mammary stem cells can be sustained in short-term culture and that they can be enriched by manually selecting for basal-like morphology.

Bovine Skeletal Muscle Satellite Cells: Isolation, Growth, and Differentiation.

Skeletal muscle in cattle occupies a large part of the animal's body mass and develops into an important source of nutrients for human nutrition. Recently, the attention on bovine myogenic cells is increased to develop strategies of cultured in vitro meat as an alternative food source, more sustainable, ethical, and healthy than traditional meat production. At present, investigating the proliferation and differentiation of bovine skeletal muscle myogenic cells in vitro maintains its importance in the study of the mechanisms underlying the physiological and pathological events affecting the skeletal muscle, but it is of particular interest in animal husbandry and the food industry fields.In cell-based biological research, cell lines are one of the favored experimental tools because a population of cells could proliferate indefinitely in vitro under different stimuli, but they are limited to addressing the relevant biological properties of a cell population. On the other hand, primary cells from normal animal tissues undergo a limited number of divisions in vitro before they enter senescence but preserve their original characteristics and functions, and researchers can acquire the opportunity to study the individual donors and not just cells.In this chapter, we provide a basic protocol to isolate satellite cells from the skeletal muscle of cattle to obtain a good number of myogenic cells that can grow in in vitro conditions and undergo multiple rounds of cell division (myoblasts) before entering differentiation (myotubes). Furthermore, the robust expansion of these cells leads to the possibility to investigate physiological events or disorders related to the skeletal muscle tissue.

Ovine Trophoblast Cells: Cell Isolation and Culturing from the Placenta at the Early Stage of Pregnancy.

Embryo development is dependent upon the exchange of oxygen and nutrients through the placenta, mainly composed of peculiar epithelioid cells, known as trophoblast cells. Normal trophoblast functionality plays a key role during the whole pregnancy, especially in the first stage of placentation. This chapter explains the techniques to obtain sheep primary trophoblast cells from the early placenta. Overall, procedures for cell isolation, culture, characterization, and cryopreservation are described.

Functional effect of mir-27b on myostatin expression: a relationship in Piedmontese cattle with double-muscled phenotype.

BACKGROUND: In Piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the myostatin (MSTN) gene. The Piedmontese MSTN missense mutation G938A is translated to C313Y myostatin protein. This mutation alters MSTN function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. MiRNAs could play a role in skeletal muscle hypertrophy modulation by down-regulating gene expression. RESULTS: After identifying a 3'-UTR consensus sequence of several negative and positive modulator genes involved in the skeletal muscle hypertrophy pathway, such as IGF1, IGF1R, PPP3CA, NFATc1, MEF2C, GSK3b, TEAD1 and MSTN, we screened miRNAs matching to it. This analysis led to the identification of miR-27b, miR-132, miR-186 and miR-199b-5p as possible candidates. We collected samples of longissimus thoracis from twenty Piedmontese and twenty Friesian male bovines. In Piedmontese group miR-27b was up-regulated 7.4-fold (p < 0.05). Further, we report that the level of MSTN mRNA was about 5-fold lower in Piedmontese cattle vs Friesian cattle (p < 0.0001) and that less mature MSTN protein was detected in the Piedmontese one (p < 0.0001). Cotransfection of miR-27b and psi-check2 vector with the luciferase reporter gene linked to the bovine wild-type 3'-UTR of MSTN strongly inhibited the luciferase activity (79%, p < 0.0001). CONCLUSIONS: These data demonstrate that bovine MSTN is a specific target of miR-27b and that miRNAs contribute to explain additive phenotypic hypertrophy in Piedmontese cattle selected for the MSTN gene mutation, possibly outlining a more precise genetic signature able to elucidate differences in muscle conformation.

Adaptation Response in Sheep: Ewes in Different Cortisol Clusters Reveal Changes in the Expression of Salivary miRNAs.

Farm procedures have an impact on animal welfare by activating the hypothalamic-pituitary-adrenal axis that induces a wide array of physiological responses. This adaptive system guarantees that the animal copes with environmental variations and it induces metabolic and molecular changes that can be quantified. MicroRNAs (miRNAs) play a key role in the regulation of homeostasis and emerging evidence has identified circulating miRNAs as promising biomarkers of stress-related disorders in animals. Based on a clustering analysis of salivary cortisol trends and levels, 20 ewes were classified into two different clusters. The introduction of a ram in the flock was identified as a common farm practice and reference time point to collect saliva samples. Sixteen miRNAs related to the adaptation response were selected. Among them, miR-16b, miR-21, miR-24, miR-26a, miR-27a, miR-99a, and miR-223 were amplified in saliva samples. Cluster 1 was characterized by a lower expression of miR-16b and miR-21 compared with Cluster 2 (p < 0.05). This study identified for the first time several miRNAs expressed in sheep saliva, pointing out significant differences in the expression patterns between the cortisol clusters. In addition, the trend analyses of these miRNAs resulted in clusters (p = 0.017), suggesting the possible cooperation of miR-16b and -21 in the integrated stress responses, as already demonstrated in other species as well. Other research to define the role of these miRNAs is needed, but the evaluation of the salivary miRNAs could support the selection of ewes for different profiles of response to sources of stressors common in the farm scenario.

Effects of Saccharomyces boulardii Supplementation on Nutritional Status, Fecal Parameters, Microbiota, and Mycobiota in Breeding Adult Dogs.

The aim of this study was to evaluate the effect of the administration of Saccharomyces boulardii on the nutritional, immunological, inflammatory, and stress status and on the composition of the gut microbiota and mycobiota in healthy adult dogs. A total of 25 American Staffordshire Terrier dogs were selected and randomly assigned to two groups: control (CTR, n = 12) and treated (TRT, n = 13) groups. No significant differences were found between the two groups regarding body weight, body condition score, and fecal score. No significant differences in microbiota/mycobiota, short chain fatty acids, indole/skatole, histamine, zonulin, or lactoferrin were detected. Indeed, supplementation with S. boulardii significantly decreased fecal calprotectin Immunoglobulin A, indicating an improvement in the gut well-being. Interestingly, fecal cortisol significantly decreased in dogs belonging to the TRT group compared to the CTR, suggesting both an improvement of the intestinal status and a reduction of stress, a common condition affecting animals managed in a breeding environment.

External and internal EGFR-activating signals drive mammary epithelial cells proliferation and viability.

During puberty, the mammary gland undergoes an intense growth, dependent on the interplay between the Epidermal Growth Factor Receptor (EGFR) in the stroma and different mammary epithelial receptors. We hypothesize that EGFR expressed in the mammary epithelium also has a role in puberty and the epithelial cells can self-sustain by EGFR-mediated autocrine signaling. We adopted mammary cell lines from different species, as in vitro model for the epithelium, and we observed that EGFR-signaling positively affects their survival and proliferation. Once deprived of external growth factors, mammary cells still showed strong Erk 1/2 phosphorylation, abolished upon EGFR inhibition, coupled with a further reduction in survival and proliferation. Based on gene expression analysis, three EGFR-ligands (AREG, EREG and HBEGF) are likely to mediate this autocrine signaling. In conclusion, internal EGFR-activating signals sustain mammary epithelial cell proliferation and survival in vitro.

Circulating skeletal muscle related microRNAs profile in Piedmontese cattle during different age.

Piedmontese cattle is known for double-muscle phenotype. MicroRNAs (miRNAs) play important role as regulators in skeletal muscle physiological processes, and we hypothesize that plasma miRNAs expression profiles could be affected by skeletal muscle growth status related to age. Plasma samples of cattle were collected during four different ages from first week of life until the time of commercial end of the fattening period before slaughter. Small-RNA sequencing data analysis revealed the presence of 40% of muscle-related miRNAs among the top 25 highly expressed miRNAs and, 19 miRNAs showed differential expression too. Using qRT-PCR, we validated in a larger bovine population, miRNAs involved in skeletal muscle physiology pathways. Comparing new-born with the other age groups, miR-10b, miR-126-5p, miR-143 and miR-146b were significantly up-regulated, whereas miR-21-5p, miR-221, miR-223 and miR-30b-5p were significantly down-regulated. High expression levels of miR-23a in all the groups were found. Myostatin, a negative regulator of skeletal muscle hypertrophy, was predicted as the target gene for miR-23a and miR-126-5p and we demonstrated their direct binding. Correlation analysis revealed association between miRNAs expression profiles and animals' weights along the age. Circulating miRNAs could be promising for future studies on their biomarker potentialities to beef cattle selection.

Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis.

Laminitis is one of the most devastating diseases in equine medicine, and although several etiopathogenetic mechanisms have been proposed, few clear answers have been identified to date. Several lines of evidence point towards its underlying pathology as being metabolism-related. In the carbonyl stress pathway, sugars are converted to methylglyoxal (MG)-a highly reactive α-oxoaldehyde, mainly derived during glycolysis in eukaryotic cells from the triose phosphates: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. One common hypothesis is that MG could be synthesized during the digestive process in horses, and excessive levels absorbed into peripheral blood could be delivered to the foot and lead to alterations in the hoof lamellar structure. In the present study, employing an ex vivo experimental design, different concentrations of MG were applied to hoof explants (HE), which were then incubated and maintained in a specific medium for 24 and 48 h. Macroscopic and histological analyses and a separation force test were performed at 24 and 48 h post-MG application. Gene expression levels of matrix metalloproteinase (MMP)-2 and -14 and tissue inhibitor of metalloproteinase (TIMP)-2 were also measured at each time point for all experimental conditions. High concentrations of MG induced macroscopic and histological changes mimicking laminitis. The separation force test revealed that hoof tissue samples incubated for 24 h in a high concentration of MG, or with lower doses but for a longer period (48 h), demonstrated significant weaknesses, and samples were easily separated. All results support that high levels of MG could induce irreversible damage in HEs, mimicking laminitis in an ex vivo model.

met oncogene activation qualifies spontaneous canine osteosarcoma as a suitable pre-clinical model of human osteosarcoma.

The Met receptor tyrosine kinase (RTK) is aberrantly expressed in human osteosarcoma and is an attractive molecular target for cancer therapy. We studied spontaneous canine osteosarcoma (OSA) as a potential pre-clinical model for evaluation of Met-targeted therapies. The canine MET oncogene exhibits 90% homology compared with human MET, indicating that cross-species functional studies are a viable strategy. Expression and activation of the canine Met receptor were studied utilizing immunohistochemical techniques in 39 samples of canine osteosarcoma, including 35 primary tumours and four metastases. Although the Met RTK is barely detectable in primary culture of canine osteoblasts, high expression of Met protein was observed in 80% of canine osteosarcoma samples acquired from various breeds. Met protein overexpression was also concordant with its activation as indicated by phosphorylation of critical tyrosine residues. In addition, Met was expressed and constitutively activated in canine osteosarcoma cell lines. OSA cells expressing high levels of Met demonstrated activation of downstream transducers, elevated spontaneous motility, and invasiveness which were impaired by both a small molecule inhibitor of Met catalytic activity (PHA-665752) and met-specific, stable RNA interference obtained by means of lentiviral vector. Similar to observations in human OSA, these data suggest that Met is commonly overexpressed and activated in canine OSA and that inhibition of Met impairs the invasive and motogenic properties of canine OSA cells. These data implicate Met as a potentially important factor for canine OSA progression and indicate that it represents a viable model to study Met-targeted therapies.

MicroRNAs as Biomarkers for Animal Health and Welfare in Livestock.

MicroRNAs (miRNAs) are small and highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through the post-transcriptional regulation of gene expression. An intriguing aspect in identifying these molecules as biomarkers is derived from their role in cell-to-cell communication, their active secretion from cells into the extracellular environment, their high stability in body fluids, and their ease of collection. All these features confer on miRNAs the potential to become a non-invasive tool to score animal welfare. There is growing interest in the importance of miRNAs as biomarkers for assessing the welfare of livestock during metabolic, environmental, and management stress, particularly in ruminants, pigs, and poultry. This review provides an overview of the current knowledge regarding the potential use of tissue and/or circulating miRNAs as biomarkers for the assessment of the health and welfare status in these livestock species.

A New Approach to LCA Evaluation of Lamb Meat Production in Two Different Breeding Systems in Northern Italy.

Lamb meat production provides vital landscape-management and ecosystem services; however, ruminant farming produces a considerable share of the world's greenhouse gas emissions. To measure and compare the advantages and disadvantages of the intensification of livestock farming, an integrative analysis was conducted in this study by combining environmental impact analysis and animal welfare assessment. This approach is the first of its kind and is the innovative aspect of this paper. The methodology of Life Cycle Assessment (LCA) entails the holistic analysis of various impact categories and the associated emission quantities of products, services, and resources over their life cycle, including resource extraction and processing, production processes, transport, usage, and the end of life. The outlines of LCA are standardized in DIN EN ISO 14040/14044. To assess the environmental impacts of the production of lamb meat in northern Italy, two case studies were undertaken using the LCA software GaBi. The analysis is based on primary data from two sheep-breeding systems (semi-extensive and semi-intensive in alpine and continental bioregions, respectively) combined with inventory data from the GaBi database and data from the literature. The assessment was conducted for the functional unit of 1 kg of lamb meat and focuses on the impact categories global warming potential, acidification potential, and eutrophication potential. For an overall evaluation of the supply chain, we have also considered a parameter indicating animal welfare, in keeping with consumer concerns, employing an analysis of chronic stress as shown by cortisol accumulation. The goal is to derive models and recommendations for an efficient, more sustainable use of resources without compromising animal welfare, meat quality, and competitiveness. The aim of this study is to provide a standard for individualized sustainability analyses for European lamb production systems in the future. From the LCA perspective, the more intensive case-study farm showed a lower impact in global impact factors and a higher impact in local impact categories in comparison with the more extensively run farm that was studied. From the animal welfare perspective, lower amounts of the stress hormone cortisol were found on the extensively managed case-study farm.

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models.

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR-2, PDGFRs and c-Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c-Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.

An in vivo model of Met-driven lymphoma as a tool to explore the therapeutic potential of Met inhibitors.

PURPOSE: Met, the tyrosine kinase receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Recent evidence indicates that Met amplification may confer resistance to treatments directed toward other receptor tyrosine kinases. Thus, there is a need to develop Met inhibitors into therapeutic tools, to be used alone or in combination with other molecularly targeted drugs. Preclinical validation of Met inhibitors has thus far been done in nude mice bearing cancer cells xenografts. A far superior model would be a transgenic line developing spontaneous Met-driven tumors with high penetrance and short latency. EXPERIMENTAL DESIGN: To this end, we introduced into the mouse genome TPR-MET, the oncogenic form of MET. The Tpr-Met protein ensures deregulation of Met signaling because dimerization motifs in the Tpr moiety cause ligand-independent activation of the Met kinase. RESULTS: Here, we describe a TPR-MET transgenic line that develops thymic T-cell lymphoma with full penetrance and very short latency. In the tumors, Tpr-Met and its effectors were phosphorylated. Treatment of tumor-derived T lymphocytes with the selective Met inhibitor PHA-665752 at nanomolar concentrations abolished phosphorylation of Met and downstream effectors and led to caspase-mediated apoptosis. I.v. administration of PHA-665752 to transgenic mice bearing lymphomas in exponential growth phase led to a significant decrease in tumor growth and, in some cases, to tumor regression. CONCLUSIONS: Our transgenic line, which within 2 months reliably develops Tpr-Met-driven T-cell lymphoma, represents a valuable tool to explore the efficacy and therapeutic potential of Met kinase inhibitors as anticancer drugs.

Bortezomib-mediated proteasome inhibition as a potential strategy for the treatment of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, divided into two major histological subtypes, embryonal (ERMS) and alveolar (ARMS). To explore the possibility that the proteasome could be a target of therapeutic value in rhabdomyosarcoma, we treated several RMS cell lines with the proteasome inhibitor bortezomib (Velcade or PS-341) at a concentration of 13-26 nM. RMS cells showed high sensitivity to the drug, whereas no toxic effect was observed in primary human myoblasts. In both ERMS and ARMS cells bortezomib promoted apoptosis, activation of caspase 3 and 7 and induced a dose-dependent reduction of anchorage-independent growth. Furthermore, bortezomib induced activation of the stress response, cell cycle arrest and the reduction of NF-kappaB transcriptional activity. Finally, bortezomib decreased tumour growth and impaired cells viability, proliferation and angiogenesis in a xenograft model of RMS. In conclusion, our data indicate that bortezomib could represent a novel drug against RMS tumours.

Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling.

Curcumin (from the rhizome of Curcuma longa) is well documented for its medicinal properties in Indian and Chinese systems of medicine where it is widely used for the treatment of several diseases. Epidemiological observations are suggestive that curcumin consumption may reduce the risk of some form of cancers and provide other protective biological effects in humans. These biological properties have been attributed to curcuminoids that have been widely studied for their anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects. In this study we have investigated on the effect of a curcumin phospholipid complex on mammary epithelial cell viability. HC11 and BME-UV cell lines, validated models to study biology of normal, not tumoral, mammary epithelial cells, were used to analyse these effects. We report that curcumin acts on STAT-3 signal pathway to reduce cell viability and increase apoptosis evaluated by the the amount of activated caspase 3. Further it reduces MAPK and AKT activations. JSI-124, a STAT-3 inhibitor (100 nM) was able to block the negative effect of curcumin on cell viability and caspase 3 activation. Finally the negative effect of cucumin on cell viability has been impaired in STAT-3i HC11, where STAT-3 protein was greatly reduced by shRNA-interference. These results indicate that curcumin presents a potential adverse effect to normal mammary epithelial cells and that it has a specific effect on signal trasduction in mammary epithelium.

Validation of met as a therapeutic target in alveolar and embryonal rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly malignant soft-tissue tumor of childhood deriving from skeletal muscle cells. RMS can be classified in two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS), the latter being characterized by the PAX3/7-FKHR translocation. Here we first investigated whether the Met receptor, a transcriptional target of PAX3 and PAX7, has a role in PAX3-FKHR-mediated transformation. Following PAX3-FKHR transduction, Met was up-regulated in mouse embryonal fibroblasts (MEF), NIH 3T3 and C2C12 cells, and they all acquired anchorage independence. This property was lost in low serum but addition of hepatocyte growth factor/scatter factor (HGF/SF) rescued soft-agar growth. Genetic proof that Met is necessary for this PAX3-FKHR-mediated effect was obtained by transducing with PAX3-FKHR MEFs derived from Met mutant (Met(D/D)) and wild-type (Met(+/+)) embryos. Only Met(+/+) MEFs acquired anchorage-independent growth whereas PAX3-FKHR-transduced Met(D/D) cells were unable to form colonies in soft agar. To verify if Met had a role in RMS maintenance, we silenced the receptor by transducing ERMS and ARMS cell lines with an inducible lentivirus expressing an anti-Met short hairpin RNA (shRNA). Met down-regulation significantly affected RMS cells proliferation, survival, invasiveness, and anchorage-independent growth. Finally, induction of the Met-directed shRNA promoted a dramatic reduction of tumor mass in a xenograft model of RMS. Our data show that both ARMS- and ERMS-derived cell lines, in spite of the genetic drift which may have occurred in years of culture, seem to have retained an "addiction" to the Met oncogene and suggest that Met may represent a target of choice to develop novel therapeutic strategies for ARMS.