Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR-101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF-beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.

Analysis of microRNA expression in canine mammary cancer stem-like cells indicates epigenetic regulation of transforming growth factor-beta signaling.

Cancer stem cells (CSCs) display both unique self-renewal ability as well as the ability to differentiate into many kinds of cancer cells. They are supposed to be responsible for cancer initiation, recurrence and drug resistance. Despite the fact that a variety of methods are currently employed in order to target CSCs, little is known about the regulation of their phenotype and biology by miRNAs. The aim of our study was to assess miRNA expression in canine mammary cancer stem-like cells (expressing stem cell antigen 1, Sca-1; CD44 and EpCAM) sorted from canine mammary tumour cell lines (CMT-U27, CMT-309 and P114). In order to prove their stem-like phenotype, we conducted a colony formation assay that confirmed their ability to form colonies from a single cell. Profiles of miRNA expression were investigated using Agilent custom-designed microarrays. The results were further validated by real-time rt-PCR analysis of expression of randomly selected miRNAs. Target genes were indicated and analysed using Kioto Encyclopedia of Genes and Genomes (KEGG) and BioCarta databases. The results revealed 24 down-regulated and nine up-regulated miRNAs in cancer stem-like cells compared to differentiated tumour cells. According to KEGG and BioCarta databases, target genes (n=240) of significantly down-regulated miRNAs were involved in transforming growth factor-beta signaling, mitogen-activated protein kinases (MAPK) signaling pathway, anaplastic lymphoma receptor tyrosine kinase (ALK) and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1A) pathways. The analysis of single-gene overlapping with different pathways showed that the most important genes were: TGFBR1, TGFBR2, SOS1, CHUK, PDGFRA, SMAD2, MEF2A, MEF2C and MEF2D. All of them are involved in tumor necrosis factor-beta signaling and may indicate its important role in cancer stem cell biology. Increased expression of TGFBR2, SMAD2, MEF2A and MEF2D in canine mammary cancer stem-like cells was further confirmed by real-time-qPCR. The results of our study point at epigenetic differences between cancer stem-like cells and differentiated tumour cells, which may be important not only for veterinary medicine but also for comparative oncology.

Canine mammary carcinoma cell line are resistant to chemosensitizers: verapamil and cyclosporin A.

Cancer chemotherapy can fail in many ways. One of the most significant is the development of multiple drug resistance (MDR), which constitutes a serious clinical problem. The development of MDR relates to the expression of a major membrane pump, P-glycoprotein (P-gp). Thus, currently one of the goals of experimental and clinical oncology is to decrease its activity. So far, many different P-gp inhibitors are available, but their efficacy is still questionable and requires further study. The aim of our study was to assess an impact of classical P-gp inhibitors (verapamil and cyclosporin A) in the reversion of multidrug resistance in canine mammary cancer cells. We used two cell lines isolated from mammary tumors and two cell lines isolated from their lung metastases. All of them showed P-gp over-expression confirmed using Real-time rt-PCR, Skan(R) screening station and confocal microscopy. The FACS analysis showed that in three of the examined cell lines, treatment with verpamil/cyclosporin A was ineffective to reverse cancer chemoresistance. However, more studies in this field are required.

Bone marrow-origin stem/progenitor cells in the mammary gland of heifers.

The aim of the study was to estimate the size of bone marrow-origin stem/progenitor population in 2-year old nonpregnant Holstein-Friesian heifers. Quantitative and qualitative analysis was done using scanning cytometry and confocal microscopy of mammary tissue slices labelled with the combination of two markers: Sca-1 (marker of stem-progenitor cells) and CD45 (marker of hematopoietic cells). The average (+/- SEM) percentage of Sca-1POS CD45 POS cells was 0.89 +/- 0.21. They were localized mainly outside of mammary ducts, in the stroma and sometimes intraluminally. Our results indicate that the subpopulation of Sca-1POS cells bearing CD45 antigen may enrich the niche of mammary stem/progenitor cells from the bone marrow and participate in the growth of the mammary gland in post-pubertal heifers.

Transcriptional background of beef marbling - novel genes implicated in intramuscular fat deposition.

The purpose of this study was to identify novel marbling-related genes by comparison of the global gene expression in semitendinosus muscle of 15-month-old Limousin (LIM), Holstein-Friesian (HF) and Hereford (HER) bulls. Muscle of LIM was lean with low intramuscular fat (IMF) content (0.53%) unlike the marbled muscles of HER and HF characterized by higher amounts of IMF (1.10 and 0.81%, respectively). The comparison of muscle transcriptional profile between marbled and lean beef revealed significant differences in expression of 144 genes, presumably involved in consecutive stages of adipose tissue development, such as preadipocyte proliferation and differentiation, adipocyte maturation, lipid filling and lipid metabolism leading to increased IMF deposition and marbling development. Correlation coefficients and regression analysis for nine of them (gadd45a, pias3, ccrn4l, diras3, pou5f1, hoxa9, atp2a2 and pim1) validated by real-time qPCR confirmed their moderate-high correlation with IMF% and explained up to 70.5% of the total variability in IMF deposition in the bulls.

Myogenic cells applications in regeneration of post-infarction cardiac tissue.

Skeletal myoblasts (SMs) transplantations are the focus of intense investigation due to their therapeutic potential for cardiac repair. Heart diseases are the main problem in humans worldwide, resulting very often in death of patients. Consequently, investigators sought methods which could improve myocardial function in patients with cardiac failure. Stem cells therapy belongs to one of the highly promising and the most realistic methods of treating myocardial infarction. Despite the fact that many cell types were transplanted into damaged myocardium, autologous skeletal myoblasts seem to be the most encouraging cell source for myocardial repair due to their myogenic and contractile phenotype, biochemical and functional similarities to cardiac cells, high proliferative capacity in vitro and resistance to ischemia. Moreover, engrafted myoblasts could repopulate post-infarction scar and improve cardiac function. Unfortunately, transplanted SMs do not functionally or electrically integrate with the host myocardium, which could also cause ventricular arrhythmias, therefore, the use of these cells still remains a subject of intensive studies aiming at improvement of this therapeutic method. In this paper we recapitulate the current state of knowledge concerning the use of skeletal myoblasts in the treatment of post-infarction scar tissue, and discuss the problems resulting from their applications.

IGF-I retards proper development of acinar structures formed by bovine mammary epithelial cells via sustained activation of Akt kinase.

Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17ß-estradiol. The addition of 17ß-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17ß-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.

Expression of inflammation-mediated cluster of genes as a new marker of canine mammary malignancy.

Because canine mammary tumours constitute a serious clinical problem and there are no good prognostic markers (only histopathological variables are used), the aim of the presented study was to find new malignancy markers as well as to identify intracellular pathways and biological processes characteristic for canine mammary malignancy. We compared gene expression of the most malignant mammary tumours (poorly differentiated cancers of the 3rd grade of malignancy) with less malignant tumours (well differentiated cancers of the 1st grade of malignancy). The results of our study indicated that in dogs the number of tumour-infiltrating myeloid cells or expression of myeloid-specific antigens by cancer cells is related to the cancer progression and may constitute a new marker of malignancy, however further studies in this field are required.

Derivation of feline vaccine-associated fibrosarcoma cell line and its growth on chick embryo chorioallantoic membrane - a new in vivo model for veterinary oncological studies.

Feline vaccine associated fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated fibrosarcoma cell line and its growth on CAM is described. The cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.

A role of ghrelin in cancerogenesis.

Ghrelin is a 28 amino-acid multi-functional peptide hormone, which was identified as a natural ligand of the growth hormone secretagogue receptor (GHS-R). Pituitary growth hormone-releasing activity in both animals and humans has been well documented. It has various biological functions, including regulation of appetite and body weight, control of energy homeostasis, modulation of cardiovascular and gastrointestinal system and anti-inflammatory effect. However, both ghrelin and its receptor (GHS-R) are widely distributed in various tumors, which strongly implies their role in neoplastic cell growth trough autocrine/paracrine mechanism. Multiple studies have demonstrated the role of ghrelin in cancer cells proliferation, differentiation, invasiveness and apoptosis inhibition. The ghrelin axis is more complex than it was originally thought and consist of several compounds that might interact with each other and affect ghrelin activities. Here, we provide an overview of the ghrelin and its receptor role in tumor progression.

Growth hormone receptor (GHR) RNAi decreases proliferation and enhances apoptosis in CMT-U27 canine mammary carcinoma cell line.

Canine mammary gland has been identified as a major site of the extrapituitary growth hormone (GH) production. This finding is linked to its role in tumourigenesis of the mammary gland. Our previous studies indicated the role of GH and GH receptor (GHR) in regulation of proliferation and apoptosis. Thus, we have optimized the ghr RNA interference method in canine mammary carcinoma cell line CMT-U27. We have analysed the effect of GHR reduction on the intracellular signalling and the cell cycle and apoptosis. The results showed that GHR reduction decreased the p-ERK1/2 expression and caused increase of apoptosis and decrease in number of cells at S and G2M phases. This study indicates that GHR besides proliferative effect promotes growth by increasing cell survival. It can tilt the balance between proliferation and death in cancer cells.

Mismatch between transcriptomic and histopathologic picture of canine lymphomas.

Lymphoma is one of the most common malignant tumours occurring in dogs. Since there is a constant need for new, more comprehensive laboratory diagnostic tools which permit the precise determination of many tumour-related factors we decided to verify whether the use of microarray analysis could be helpful in classifying lymphomas. The study was performed on samples collected from 7 dogs in which multicentric lymphoma was recognized. Among this group we were able to identify one sub-cluster of transcriptionally similar tumours, which completely differed in terms of the histopathological examination. Among them there were one diffuse large B cell lymphoma, one diffuse macronucleolated medium-sized cell lymphoma and one pleomorphic mixed small and large T-cell lymphoma. The lymphomas belonging to the sub-cluster differed from other analysed tumours in the expression of more than 100 genes of which only 18 were described earlier in regard to lymphomas.

Role of extracellular matrix and prolactin in functional differentiation of bovine BME-UV1 mammary epithelial cells.

Interactions between extracellular matrix (ECM) and epithelial cells are necessary for proper organisation and function of the epithelium. In the present study we show that bovine mammary epithelial cell line BME-UV1 cultured on ECM components, commercially available as Matrigel, constitutes a good model for studying mechanisms controlling functional differentiation of the bovine mammary gland. In contact with Matrigel BME-UV1 cells induce apicobasal polarity, and within 16 days form three dimensional (3D) acinar structures with a centrally localized hollow lumen, which structurally resemble mammary alveoli present in the functionally active mammary gland. We have shown that the 3D culture system enables a high expression and proper localisation of integrin receptors and tight junction proteins in BME-UV1 cells to be induced. This effect was not obtained in cells grown in the classical 2D culture system on plastic. Moreover, ECM highly stimulated the synthesis of one of the major milk proteins, beta-casein, even in the absence of prolactin. Our results show that contact with ECM plays an important role in the lactogenic activity of bovine MECs, however, prolactin is necessary for the efficient secretion of milk proteins.

Relationship between receptors for insulin-like growth factor- I, steroid hormones and apoptosis-associated proteins in canine mammary tumors.

In the veterinary literature there are few data concerning the expression of insulin-like growth factor type I (IGF-IR) in the canine mammary gland tumors. The aim of the present study was the evaluation of IGF-IR expression and its correlation to the expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), proteins: Bcl-2, Bax, p53 in canine mammary gland tumors, and also a correlation with other features: bitch's age, tumor diameter, histologic type of tumor, degree of histologic malignancy, proliferate activity. The study was done on 112 epithelial neoplasms: 21 (19%) were adenoma, 38 (34%) complex carcinoma (adenocarcinoma), 47 (42%) simple carcinoma (adenocarcinoma) and 6 (5%) solid carcinoma. Histochemistry and immunohistochemistry methods were employed. It was shown that more common and/or higher IGF-IR expression in cells of canine mammary gland tumors was related to the histologic type of cancer of worse prognostic (solid and simple carcinoma), high histologic degree of malignancy (III degrees) but the statistical analysis did not reveal any significant differences. We observed the high degree of IGF-IR expression in tumors which displayed the high ERalpha and PR expression. These results suggest the involvement of IGF-IR in the development of hormonosensitive canine mammary tumors. Additionally, the significant positive correlation between expression of IGF-IR and p53, Bax was found. Our study provides some evidence that interactions exist between the IGF-IR and these apoptosis-associated proteins may contribute to the development and progression of canine mammary gland tumors. These results require further investigations.

Density of tumor-associated macrophages (TAMs) and expression of their growth factor receptor MCSF-R and CD14 in canine mammary adenocarcinomas of various grade of malignancy and metastasis.

Several years ago, the presence of macrophages in the tumor microenvironment was thought to be an inflammatory response to kill the cancer cells. Now, this is clear that the inflammatory cells that exit blood vessels and migrate to the tumor tissue play an important role in cancer progression. Various cells present in the tumor microenvironment enhance cancer growth and invasiveness by secretion of tumor-enhancing products. That is why tumors should not be treated as only aggregates of cancer cells but as separate structures. Macrophages form a major component of the inflammatory infiltration in tumors, where they are termed tumor-associated macrophages (TAMs). To the best of our knowledge, up-to-date there were no studies on tumor associated macrophages and the role of the tumor microenvironment in tumor invasion/metastasis in dogs. This is the first study performed to asses if the number of TAMs and expression of MCSF-R (macrophages colony stimulating factor receptor) and CD14 (LPS co-receptor) are associated with the grade of tumor malignancy and its ability to metastasize. We have performed immunohistochemical analysis of 50 canine mammary adenocarcinomas of various grade of malignancy (1st, 2nd, 3rd) and tumors that gave local or distant metastases. The results indicate that in dogs, similarly to humans and mice, the number of tumor associated macrophages is related to the cancer ability to metastasize. Our results also indicate that the expression of MCSF-R and, what is particularly new finding, CD14 is associated with tumor malignancy and its ability to metastasize. Hence, these molecules play a role in tumor progression, metastasis and microenvironment interactions. These results show that in dogs we should treat the tumor as a whole organ rather than just try to eliminate the cancer cells.

Gene expression pattern in canine mammary osteosarcoma.

Canine mammary sarcomas are usually very aggressive and easily metastasize. Unfortunately the biology of this type of tumor is not well known because they are a very rare type of tumors. The aim of this study was to find differences in gene expression patterns in canine mammary osteosarcomas (malignant) versus osteomas (benign) using DNA microarrays. Our microarray experiment showed that 11 genes were up-regulated in osteosarcoma in comparison to osteoma whereas 36 genes were down-regulated. Among the up-regulated genes were: PDK1, EXT1, and EIF4H which are involved in AKT/PI3K and GLI/Hedgehog pathways. These genes play an important role in cell biology (cancer cell proliferation) and may be essential in osteosarcoma formation and development. Analyzing the down-regulated genes, the most interesting seemed to be HSPB8 and SEPP1. HSPB8 is a small heat shock protein that plays an important role in cell cycle regulation, apoptosis, and breast carcinogenesis. Also SEPP1 may play a role in carcinogenesis, as its down-regulation may induce oxidative stress possibly resulting in carcinogenesis. The preliminary results of the present study indicate that the up-regulation of three genes EXT1, EIF4H, and PDK1 may play an essential role in osteosarcoma formation, development and proliferation. In our opinion the cross-talk between GLI/Hedgehog and PI3K/AKT pathways may be a key factor to increase tumor proliferation and malignancy.

The transcriptomic signature of myostatin inhibitory influence on the differentiation of mouse C2C12 myoblasts.

GDF8 (myostatin) is a unique cytokine strongly affecting the skeletal muscle phenotype in human and animals. The aim of the present study was to elucidate the molecular mechanism of myostatin influence on the differentiation of mouse C2C12 myoblasts, using the global-transcriptome analysis with the DNA microarray technique. Treatment with exogenous GDF8 strongly affected the growth and development of C2C12 mouse myoblasts. This was manifested by the inhibition of proliferation and differentiation as well as the impairment of cell fusion. DNA microarray analysis revealed 778 genes regulated by GDF8 in differentiating myoblasts (436 down-regulated and 235 up-regulated). Ontological analysis revealed their involvement in 17 types of biological processes, 10 types of molecular functions and 68 different signalling pathways. The effect of GDF8 was mainly mediated by the disruption of the cell cycle, calcium and insulin signalling pathways and expression of cytoskeletal and muscle specific proteins. The identified key-genes that could play a role as GDF8 targets in differentiating myoblasts are: Mef2, Hgf, Ilbl, Itgb1, Edn1, Ppargc1a.

Why chemotherapy can fail?

There are many reasons that lead to failure of cancer chemotherapy. Cancer has the ability to become resistant to many different types of drugs. Increased efflux of drug, enhanced repair/increased tolerance to DNA damage, high antiapoptotic potential, decreased permeability and enzymatic deactivation allow cancer cell survive the chemotherapy. Treatment can lead to the death of most tumor cells (drug-sensitive), but some of them (drug-resistant) survive and grow again. These tumor cells may arise from stem cells. There are many studies describing human experiments with multidrug resistance, especially in breast cancer. Unfortunately, studies of canine or feline ABC super family members are not as extensive as in human or mice and they are limited to several papers describing PGP in mammary cancer, cutaneous mast cell tumors and lymphoma. Multidrug resistance is one of the most significant problems in oncology today. The involvement of many different, not fully recognized, mechanisms in multidrug resistance of cancer cells makes the development of effective methods of therapy very difficult. Understanding the mechanisms of drug resistance in cancer cells may improve the results of treatment. This review article provides a synopsis of all aspects that refer to cancer cell resistance to antitumor drugs.

Gene expression profiling in peripheral blood nuclear cells in patients with refractory ischaemic end-stage heart failure.

Functional analysis of up- and down-regulated genes might reveal whether peripheral blood cells may be considered as a material of diagnostic or prognostic value in patients with end-stage heart failure (HF). The aim of the present study was to compare the transcriptomic profile of peripheral blood nuclear cells from 6 male patients with ischaemic end-stage HF with those of 6 male patients with asymptomatic cardiac dysfunction. The expression of genes in peripheral blood nuclear cells in both groups of patients was measured using whole-genome oligonucleotide microarrays utilizing 35 035 oligonucleotide probes. Microarray analyses revealed 130 down-regulated genes and 15 up-regulated genes in the patients with end-stage HF. Some of the down-regulated genes belonged to the pathways that other studies have shown to be down-regulated in cardiomyopathy. We also identified up-regulated genes that have been correlated with HF severity (CXCL16) and genes involved in the regulation of expression of platelet activation factor receptor (PTAFR, RBPSUH, MCC, and PSMA7). In conclusion, the identification of genes that are differentially expressed in peripheral blood nuclear cells of patients with HF supports the suggestion that this diagnostic approach may be useful in searching for the molecular predisposition for development of severe refractory HF in patients with post-infarction asymptomatic abnormalities and remodelling of the left ventricle. These results need further investigation and validation.

Transcriptomic "portraits" of canine mammary cancer cell lines with various phenotypes.

In light of the high incidence of mammary cancer in dogs and completion of the canine genome sequencing, the new possibilities of gene profiling by using DNA microarrays give hope to veterinary oncology. The cell lines isolated from mammary tumors are a valuable tool in developing and testing new pathway-specific cancer therapeutics. Differential cytometric analysis of 6 canine mammary cancer cell lines was performed. We divided cell lines into 3 groups based on their phenotype: 2 lines with high proliferative potential, 2 lines with high antiapoptotic potential, and 2 lines with high metastatic potential. DNA microarray analysis revealed common genes for cell lines of each group. We found that genes encoding the receptors for growth hormone and ghrelin are related to high proliferation rate, while ABR (active BCR-related) and TMD1 (TM2 domain containing 1) genes are related to a high antiapoptotic potential of the cancer cells. Metastatic properties of mammary cancer cells seem to be associated with elevated expression of PGP (P glycoprotein), SEMA3B (semaphorin 3B), and STIM1 (stromal interaction molecule 1).