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.


Introduction
Spontaneous mammary tumours are the most prevalent type of malignant neoplasm in the bitch and woman with the three times over incidence in dog (MacEwen 1990). About 50 % of all the mammary tumours are malignant (Misdorp 2002). The aetiology of mammary cancer is very complex and not clearly understood. The known mediators of tumourigenesis in both species are: genetic, hormonal, dietary, environmental and carcinogenic factors (Russo and Russo 1998). Moreover, both species live in the same conditions, thus the dog is a good model for breast cancer studies.
The role of oestrogens, progestins and growth hormone in canine mammary cancer development has been documented . That is why mainly affected are not spayed female dogs in the middle age. The early ovariectomy reduces risk of mammary cancer development (Misdorp 1991). However, the high morbidity and mortality rate, which is caused by poor diagnostics and ineffective treatment strategies makes this problem still actual in both humans and dogs. The conventional approach to cancer therapy provide treatment according to the organ in which the cancer originates. However, different intracellular signalling pathways are perturbed in the various cancers even if they represent the same type. Thus, the patients with the same type of cancer often have dissimilar genetic defects in their tumours and respond in a heterogeneous manner to anticancer agents (Veer van't and Bernards 2008). Moreover, the diagnostic methodologies available in veterinary oncology may still be considered to be in progress. So far, only histopathological variables (tumour size, lymph node status, vascular invasion and tumour grade of differentiation) are used as prognostic parameters (Manuali et al. 2012).
Thus, the aim of the presented study was to find 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) in order to find new diagnostic and prognostic markers.

Tissue samples
Tumour samples of canine mammary cancers were obtained from patients subjected to surgery. The tumours then, were divided into two equal halves, one of them was fixed in 10 % neutral buffered formalin and routinely embedded in paraffin to perform histological assay. The other was snap frozen in liquid nitrogen and stored in −80°C. Four μm samples from paraffin blocks were fixed on glass slides, stained with haematoxylin-eosin (HE) and examined by certified pathologists (prof. Dr. hab. Elżbieta Malicka and Dr. Izabella Dolka, both from the Warsaw University of Life Sciences, Poland). The immunohistochemical examination of cytokeratin, vimentin, smooth muscle actin, s100 protein and p63 Table 1 Primers sequences used in this study and their annealing optimal temperature and time. The mRNA sequences of key genes were obtained from NCBI database. Primers were designed using PRIMER3 software (free on-line access) and checked using Oligo Calculator (free on-line access) and Primer-Blast (NCBI database). Rps19 gene was used as a non-regulated reference gene for normalization of target gene expression (Brinkhof et al. 2006;Etschmann et al. 2006  protein expression was performed (data not shown). The tumour types of specimens were classified based on the World Health Organization (WHO) Histological Classification and Mammary Tumours of the Dog and Cat classification (Misdorp et al. 1999). Histological tumour grading was conducted on HE-stained sections using a Misdorp classification (2002). The mammary carcinoma grading was assessed in respect to tubule formation, degree of differentiation and mitotic index as. All the tumours examined were classified as the 1st grade of malignancy or the 3rd grade of malignancy (6 tumours in each group). Unfortunately survival data of these dogs is unavailable.

Microarray analyses
The total RNA from the samples was isolated using a Total RNA kit (A&A Biotechnology, Poland) according to the manufacturer's protocol. Isolated RNA samples were dissolved in RNase-free water. The quantity of RNA was measured using NanoDrop (NanoDrop Technologies USA). The samples with adequate amounts of RNA were treated with DNaseI to eliminate a possibility of DNA contamination. The samples were subsequently purified using RNeasy MiniElute Cleanup Kit (Qiagen, Germany). Finally RNA samples were analyzed using BioAnalyzer (Agilent, USA) to measure the final RNA quality and integrity. The Quick Amp Labeling Kit (Agilent, USA) was used to amplify and label target RNA to generate complementary RNA (cRNA) for oligo microarrays used in gene expression profiling and other downstream analyses. The gene expression of the poorly differentiated, most malignant tumours was compared against the gene expression of the well differentiated tumours (the 1st grade of malignancy). Samples were examined in four repetitions (two dye-swaps to eliminate the effect of label factor). Thus, each biological condition was labelled once by Cy3 and once by Cy5. Taking the average of all labelled arrays, the dye effect on any particular gene was cancelled. The hybridization was performed with canine-specific AMADID Release GE 4x44K microarrays (Agilent, USA) using Gene Expression Hybridization Kit (Agilent, USA) according to the manufacturer's protocol.

Signal detection, quantification and analysis
Acquisition and analysis of hybridization intensities were performed using DNA microarray scanner (Agilent, USA). Then, the results were extracted using Agilent's Feature Extraction Software with normalization and robust statistical analyses. Results were analyzed for statistical purposes using Future Extraction and Gene Spring software (Agilent, USA). The unpaired t-test with Benjamin-Hochberg FDR<5 % (false discovery rate) correction was applied (with p value cut-off <0.01). For further analysis we chose only these genes with values within upper and lower cut-off (100.00 and 20.00, respectively) in each of the slide, whose expression changed at least 1.5fold in each of examined slide. The area of the analyses covered in this publication has been deposited in NCBI's Gene Expression Omnibus and is accessible via GEO Series accession number GSE 44033.
Gene function was identified using the PANTHER pathway analysis software (Mi et al. 2005) and Pathway Studio software (Agilent, USA). PANTHER online platform allowed for wide analysis of the Canis familiaris regulated genes and also for statistical analysis of number of regulated genes involved in specific pathways or biological functions compared to the normal healthy cell of this specie.

Real-time qPCR
The mRNA sequences of the key genes were obtained from NCBI database. Primers were designed using PRIMER3 software (free on-line access) and checked using Oligo  Calculator (free on-line access) and Primer-Blast (NCBI database). Primers' sequences are listed in Table 1. Rps19 gene was used as a non-regulated, reference gene for normalization of target gene expression (Brinkhof et al. 2006;Etschmann et al. 2006). Quantitative RT-PCR was performed using fluorogenic Lightcycler Fast Strand DNA Sybr Green (Roche) and the Light Cycler (Roche). The results were analyzed using comparative Ct method (Schmittgen and Livak 2008). Relative transcript abundance of the gene equals ΔCt values (ΔCt = Ct reference -Ct target ). Relative gene expression is expressed as ΔΔCt value (ΔΔCt=2 -ΔCt ). The experiment was conducted in triplicates.
Then, to visualize the PCR product it was dedicated for electrophoresis in 2 % agarose gel (Sigma Aldrich), stained with ethidium bromide (Sigma Aldrich) and run for 60 min at 90 mV in 1× tris-borate-EDTA buffer. Then, the gel was visualized under UV light.

Gene expression in canine mammary malignancy
The microarray-based transcriptional profile of the canine mammary cancers of the 3rd grade of malignancy was Fig. 2 Classification of up-regulated genes in canine mammary cancers of the 3rd grade of malignancy (a.) and in canine mammary cancers of the 1st grade of malignancy (b.) according to their involvement in biological processes (based on the PANTHER Database, www.pantherdb.org) compared to the canine mammary cancers of the 1st grade of malignancy used as a reference. For each comparison 2 separate dye-swap experiments were performed. This study showed 70 statistically significant (p<0.005; Fold change = 1.5) regulated genes (Fig. 1). Further analysis showed 39 up-regulated genes ( Table 2) and 31 down-regulated genes (Table 3) in canine mammary cancer of the 3rd grade of malignancy.

Real-time qPCR gene expression
For the purpose of microarray data validation, we have randomly selected 4 genes: il15, ergic2, elspb1 and extl3. Realtime qPCR results showed similar trends in gene expression changes as were observed in microarray studies (Fig. 3). The expression of examined genes was higher in the most malignant canine mammary cancers than in the tumours of the 1st grade of malignancy.

Discussion
Canine mammary cancer constitutes a serious clinical problem. That is a reason why its molecular biology has been systematically examined during the last few years (Rao et al. 2009;Pawłowski et al. 2011, Klopfleish et al. 2010Pawłowski et al. 2013).
The very interesting study was conducted by Klopfleisch et al. (2010) who identified a gene expression profile in canine mammary tumours that was associated with early metastatic spread to the lymph nodes. Based on the gene expression pattern of these tumours the authors were able to discriminate carcinomas with divergent metastatic potential despite similar histological features. Moreover, a partial overlap was found between the canine mammary "metastatic" gene expression profile and similar metastasis-associated gene expression "signature" of breast cancer (Veer et al. 2002).
Our previous study of gene expression in canine mammary tumours of various grade of malignancy showed that histological diagnosis was distinct from molecular diagnosis . We have also identified cellular pathways and biological processes in which the most Fig. 3 Results for agarose gel electrophoresis of examined gene's PCR products following real time Sybr green amplification (a.). The RT-qPCR expression of examined genes (n=3) (b.). Estimated relative gene expression for each gene was compared between canine mammary carcinomas of the 3rd grade of malignancy and of the 1st grade of malignancy (ANOVA and Tukey test; Graph Pad Prism 3.0, USA). The p value <0.05 was regarded as significant and marked as *, p<0.001 was regarded as highly significant and marked as ** significant up-regulated genes were involved. In the tumours of the 3rd grade of malignancy we identified interesting upregulated cluster of genes related to immunological system. Their higher expression found in the most malignant cancers might be related with increased recruitment of hematopoietic cells into the tumour mass. Although the tumour is composed of various cells depending on the tumour type, myeloid cells seem to form a major component (Bingle et al. 2002). Clinical studies have shown a correlation between the number of myeloid cells (mainly macrophages) and a poor prognosis in many human cancers (e.g. breast, prostate, ovarian, etc.) (Jadus et al. 1996). Our own studies conducted on canine mammary cancers have not shown any correlation between number of macrophages in tumour mass and a grade of tumour malignancy . However, interestingly we observed expression of myeloid cell antigens in cancer cell lines and tissues  which increased upon the co-culture of these both types of cells . We have shown that expression of typical macrophage antigens (CD14, CSF-1R) in canine mammary cancer tissues correlated with the tumour grade of malignancy . Similarly, Dr. Pollard (2008) described that a gene expression signature characteristic for macrophages was an independent predictor of poor outcome in follicular lymphoma. Thus, these genes were typed as new malignancy markers.
Based on these results, the aim of the presented study was two-fold: 1) to compare gene expression in canine mammary tumours of the 1st and the 3rd grade malignancy in order to find new possible prognostic markers and 2) to validate whether genes characteristic for immunological system can constitute new markers of malignancy.
Similarly to our previous study ) we showed significant over-manifestation of genes related with chemokine and cytokine mediated signalling pathway (HLA-DQB1, NELL1, LYZL6, S100P, TFPI2, TREM1, EMR3, IL6, IL8, IL15, MARCO, CAMP) (Fig. 2, Tables 2 and 3). A few of these genes seemed to be particularly interesting. For example, S100P calcium binding protein (which expression is regulated by androgens and IL6another up-regulated gene in the most malignant canine mammary tumours) is though as a new prognostic factor (Parkkila et al. 2008). A correlation was found between its increased expression and poor survival, cancer proliferation and increased resistance to chemotherapy (Maciejczyk et al. 2013). Our results are in accordance with clinical data as the cancers of high grade of malignancy (which express higher levels of S100P) are associated with an increased risk of death within 2 years after mastectomy (Karayannopoulou et al. 2005).
In the most malignant canine mammary cancers an increased expression of two metalloproteinases (MMPs): 1 and 3 was observed (Table 2). MMPs comprise a structurally and functionally related family degrading extracellular matrix and basement membrane barriers. That is why they are thought to play a key role in angiogenesis, inflammatory processes, cancer development and metastasis, as well as in proliferation and apoptosis (Sauter et al. 2008). Because of their role in the degradation of the extracellular matrix leading to tumor invasion and metastasis, they may also serve as prognostic markers (Pardo andSelman 2005, Brickerhoff andMatrisian 2002). In this context, MMPs have been focused on as targets for therapeutic strategies.
The metalloproteinases are also linked to specific aspects of an inflammatory or immune response, such as the generation of chemokine gradients or immune cell influx (Hojilla et al. 2008). In addition to the metalloproteinase-mediated generation of inflammation triggers, metalloproteinases are, in turn, utilized by immune cells to further propagate the inflammatory reaction. In breast cancer samples, MMPs are found in neutrophils, macrophages, and T lymphocytes as well as in cancer cells (Benaud et al. 1998).
Cancer development is a complex process. In addition to the cancer cell intrinsic factors, the cancer microenvironment composed of various cells influences the behavior of cancer cells. The results of our study indicate 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.