Canine mammary cancers in bulk arise from epithelial cells. Several genetic alterations have been detected, that may predispose these cells to the malignant transformation [10
]. However, researches over the last few years suggested that concomitant changes also occur in stromal cells that form the tumour microenvironment [1
]. The hypothesis of stromal cells involvement in tumorigenesis is based on a study of embryological development where interactions between various cells are necessary for programming and maintaining epithelial structure and function. The embryonic epithelial and stromal cells of mesenchymal origin engage in a molecular dialogue that ensures the proper organ development and function [3
The study showed in cancer cells after a co-culture with CAFs an up-regulation of 23 genes (Table ) involved in developmental processes (a nervous system development, an embryonic development, a mesoderm and ectoderm development).
The involvement of fibroblasts in the malignant transformation of epithelial cells has previously been documented [3
]. Moreover, the histology and growth characteristics of CAFs were found different from those of the fibroblasts associated with normal epithelial cells [3
]. Mishra et al. [29
] have proposed bidirectional cross-talk between the CAFs and the cancer cells which release proteins that increase the fibroblasts ability to secrete a variety of tumour-promoting factors, which then act back on the malignant cells to change their gene expression and promote their proliferative, migratory, and invasive properties. On the other hand, other studies showed that only direct contact of fibroblasts with cancer cells is able to cause changes in their gene expression and biology [30
So far several papers have been published about gene expression in tumour microenvironment. Most of them describe gene expression in fibroblasts, but not in cancer cells, however there are some papers available about the changes in gene expression in cancer cells [33
]. These reports indicated up-regulation of genes involved in angiogenesis, EMT and migration in cancer cells grown with fibroblasts. Surprisingly, some of the genes identified, even though functionally identical turned out to be of different names. The studies have been conducted using various cancer models (various species) and various cell lines, so the differences are possible.
The results of the study hereby revealed increased expression of 13 genes involved in cell adhesion (Table ) among cancer cells co-cultured with the CAFs. As much as 10 of them are involved in developmental processes as well. These genes seem to be particularly significant because the cell adhesion is responsible for tumour progression and metastasis, detachment from the primary tumour and spreading to the circulatory system. Moreover, the up-regulated genes responsible for adhesion are by rule involved in angiogenesis and lymphangiogenesis. For example, the vascular cell adhesion molecule-1 (VCAM-1) up-regulated in cancer cells grown as a co-culture with CAFs may be involved in tumor progression and metastasis particularly via lymphangiogenesis promotion [37
]. It also has previously been demonstrated that the VCAM-1 plays a crucial role in the endothelial-carcinoma cell adhesion [38
The study also revealed an up-regulation of desmoplakin (DSP), which is a key component of cellular adhesion junctions known as desmosomes. These junctions are found at contact sites between endothelial cells that form capillaries, thus DSP play a role in de novo
capillary formation and branching during tumourigenesis, embryonic development and cardiovascular development [39
]. Moreover, desmoplakin isoform 2 was only detected in tumours associated with a poor clinical outcome. It may suggest its potentially specific function in the regulation of cancer cells proliferation, differentiation, invasion and metastasis [40
Moreover, desmosomes may also be important in the epithelium-mesenchymal transition (EMT). The epithelium-mesenchymal transition is an indispensable mechanism for morphogenesis during embryonic development, and is implicated in conversion of early-stage tumours into invasive cancers. During EMT, epithelial cells undergo changes in morphology and acquire the migratory and invasive characteristics of mesenchymal cells [30
]. EMT is also promoted by the FOXQ1, another up-regulated gene in cancer cells grown under co-culture conditions with the CAFs [42
]. It also increases expression of several junction proteins promoting cancer cells to gain the stem-cell-like properties and ensuring resistance to apoptosis [42
]. Moreover, the down regulation of keratin 20 (Table ) in cancer cells following the co-culture with the CAFs may indicate the EMT induction [45
Interestingly, another up-regulated gene in cancer cells grown with the CAFs, which contributes to cancer invasion is myelin-associated glycoprotein (MAG) that binds to the oncogenic glycoprotein MUC1 [47
]. Swanson et al. [47
] described an interaction between the MUC1 and the MAG in cancers that invade perineurally, including prostate, salivary, and breast carcinomas. Furthermore, breast cancers may metastasize to the brain where the MAG is abundantly expressed. Interactions between the MUC1 and the MAG have not fully been defined yet. We confirmed the MUC1 expression in all of the examined cell lines (Figure ). Thus, based on our own observations and those of Swanson et al. [47
], we suppose that the MAG up-regulation in cancer cells grown with the CAFs and its binding to the MUC1 may contribute to the adhesion between tumour cells and Schwann cells promoting metastasis to the nervous system.
We also found a down-regulation of 5 key genes associated with adhesion. Subject literature suggests 3 of them play a role in cancer development. The down-regulation of these genes is associated with poor prognosis and cancer metastases. One of these genes is the ADAMTS15 (a disintegrin and metalloproteinase with thrombospondin motif 15) which is an anti-angiogenic factor [48
]. Our study also revealed a down-regulation of the CADM4. Nagata et al. [49
] found decrease in the CADM4 expression in most of renal cell carcinomas and the cancer cell lines. Moreover, the CADM4 expression was decreased in carcinomas with vascular infiltration, suggesting that loss of the CADM4 is involved in tumour angiogenesis and invasion. We have also found a down-regulation of the MATN1 gene which has been defined an angiogenesis inhibitor [50
In the current study we showed a significant over-manifestation of genes involved in the oxytocin receptor mediated signaling pathway, the thyrotropin-releasing hormone receptor signaling, the Beta 2 and Beta1 adrenergic receptor signaling, and the histamine H1 receptor mediated signaling in cancer cells grown with the CAFs. Entschladen et al. [51
] described the role of neurotransmitters in cancer progression and metastasis. They found that similarly to chemokines, neurotransmitters are regulators of cell migration. Sadly though, we noticed that only a few results are available on the expression of neurotransmitter receptors in tumour tissues. Among them the best understood is the role of catecholamines in carcinogenesis and tumour progression. These are the stress hormones, whereas stress in turn is a major risk factor for the development of cancer. Norepinephrine has been shown to strongly induce the migration of tumor cells [52
], whereas epinephrine was found a modulator for the carcinogenesis in the lung [54
An interesting gene in cancer cells grown as a co-culture with the CAFs is the up-regulated protocadherin 19 (PCDH19) (Table ). Up-to-date there is no information available on the involvement of this gene in tumor progression or metastasis. However, a PCDH19 mutation was found to be responsible for epilepsy and mental retardation confined to females (EFMR) [55
]. There has been an on-going debate about the relationship between epilepsy and cancer. It has been hypothesized that the incidence of cancer is increased in people with epilepsy owing to the cancer promotion by antiepileptic drugs [56
]. Perhaps the increased risk of cancer in epileptic patients is caused by the PCDH19 mutation and over-expression, not however related to drugs toxicity. This hypothesis requires further studies.