Companion Animals as Models for Human Mammary Cancer Research

Abstract

In both humans and companion animals, cancer is one of the leading causes of death worldwide. Given the increasing incidence in humans and dogs, there is an urgent need to find or improve strategies for diagnosis, treatment and prognosis. Hence, the importance of having very similar study models. Both canine and feline models have advantages over their murine counterparts in the study of breast cancer and cancer in general. Among other things, at the molecular and genetic levels, in terms of risk factors, spontaneous disease onset and tumour heterogeneity, domestic animals share greater similarities with the human species than the murine model. In addition, they share environmental and socioeconomic factors. Another advantage is their similar response to chemotherapy treatment, and rapid imaging results can be obtained with the same screening techniques used in humans. Finally, this chapter discusses the main features that make the canine and feline model the main source for the study of breast cancer in vitro and in vivo.

1. Introduction

Breast cancer is one of the most common cancers in women worldwide. The disease is often hormonally regulated, with estradiol and some selective oestrogen receptor modulators (SERMs) influencing the development and/or progression of many breast tumours due to their association with nuclear and cytoplasmic oestrogen receptors (ERs). Binding of SERMs to these receptors leads to genomic and “non-genomic” effects (rapid activation of cellular phenomena, e.g., signalling cascades) and affects the development of some tumours in different ways. Treatment of breast cancer with anti-oestrogens or aromatase inhibitors (AIs) is often effective in patients with “ER -positive” breast cancer; unfortunately, not all cancers respond to such treatments. In addition, some treated tumours often develop resistance to these therapies.

Importantly, breast cancer can be classified into the following subtypes according to the presence and/or absence of various receptors: luminal A (ER+ and/or progesterone receptor (PR)+, EGFR2-), luminal B (ER+ and/or PR+, EGFR2+), EGFR2 over-expressed (ER-, PR-, EGFR2+), basal-like or triple negative (ER-, PR-, EGFR2-, cytokeratin (CK) 5/6 positive and/or epidermal growth factor receptor/EGFR positive) and normal breast-like tumours [1]. Luminal tumours have been associated with the most favourable prognoses, while EGFR2-overexpressing and basal-like tumours have been associated with the worst prognoses [1]. Approximately two out of three women with the disease have a tumour characterised by ER (“ER-positive”) [2].

In other species, spontaneous tumours in cats and dogs are similar to human cancer. These animals share the same lifestyle as humans, and their tumours are genetically heterogeneous, as are randomly selected groups of cancer patients [3].

For research purposes, both the canine and feline models of breast cancer are of great importance for the study of this pathology due to a number of similarities at the molecular, histological and etiological levels.

2. Breast cancer in women

Worldwide, the number of women with breast cancer is steadily increasing. In 2019, 2,002,350 million new cases were found, resulting in 700,660 deaths from the disease [4]. As a result, it has become one of the most common cancers affecting women. In developed countries, the disease affects one in eight women, with an average age at diagnosis of 61 years. Approximately 2% of breast tumours occur in young women aged 20–34, 11% between the ages of 35–44 and 87% in patients aged 45 and older. Most tumours are diagnosed at an early stage, without spread to the contralateral breast or axillary lymph nodes (i.e. ductal carcinomas in situ and stages I, IIA, IIB and IIIA) and are considered potentially curable. It is encouraging to note that the survival of those patients is improving, reaching a survival rate of around 88% in the following five years of treatment. The prognosis appears to be worse in women aged ≤35 years at diagnosis [5]. Although characterised by cellular, molecular and clinical heterogeneity [1], it is accepted that reproductive endocrine factors, especially those related to increased exposure to oestrogens and/or progestogens, are among the major risk factors for this disease [6].

3. Canine mammary cancer

Within veterinary clinical practice, it is common to have patients presenting with mammary cancer. Canine bitches have a high percentage of mammary tumours, with malignant neoplasms accounting for 30–50%, of which 50–75% recur or metastasise within one to two years [7]. Most malignant breast tumours are classified as epithelial tumours or carcinomas. Pure sarcomas represent a minority. Benign tumours include simple/complex adenomas, fibroadenomas and benign mixed tumour [8].

Comparatively, some prognostic factors are similar for human and canine species, although regional lymph node metastasis does not seem to be of major importance in canines. Metastatic spread is similar in both species, except that liver and bone metastases are not common in canines as they are in humans [9].

As in humans, the risk of canine mammary neoplasia is affected by oestrogen exposure during early mammary development [10]. Factors influencing females such as advanced age, treatment with progesterone or synthetic progestins, obesity at an early age, nulliparity, breed (heredity) and diet also increase the predisposition to develop mammary tumours in bitches [11]. Epidemiological studies in humans have shown that a high-fat diet and obesity increase the risk of mammary cancer. A study conducted in the United States showed that among neutered canines, the risk of developing mammary cancer was reduced if the dogs were lean (determined by body condition score) at nine to twelve months of age [12]. Consumption of homemade diets (compared to commercial foods) was also associated with an increased risk [13].

Several international studies have estimated that the incidence of obesity in the canine population varies between 22% and 40% [14], with the incidence of feline obesity being of similar frequency [15]. Several researchers agree that, similar to the trend in humans, the incidence of obesity in the pet population is increasing [16, 17].

Obesity is known to be a risk factor for mammary cancer as adipocytes have a high capacity to produce oestrogen from androgens, so cells in a mammary gland with a large amount of adipose tissue will be exposed to high concentrations of oestrogen. In animal models, biomarkers such as increased concentrations of IGF-1, leptin and sex hormones, as well as decreased concentrations of adiponectin, have been shown to promote tumour development. The same has been observed in humans [17].

The average age of presentation of mammary tumours is from ten to eleven years, with rare occurrence in canines younger than four years. Several spaniel breeds and, according to some studies, poodles and dachshunds seem to be predisposed to this condition [18]. Studies have reported that 25% of small breed dogs manifest histologically malignant tumours compared to 58% of medium to large breed patients [19]. A higher predisposition to mammary tumours has also been found in purebred dogs compared to mongrels [20].

One in four non-ovariohysterectomised bitches over four years of age is expected to develop mammary neoplasia [20]. Therefore, one of the control measures would be to perform ovariohysterectomies before the first, second or third oestrous cycle, as they are known to have a positive effect in decreasing the relative risk of developing mammary tumours to 0.5%, 8% or 26%, respectively [21]. In support of the above, a positive effect of ovariectomy in females is recognised, and its performance is recommended before the age of forty years [22].

The presence of oestrogen receptors (ERs), progesterone receptors (PRs) and epidermal growth factor receptors (EGFR) in both normal mammary tissue and mammary tumour tissue has been described in all species, including the canine species. The amount of steroid receptors in healthy tissue varies significantly with age (older canines have more ERs), location (posterior glands have higher concentrations of ERs) and the stage of the cycle (the highest number of ERs is found in the mid-luteal phase and the lowest concentrations of ERs in the early luteal phase). The EGFR content in the normal gland varies according to proliferative (oestrus, early and mid-luteal phase) and non-proliferative (early pro-estrus and anestrus) status. Most benign tumours contain ERs and PRs, the latter existing at levels similar to the normal gland [23]. In contrast, carcinomas, devoid of remnants of normal mammary epithelium, contain ERs and PRs in decreased numbers, with rare occurrence of metastases [24]. In a study by Millanta and colleagues, no significant differences in quantitative ER expression were found between normal, dysplastic, benign and carcinoma in situ tissue, while ER expression in invasive carcinomas was significantly lower [25].

Other research has revealed overexpression of messenger ribonucleic acid (mRNA) for the oncogene epidermal growth factor receptor 2 (also called EGFR2, HER2, erbB-2 or neu) in most malignant breast tumours (not so in benign ones) although without local invasion or regional metastatic disease. This suggests that EGFR2 overexpression may play a role in favouring the malignant process [26]. However, another study indicates that these proteins were more often expressed in benign tumours (50%) than in malignant tumours (19%) [27]. Dutra et al. detected EGFR2 protein expression in 35.4% of the malignant tumours analysed, while none of the benign tumours contained EGFR2 [28]. All these results show the heterogeneity of breast tumours and that, despite so much research, the importance of this receptor in the pathogenesis of this disease has not yet been fully elucidated.

Antuofermo et al. found that the canine model of intraepithelial mammary lesions, which is histologically and in the expression pattern of REα and HER-2 very similar to the human species, is ideally suited for the study of RE-negative (HER-2 + or -) human breast cancer [10].

The p53 tumour suppressor gene is the most frequently mutated gene in human cancer. Trials in canine mammary cancer found that 17% of carcinomas tested had mutations in p53 [29]. The p53 gene could contribute to prognostic assessment in canine mammary carcinomas, as it does in human tumours. Alterations of a second tumour suppressor gene, BRCA2, which is partly responsible for human hereditary mammary tumours, have been described in canine mammary neoplasms. These mutations appear to affect the interaction with RAD51 and thus DNA repair [30].

4. Feline mammary cancer

Mammary tumours are the third most common tumour in felines, after haematopoietic neoplasms and skin tumours [20].

The incidence of mammary tumours is less than half that seen in humans and dogs, accounting for 12% of all tumours affecting felines and 17% of neoplasms in cats [20]. Mammary tumours have also been reported in males, although less frequently (1–5% of feline mammary tumours) [31]. In contrast to humans and canines, 85–93% of feline mammary tumours are malignant [32]. Invasion of the lymphatic system and lymph nodes is common. In several studies, more than 80% of cats with mammary malignancy had metastases to one or more of the following organs at the time of death: lymph nodes, lungs, liver, adrenal glands and kidneys [33].

More than 80% of feline mammary tumours are histologically classified as adenocarcinomas, the same predominant type of human mammary cancer [7]. Sarcomas, squamous cell carcinomas and mucinous carcinomas are less common malignancies. Approximately 15% of breast masses are benign neoplasms or dysplasias, including simple/complex adenomas and fibroadenomas. In addition, there are three types of non-inflammatory hyperplasia of the feline mammary gland: ductular hyperplasia, lobular hyperplasia and fibroepithelial hyperplasia [34].

There is evidence associating the presentation of mammary tumours with breed; domestic shorthair and Siamese cats appear to have higher incidence rates [32].

The mean age of presentation for feline mammary tumours is 10–12 years, with cases occurring from 9 months to 23 years of age [32].

Dorn et al. described that female cats spayed at 6 months of age had an approximately seven times lower risk of mammary cancer than intact female cats [20]. A case–control study by Overly and colleagues showed that cats spayed before 6 months of age had a 91% reduced risk of mammary carcinoma compared to intact cats, and those spayed before 1 year of age had an 86% reduced risk [35].

A strong association has also been documented between prior use of drugs containing synthetic progestins or oestrogen-progestin combinations and the development of benign or malignant mammary tumours in female cats. In both cases, the risk was three times higher in treated than in untreated cats [36].

Both normal tissue and benign proliferative lesions express low levels of ERs and moderate levels of PRs. Comparison of steroid receptor expression in human and feline mammary cancer indicates that the levels of ERs and PRs are decreased in feline mammary carcinoma. This could signify a loss of steroid hormone dependence during malignant progression, which would occur at earlier stages in feline mammary cancer than in human [37].

Feline mammary carcinoma has been proposed as a useful model for comparison with hormone-independent human mammary carcinomas because they share similar features such as equivalent age of incidence, histopathology and patterns of metastasis [3]. To this end, molecular studies have evaluated the overexpression of epidermal growth factor receptor 2 (also called EGFR2, HER2, erbB2 or neu) in feline mammary carcinomas. When this oncogene is amplified and overexpressed in human mammary cancer, EGFR2 is associated with clinically aggressive tumours and a poor overall prognosis [3] and may also predict poor response to hormone therapy and standard chemotherapy regimens [38]. This overexpression is reported to occur in 10–40% of human mammary carcinomas [3]. Millanta and colleagues described the same overexpression in 59% of feline mammary carcinoma cases studied and found that it was associated with shorter overall survival [25]. A second study showed that the feline EGFR2 gene domain had 92% homology with its human counterpart and found EGFR2 overexpression in 39% of the mammary carcinomas tested [3].

5. Oestrogen receptor

The response of target cells to oestrogen, primarily 17β-estradiol E2, is primarily determined by their oestrogen receptor (ER) content, proteins belonging to the steroid, thyroid and vitamin D receptor superfamily. Oestradiol (E2) diffuses across the plasma membrane of target cells and signals through hormone-specific oestrogen receptors. Two types of signalling can result from such binding, a genomic or classical pathway and a non-genomic or non-genotropic pathway (represented by rapidly activating cellular phenomena, e.g. signalling cascades). In the genomic pathway, oestrogens bind to the ER, inducing a conformational change of the ER that causes its dissociation from chaperones, followed by receptor dimerisation and activation of the transcriptional domain of the receptor [39]. Therefore, these receptors are considered ligand-inducible transcription factors [40]. The normal or canonical model for ER-mediated regulation of gene expression involves direct binding of the dimerised ER to DNA sequences known as “oestrogen response elements” (EREs) [41]. In either case, ER interaction with E2 leads to transcriptional activation of associated genes by recruiting co-activators and components of the basal transcriptional machinery [42].

Initially thought to be found only at the nuclear level, several studies have revealed the existence of these receptors at the plasma membrane, in the cytoplasm and even in the mitochondrion. The plasma membrane-associated ER is involved in the non-genomic oestrogen signalling pathway, which can lead to cytoplasmic alterations and/or regulation of gene expression [43].

For many years, it was believed that there was a single ER. However, in 1995, a second ER, REβ (so named to differentiate it from REα, an ER previously cloned from rat uterus), was cloned from rat prostate complementary DNA (cDNA) [44]. These two isoforms, ER alpha (REα) and ER beta (REβ), have similarities in size, estradiol affinity constants and structure but are encoded in different genes [45].

Despite the beneficial effects of oestrogens at the bone, cardiovascular and nervous system levels [40], several studies have demonstrated their involvement in the development of the breast cancer due to the effects of their signalling pathways and on the selective expression of REα and REβ during tumour genesis and progression [6].

6. Treatments for ER-positive breast cancer

The most commonly employed breast cancer treatments consist of anti-oestrogen and/or aromatase inhibitors (AIs), which are usually effective only in patients with ER-positive breast cancer [46]. Anti-oestrogen agents were called selective oestrogen receptor modulators (SERMs) some years ago because they manifested variable biochar properties: agonist and antagonist in terms of responses triggered in different tissues [45]. Oestradiol and SERMs are now known to influence the establishment and/or progression of many mammary tumours through association with oestrogen receptors. Binding of SERMs to ER regulates genomic and/or non-genomic effects and differentially influences the development of some tumours. For more than two decades, the anti-oestrogen tamoxifen was the treatment of choice for ER-positive female breast cancer, due to its antagonistic action in the mammary gland, where it inhibits transcription of target genes [46]. While this compound exerts an antagonistic effect on oestradiol in the breast, it exerts agonist activity in other tissues such as bone and endometrial [47]. This is demonstrated by the increased risk of endometrial cancer observed with prolonged tamoxifen treatment [48].

ICI 182,780 (also known as fulvestrant) is a “pure” steroidal anti-oestrogen, which differs from tamoxifen in its mechanism of action and in the lack of agonist activity in ER-containing tissues other than the breast. At the cellular level, apart from binding to the ER and increasing its degradation, ICI 182,780 would recruit cancer cells in G0/G1 by inducing the expression of a cyclin-dependent kinase inhibitor, p21kip1 [49]. Fulvestrant is also known as the first SERD (selective oestrogen receptor degrader) [50].

Several non-steroidal aromatase inhibitors, including anastrozole and letrozole, have been available for almost two decades and are efficient alternatives for the management of anti-oestrogen-resistant breast cancer in post-menopausal women [46]. It is worth mentioning that not all ER-positive breast tumours react to these treatments, and in many cases, resistance can develop; this can be “intrinsic” (never responds to treatment) or acquired (initially remits, but then progresses again) [2]. The development of resistance appears to be related to the activation of ER-dependent signalling pathways by agents not considered to date as conventional ligands [51]. Other mechanisms associated with the resistance phenomenon include the epidermal growth factor receptor (EGFR), which is overexpressed in many tumours that are unresponsive to the aforementioned therapies. Amplification of the epidermal growth factor receptor 2 (EGFR2/HER2/erbB2/neu) gene, resulting in overexpression of EGFR2, has been found in 15–25% of human breast cancers, a frequency of genetic alteration second only to p53 mutations [52]. EGFR2 overexpression is an important predictive marker as its presentation generally represents an aggressive cancer with poor prognosis, as well as a unique target for molecular targeted therapy. Resistance is driven by intense signalling that activates the “downstream” cascade of kinases, which in turn stimulate the ER, increasing its transcriptional activity. This is known as “crosstalk” between ER and EGFR tyrosine kinases [53].

7. Mammary cancer signalling pathways

The main signalling pathways linked to mammary cancer, and shared by human and canine species, are those related to oestrogen, MAPK, PI3K/AKT, KRAS, PTEN and Wnt/β-catenin [54].

Cyclins are a family of proteins that control the progression of cells through the cell cycle by activating cyclin-dependent kinase (Cdk) enzymes. The cell cycle is promoted by the activation of these cyclin-dependent kinases, which are positively regulated by cyclins and negatively regulated by Cdk inhibitors (CKIs). This well-controlled expression is altered in tumour cells [55]. D-type cyclins (D1, D2, D3), regulatory subunits of Cdk4/6 kinases, function as critical mitogenic sensors that integrate growth factor-initiated signals with G1 phase progression. Mitogenic stimulus triggers the accumulation of active cyclin D1-Cdk4 complexes through increased cyclin expression, decreased cyclin proteolysis and promotion of cyclin D1-Cdk4 assembly. Mitogen-dependent expression of cyclin D1 requires growth factor-mediated activation of a transductional signalling cascade involving Ras, Raf-1 and extracellular signal-regulated protein kinases (ERK1 and 2) [56].

Overexpression of cyclin D1 has been linked to breast cancer progression and growth, as well as to the development of resistance to hormone therapy. Several hormones are involved in the proliferation of breast cancer cells, with cyclin D1 being an important target of the intracellular signalling pathways of these hormones [57].

Overexpression of cyclins A and E has been associated with poor prognosis [58] and overexpression of cyclin B1 with tumour grade (related to the fact that the higher the presence of the Ki-67 antigen, the more aggressive the tumour), mitosis and adverse clinical outcomes [59]. Cyclin D1 is overexpressed in more than 50% of human breast cancers [60]. Some studies show that cyclin D1 expression is positively correlated with ER status and negatively correlated with tumour grade and size, suggesting that cyclin D1 overexpression is a good prognostic marker, particularly when co-expressed with ER [61]. There are reports showing that cyclin D1 overexpression predicts resistance to tamoxifen treatment in breast cancer patients [58].

The importance of cyclin D1 nuclear localisation is critical for the cell cycle regulatory functions of cyclin D1-dependent kinase during the G1 phase and for the inhibition of proteolytic degradation of cyclin D1 [56]. Cyclin D1 degradation is regulated through the 26S proteosome, and efficient proteolysis requires phosphorylation of threonine-286. The ability of targeted phosphorylation of nuclear exported cyclin D1 leads to the hypothesis that cyclin D1 destruction occurs preferentially in the cytoplasm [62]. The ability of the CKIs, p27kip1 and p21cip1 to reduce the range of cyclin D1 passing into the cytoplasm is consistent with this observation [63].

Reinforcing this, a duality of CKIs has recently been described in that they are required as potent inhibitors of Cdk2 kinase and as a positive regulator of the cyclin D1-Cdk4 complex. Studies showed that inhibition of cyclin D1 nuclear export by CKIs is required for nuclear accumulation of the cyclin D1-Cdk4 complex during the G1 phase of the cell cycle [56].

Overexpression of EGFR2 has been linked to aggressive breast cancers with high metastasis and chemoresistance. For example, its presence has been shown to lead to increased resistance to tamoxifen. EGFR2 has also been shown to generate resistance to another compound used in breast cancer treatment, paclitaxel (Taxol®). Some findings have described that the mechanism of resistance is based on EGFR2 directly phosphorylating the cyclin-dependent kinase, Cdk2, and generating a transcriptional upregulation of p21cip1; however, the mechanism by which EGFR2 induces this transcriptional upregulation is not yet defined [64].

Among the classical function of p21cip1, a CKI that regulates progression through the G1 phase of the cell cycle, is to act as an effector of tumour suppressor proteins such as p53, BRCA1, WT1 and TGFβ [65]. Another important role, recently attributed, is that of attenuating epithelial-mesenchymal transition (EMT) cell characteristics in human mammary cancer cells. For example, p21cip1 antagonises the repression exerted by Twist on E-cadherin promoter activity [65].

Cadherins are glycoproteins with calcium-dependent transmembrane domains that mediate cell–cell adhesion. The cadherin family includes many different types. E-cadherin is the most studied. It is located on the surface of epithelial cells in regions of cell–cell contact known as adherens junctions. To perform their adhesive function, cadherins must form complexes with proteins of the cytoplasmic plate, called catenins, and with the actin cytoskeleton [66]. The development of malignant tumours, in particular the transition to invasive metastatic cancer, is characterised by the ability of tumour cells to overcome cell–cell adhesion and invade surrounding tissue [67]. This phenomenon is known as epithelial-mesenchymal transition (EMT). Studies in ovarian cancer have revealed that cells with low E-cadherin expression are more invasive [67, 68], and the absence of E-cadherin expression in ovarian cancer may predict short survival [69]. Several studies have shown that restoration of E-cadherin expression results in a reversion from an invasive phenotype to a sessile epithelial tumour cell phenotype, providing evidence that E-cadherin may act as an invasion suppressor molecule [70].

Factors that may play a role in the progression of canine mammary tumours include the expression of adherens junction and gap junction proteins such as E-cadherin, connexins and paxillin. In general, the most invasive, proliferative and aggressive tumour is, histologically, the one with the lowest expression of the protein in a localised and intense form, indicating a change that favours increased cell motility [71]. E-cadherin expression has been reported to be reduced or absent in 70% of feline mammary carcinomas compared to normal tissue [72].

The ability of β-catenin to act as part of the adhesive and transcriptional machinery is due to its modulation through phosphorylated serine/threonine and tyrosine residues: reduced serine/threonine phosphorylation destabilises the adhesion complex formed with cadherins and facilitates nuclear translocation of β-catenin and subsequent gene transcription. On the other hand, tyrosine phosphorylation results in its dissociation from E-cadherin and loss of association with the cytoskeleton, promoting reduced cell–cell adhesion and cell spreading. It is suggested that tyrosine phosphorylation would be promoted by EGFR2 [73]. Furthermore, the levels of complexes formed between β-catenin and EGFR2 are elevated in the most aggressive and metastatic tumour types in humans [74].

On the other hand, it is known that the amount of ERα is higher than that of ERβ in breast cancer cells. Oestrogens have a paradoxical role in human cancer as a promoter of carcinoma progression in some cases and inhibitor of cancer cell invasion in others. This could be explained by the antagonistic effect of ligand-bound ERα and ERβ on cancer pathogenesis. Studies in breast and gynaecological cancer have shown a positive effect of ERα on tumour cell proliferation and growth, while ERβ would promote apoptosis and inhibition of tumour growth [75].

One study showed that ERβ expression correlated positively with β-catenin expression at the cell membrane and negatively with β-catenin translocation to the nucleus [76]. This would imply a crosstalk between ER signalling and the Wnt/β-catenin pathway [77]. In addition, ERβ was positively correlated with the maintenance of E-cadherin in the plasma membrane of mammary tumour cells [76]. Another study using a prostate cancer cell line endogenously expressing REα and REβ showed that induction of EMT with TGFβ or exposure to hypoxia concomitantly led to a reduction in the number of REβ, suggesting that loss of REβ promotes EMT in prostate cancer cells [78].

8. Conclusion

The increasing incidence of mammary cancer in humans and dogs has forced the search for better diagnostic, treatment and/or prognostic strategies. Alternatively, several authors compare and highlight the similarities that allow postulating the canine and feline species as models for the study of such cancer and thus replace the murine model. Thus, important advances have been made in this area with promising results. However, given the complex nature of malignant mammary tumours, much remains to be studied.

Conflict of interest

The author declares no conflict of interest.

Nomenclature

ER	oestrogen receptor
ERα	oestrogen receptor alpha
ERβ	oestrogen receptor beta
SERMs	selective oestrogen receptor modulators
AIs	aromatase inhibitors
PR	progesterone receptor
EGFR	epidermal growth factor receptor
CK	cytokeratin
EGFR2/HER2/erbB2/neu	epidermal growth factor receptor 2
ICI 182,780	fulvestrant
SERD	selective oestrogen receptor degrader
Cdk	cyclin-dependent kinase
CKIs	Cdk inhibitors
EMT	Epithelial-mesenchymal transition