Features of the Expression of Some Interferon- and Tumour-Suppressor Genes in Cats with Benign and Malignant Neoplasms of the Mammary gland

Laboratory methods for assessing the expression of oncogenes and tumour suppressors are currently increasingly in demand in the practices of veterinarians working with oncological patients. Despite this diagnostic approach long being popular in clinical practice, it does not always provide unambiguous information. For example, the absence has been described of mRNA of the c-kit gene in mesothelioma tissue samples, despite the fact that these tissues are characterised by a strong specific immune histochemical staining for this receptor (Horvai et al., 2003). However, in most other cases the use of such diagnostic methods is characterised by high resolution. For example, a significant decrease has been described in the mRNA content of the p53 and bcl-2 genes in the cells of breast cancer (BC) metastases in the brain compared to in the cells of the primary tumour. Moreover, these results correlate with the data of immunohistochemical staining (Stark et al., 2006).

Practitioners in the local veterinary clinic use such molecular markers as CK-Pan (pan Cytokeratin), p40 and S100 for the differential diagnosis of oncological diseases of different histogenesis. Diagnostic methods for assessing the expression of the TP63 protein are also used. This is a transcription factor from the p53 gene family, which also includes the p73 gene. The products of these genes are now regarded as essential for the control of cell differentiation, proliferation and survival in various tissues. Therefore, the study of the expression of this gene family is recommended to differentiate tumours by their histogenesis (Gatti et al., 2019; Steurer et al., 2021).

Testing the expression level of such genes may be considered as one of the methods for assessing the degree of a neoplasm malignancy (Graziano & De Laurenzi, 2011; Steurer et al., 2021) as well as a criterion for choosing the complex therapy methods of an oncological disease and evaluating the effectiveness of such therapies (Borrero & El-Deiry, 2021).

The prognostic value of assessing the expression of cell cycle control genes (p53 and gadd45g), as well as some genes of the interferon (IFN) system (ifnα7, ifnβ1, ifnγ and ifnλ1) in haemoblastosis of cats to determine the risk of the disease progression to a more aggressive stage has been confirmed in our previous studies (Tsybulsky et al., 2022).

An evaluation of the feasibility of such a diagnostic approach in relation to animals with tumour neoplasms of MG of different histogenesis and different degrees of malignancy was made in the present work. In addition to the above list of molecular biological tests, we examined the expression of important tumour suppressors such as rb1 and cdkn2a (cyclin-dependent kinase inhibitor 2A) in animals with MG tumours. The rb1 gene encodes the retinoblastoma protein (pRb), which is one of the most important antitumour suppressors. In the genome of cats, this gene has a size of 181,640 bp and consists of 30 exons. Eight transcripts have been described for this gene (Gene ID: 101101356, updated 4 Jan 2022). The presence of several alternative splicing products creates significant difficulties in assessing the correlation of this gene expression level with the pathogenesis of cancer. Nevertheless, given the exceptional importance of the products of rb1in the control of normal and pathologically altered cell cycles, the development of a methodology for assessing this parameter in veterinary practice is considered necessary. It is important to note that the disruption of the normal expression of the pRb protein has a high level of penetrance, with many types of oncological pathologies in humans and animals due to disorganisation of the cell cycle checkpoint at the level of CDK4/6 kinase activity and E2F-DP transcription factors (Chakraborty et al., 2021). Changes in the expression of the pRb protein also affect the immunological characteristics of a tumour and its microenvironment; therefore, the assessment of the expression of this tumour suppressor may be useful in predicting the dynamics of the disease and evaluating the effectiveness of a therapy (Knudsen et al., 2019).

Estimation of the cdkn2a expression is a non-trivial task. Modulation of this gene expression by alternative splicing leads to the appearance of two transcripts, the products of which differ greatly in structure; however, all of them exhibit a wide range of regulatory functions in relation to the mechanisms of antitumour surveillance. In particular, the p16INK4A (p16) transcript product is a tumour suppressor that modulates the cell cycle at the level of the pRb-regulated G1-to-S phase transition, inhibiting the kinase activity of CDK4 and CDK6 (Palmieri et al., 2009; Witkiewicz et al., 2011). Of no less interest is the p14ARF transcript variant, the product of which is an important factor for the stabilisation and protection of the TP53 protein from ubiquitin-dependent proteolysis. The product of p14ARF is a physiological inhibitor of MDM2 (an E3 ubiquitin ligase that controls P53 activity and stability). Loss of P14ARF activity may have the same effect as the loss of P53 function (Agrawal et al., 2006). However, the P14ARF transcript has not been described in cats. It is also impossible to assume its presence by analogy with human P14ARF due to the fact that during the formation of the p14ARF transcript in humans a region from outside the gene is involved. Therefore, in this study we evaluated only the expression of the p16INK4A transcript.

Domestic non-pedigree cats with a neoplasm of the MG detected for the first time during examination in the veterinary clinic. The age of cats was 7–15 years. All animals underwent the surgical treatment of unilateral mastectomy with simultaneous hysterovariectomy. According to the results of histological examination, two groups of animals were formed: (1) cats diagnosed with BC stage II (n = 4) and (2) cats with FCM (n = 3).

The healthy cats (n = 4) operated on for planned sterilisation were used as control animals. The age of the control cats was 4–7 years.

All manipulations with animals were carried out in accordance with the regulations approved for veterinary clinics in Russia.

Blood samples and operational material of the tissue of the MG and uterus were taken. The peripheral blood was taken in a volume of 2 mL from the lateral vein of the forelimb.

Haematological analysis was performed using a Cell Dyn 3700 haematological analyser (Abbott, USA).

Biochemical analysis of blood was performed using a URIT-800 VET biochemical analyser (URIT Medical Electronic Co., Ltd., China).

Histological examination of a macropreparation of operational material of MG with a tumour was performed in accordance with the diagnostic algorithms recommended for the histology of breast and soft-tissue tumours (Goldschmidt et al., 2017; Hendrick, 2017). Histological preparations were stained with haematoxylin-eosin according to the methods of Pappenheim and Papanicolaou.

Analysis of target gene expression: Extraction of the total RNA from the cats’ white blood cells (WBCs) was performed with the ExtractRNA reagent (Eurogen, Russia) according to the manufacturer’s recommendations. The concentration and quality of the isolated RNA were analysed with a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA). Reverse transcription was performed using the MMLV RT reagent kit (Eurogen) with the Oligo(dT) primer. All procedures were performed according to the manufacturer’s recommendations.

The expression of the following feline genes was analysed: (1) IFN genes: ifnα7, ifnβ1, ifnγ and ifnλ1; (2) tumour-suppressor genes: rb1, p53, cdkn2a and gadd45g; and (3) gapdh (glyceraldehyde 3-phosphate dehydrogenase) as housekeeping gene.

Primer-BLAST NCBI and SnapGene 4.1.8 were used to design the primers. The following pairs of forward (F) and reverse (R) gene-specific primers were used (Table 1).

Gene expression analysis was carried out using the qPCR mix HS SYBR (Eurogen) by qPCR on a CFX96 thermocycler (Bio-Rad, USA). The gapdh gene was used as a normaliser. Samples of cDNA derived from the mRNA of healthy cats were used as control. Relative differences in the expression parameters of each gene in the group of animals with stage II breast BC and FCM in comparison with the control group were obtained by calculating the multiplicity of expression variation (fold) according to a previously described method (Livak and Schmittgen, 2001). The expression index of the target gene in the control group was taken as a conditional 1 unit.

Statistical analysis of the data was carried out using the SPSS 11.0 statistical analysis software with the determination of the nonparametric Mann–Whitney U-test. Differences were considered significant if the significance level (a) was <0.05.

The general condition of the cats examined was characterised as relatively satisfactory. According to the results of the preoperative blood test, there were no significant changes in the cellular composition or biochemical parameters of the blood. All studied parameters were within the ranges typical for healthy cats, as adopted in the laboratory of the veterinary clinic. However, some blood parameters of animals with MG neoplasms differed significantly from those of healthy control animals (Table 2).

Especially significant were the changes in the blood formula, characterising the absolute and relative content of lymphocytes. In cats with breast neoplasms, the relative content of lymphocytes was significantly (a < 0.05) lower than in control animals. At the same time, in cats with BC such a significant decrease in the absolute content of lymphocytes suggests the significance of lymphopenia in the pathogenesis of cancer. Elevated levels of granulocytes and erythrocyte sedimentation rate (ESR) against the background of lymphopenia indicate an inflammatory process in the body of tumour-bearing animals. This process is predominantly non-specific. In animals with BC, a decrease in blood platelets was also determined, which apparently reflects the general toxic nature of the pathological process.

Table 1: Primer sequences specific for feline target genes

⃰ - The rb1 gene in F. catus consists of 30 exons. The eight transcripts are known for this gene. Primers for assessing the level of expression of this gene were developed taking into account differences in

Table 2: Results of haematological and blood biochemistry analyses of cats with MG tumours and healthy cats

In the present study, cats with FCM-type benign breast neoplasms, as well as those with stage II BC were examined. All cats had a preliminary diagnosis of BC in the operable stage of the oncological process. In all cases, the same type of surgical treatment, in the form of a unilateral mastectomy with simultaneous hysterovariectomy, was performed. The final diagnosis was made according to histological examination of the surgical material. In accordance with this, the operated animals were divided into two groups: (1) stage II BC, and (2) FCM.

Some significant differences in laboratory parameters between animals with BC and with FCM were identified.

In general, changes in hematological parameters were quite typical, reflecting the presence of a chronic inflammatory process, which usually develops in patients with tumors (Lim et al., 2018). In cats with BC and FCM, a significant increase in ESR was determined. This indicator was especially high in cats with BC, which is apparently associated with the induction of inflammatory reactions due to the formation of a large number of necrosis zones in the tumour tissue (which was shown during histological examination of the tumour tissue). The development of paraneoplastic syndrome in animals with BC is apparently associated with inhibition of thrombocytopoiesis, which is manifested by statistically significant (a < 0.05) thrombocytopenia, as well as increased levels of creatinine and urea in the blood. A general trend was established: all of these haematological and biochemical parameters were higher in cats with BC compared to those with FCM.

Particularly noticeable changes in the blood formula in tumour-bearing animals were found in relation to the lymphoid component of the blood. A statistically significant (a < 0.05) decrease in the percentage of lymphocytes was found both in animals with BC and with FCM. However, in cats with BC, the tendency to develop lymphopenia was much more pronounced: the absolute content of lymphocytes in the blood of these animals was more than 2-fold lower than in healthy control animals. In general, this is a fairly well-known haematological phenomenon, typical of oncological processes of different histogenesis (Menetrier-Caux et al., 2019). There was a decrease in the number and competence of the immune system lymphoid elements against the background of a predominance of granulocyte blood elements, which are participants in non-specific inflammatory processes occurring in the body of tumour-bearing patients (Ocana et al., 2017). Despite the differential diagnostic value of such criteria being relative, in combination with more targeted tests designed to assess immunological and other parameters characterising the mechanisms of cellular and molecular antitumour surveillance, the assessment of the above haematological criteria is quite applicable.

The expression pattern studied in this work is represented by genes, the expression of which turned out to be diagnostically significant in our previous work (Tsybulsky et al., 2022). To this gene spectrum in this study, we added an analysis of the rb1 and cdkn2a gene expression. The products of these genes are important elements of the molecular cell cycle control systems and antitumour resistance. A deficiency in the function of these factors leads to increased resistance of tumour cells to apoptosis-inducing mechanisms (Chakraborty et al., 2021).

The expression of the IFN system and tumour-suppressor genes chosen in this study differed significantly between cats with benign and malignant breast diseases. To the greatest extent, these differences were pronounced in the breast tumour tissue itself and in WBCs.

A significant (5–15-fold) increase was found in the expression of ifnα7, ifnβ1, ifnγ, cdkn2a, rb1 and p53 genes in BC tumour tissue. Expression of these genes in the MG metaplastic tissue from cats with FCM was significantly lower and, in general, did not differ significantly from that of control animals. This indicates the relative inertness of these censor systems in the tumour tissue of animals with FCM.

Along with large differences in most parameters of the expression pattern, we were able to identify a common molecular criterion that is characteristic for both groups of animals with BC pathology: inhibition of the ifnλ1 and gadd45g gene expression in tumour tissue (Figure 1).

The products of the gadd45 gene are one of the most significant components of the tumour suppression system, of which the GADD45g variant attracts special attention (Tamura et al., 2012). A decrease in the expression level of this protein is associated with a wide variety of oncological diseases of different histogenesis. With regard to BC, there is evidence that the GADD45g protein inhibits the growth of human BC cells in vivo and in vitro, acting as an antitumour agent (Zhang et al., 2021). Moreover, the antitumour effect of the GADD45g protein can be realised independently of other important molecular factors that counteract carcinogenesis (P53, pRB, etc.; Zhang et al., 2014). The ifnλ1 gene product is a type III IFN system specialised for epithelial tissues, whose role, along with protection against viral pathogens, is extremely important for maintaining the genetic stability of endothelial cells and epithelial tissues (Lasfar et al., 2016; Walker et al., 2021).

Thus, insufficient expression of the ifnλ1 and gadd45g genes may indicate a reduced activity of molecular censor systems that counteract the processes of mutagenesis and carcinogenesis. We have previously found the same changes in the expression pattern in cats with lymphoblastoses. Moreover, a decrease in the expression activity of these genes is correlated with the risk of the disease transition to a more aggressive phase (Tsybulsky et al., 2021). According to our assumptions, such changes in the expression pattern may serve as a prognostic criterion for forms of oncological pathology in cats, such as BC. It can be supposed that it is advisable to correct these indicators by methods of targeted induction of the corresponding genes. Substitution therapy with appropriate recombinant products as part of the complex therapy for oncological diseases also seems to be a promising method for disease treatment. Testing these working hypotheses requires more extensive research and the development of appropriate therapeutic technologies. These questions relate to the area of our scientific interests and the genetic engineering technologies being developed.

It must be noted that the development of such methods of targeted therapy should be more focused on the activation of the expression of these genes in tumour cells themselves or in immunocompetent cells infiltrating the tumour. This is due to the fact that the expression of the gadd45g gene is at a very high level in peripheral blood leukocytes against the background of its expression inhibition in the tumour tissue itself. With regard to the ifnλ1 gene, the need to induce the expression of this gene in situ is even more clear, since peripheral WBCs are not specialised producers of this cytokine.

(Wack et al., 2015). It is difficult to explain that a noticeable expression inhibition of the entire range of genes studied (with the exception of ifnλ1) was found in the uterine tissue of cats with BC. Moreover, in two cats with BC the expression rates of the entire group of tumour-suppressor genes were less than 50% of the corresponding indicators in the control animals. In the uterine tissue of these animals, the expression of ifnα7 and ifnγ genes was practically absent. Speculatively, these data can be interpreted as evidence of the suppression of antitumour surveillance mechanisms in the reproductive system of animals with BC, as well as evidence of the absence of tissue infiltration by immunocompetent cells and, accordingly, the absence of inflammation. To a certain extent, this can also be associated with the formation of a state of lymphopenia, which is most pronounced in cats with BC.

However, all these assumptions require appropriate experimental verification. It should be noted that the features of the expression pattern presented in this report were established precisely in animals with stage II BC, i.e., at the operable stage of the disease. A separate study requires the assessment of changes in the expression of the studied spectrum of genes in animals with BC at other clinical stages.

The differences of the IFN and a number of key tumor supressor genes expression patterns are expressed in the tumour tissue itself, in WBCs and in the tissue of the uterus in cats with BC and FCM. We consider the suppression of gadd45g and ifnλ1 gene expression in BC tumour tissue to be the most significant. The high level of p53, rb1 and cdkn2a gene expression in animals with BC can serve as a molecular criterion for the intensity of the cell cycle control mechanisms and antitumour surveillance in general. However, the obviously insufficient level of expression of the gadd45g gene makes it possible to interpret such an expression pattern as a sign of an imbalance in the molecular censor systems for protecting the health of the cellular genome.

Estimation of the expression levels of p53, rb1, cdkn2a, gadd45g and ifnλ1 genes may be useful in the differential diagnosis of benign and malignant breast diseases in cats.

Therapy based on the targeted modulation of gadd45g and ifnλ1 gene expression may be a useful addition to the complex therapy of cats with breast cancer.

This research was approved by ethical standards governing the work of veterinary clinics in Vladivostok and comprised a study of samples collected with full informed consent from owners who agreed to the use of such samples for research purposes.

This research was supported by the RFBR grant (Agreement No. 189–09060/18).

No competing financial interests exist.

Each author made substantial contributions to the conception and design of the work and the acquisition and analysis of blood and DNA/RNA samples. The first author performed the final analysis of all results and prepared the final version of this original article. All authors read and approved the final manuscript.