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. 2024 May 21;86(7):816–823. doi: 10.1292/jvms.23-0470

Reduction of phosphorylated signal transducer and activator of transcription-5 expression in feline mammary carcinoma

Keishi OWAKI 1, Mami MURAKAMI 2,*, Kana KATO 3, Akihiro HIRATA 1,3,4, Hiroki SAKAI 1,3,4
PMCID: PMC11251807  PMID: 38777776

Abstract

Signal transducers and activators of transcription (STATs) are a family of transcription factors involved in various normal physiological cellular processes. Moreover, STATs have been recently identified as novel therapeutic targets for various human tumors. STAT3, STAT5a, and STAT6 have been suggested to be involved in tumorigenesis in human breast cancer. Owing to the similarity between feline mammary carcinomas (FMCs) and human breast cancers, these factors may play an important role in FMCs. However, studies on the expression of STATs in animal tumors are limited. Therefore, in this study, we aimed to characterize the expression of total STAT5 (tSTAT5) and phosphorylated STAT5 (pSTAT5) in FMCs, feline mammary adenomas, non-neoplastic proliferative mammary gland lesions, and normal feline mammary glands using immunohistochemistry. High expression of tSTAT5 was observed in the cytoplasm of all the samples assessed in this study. Moreover, high expression of tSTAT5 was observed in the nucleus; however, its levels varied depending on the lesion. The percentage of pSTAT5-nuclear positive cells varied among normal feline mammary glands (40.1 ± 25.1%), and non-neoplastic lesions, including mammary hyperplasia (43.2 ± 28.6%) and fibroadenomatous changes (18.0 ± 13.6%). Moreover, the percentage of pSTAT5-nuclear-positive cells in feline mammary adenomas was 24.5 ± 19.2%, which was significantly reduced in feline mammary carcinomas (2.4 ± 5.6%), regardless of histopathological subtype. This study suggests that decreased STAT5 activity may be involved in the development and malignant progression of feline mammary carcinomas.

Keywords: feline mammary carcinoma, immunohistochemistry, signal transducers and activator of transcription-5

Mammary tumors are the third most common tumors in female cats, accounting for 17% of tumors and 25% of neoplasms in these animals [28, 52]. The reported incidence of malignancy in feline mammary tumors is high (80–96%) [58]. Feline mammary carcinomas (FMCs) are malignant and highly invasive to surrounding tissues with extensive lymph node or lung metastases, consequently complicating complete surgical resection of these tumors, resulting in poor prognosis and a median time from tumor detection to death of 12.3 months [28, 42, 46, 58]. While a combination of surgery and chemotherapy with doxorubicin and other chemotherapeutic agents is currently recommended for the treatment of FMCs, no previous studies have reported the advantages of such combinatorial therapies compared with surgical resection alone; therefore, it is crucial to develop more effective treatment strategies [46].

Human breast cancer is the most commonly diagnosed cancer in women, and various therapeutic strategies have been developed to treat breast cancer based on hormon receptors [17]. Among the various histological human breast cancer types, triple-negative breast cancer, which lacks the estrogen and progesterone receptors and human epidermal growth factor receptor 2 targeted for therapy, is associated with poor prognosis as compared with other breast cancer subtypes. Various treatment strategies have been proposed for triple-negative breast cancer; however, effective treatments are limited [3, 47]. FMCs are similar to human breast cancer with respect to the late age of onset, incidence, histopathological features, biological behavior, and metastasis pattern [6, 11, 58]. Specifically, FMCs possess histopathological features and clinical dynamics comparable to those of human triple-negative breast cancer; thus, FMCs are considered a model of human triple-negative breast cancer [6, 45].

In recent years, the signal transducer and activator of transcription (STAT) family of transcription factors has emerged as a novel therapeutic target in various human tumors [4, 51]. Seven STAT genes have been identified in the human genome: STAT1–4, STAT5(a, b), and STAT6. These factors transmit signals from the cell membrane to the nucleus and activate gene transcription. These signaling pathways are involved in many normal physiological cellular processes, including proliferation, differentiation, apoptosis, angiogenesis, and immune system regulation [4, 51]. The signaling pathways controlled by the STAT family of transcription factors play an important role in many physiological cellular processes downstream of the Janus Kinase (JAK)/STAT pathway. The binding of cytokines such as IL-6 to cell surface receptors activates JAK, which is associated with the intracellular domain of the receptor protein, resulting in tyrosine phosphorylation of the receptor. The phosphorylated tyrosine residue of the receptor serves as a binding site for the Src homology 2 (SH2) domain of STAT, activating STAT dimerization via the SH2 domain and the phosphorylated tyrosine residue on the carboxyl side. Once activated, the dimeric STAT complex translocates to the nucleus, binds to its target promoter regions, and triggers transcriptional activation, leading to the expression of various proteins associated with different biological processes, including the regulation of cell proliferation, differentiation, apoptosis, and the regulation of the immune system [4, 51]. The following phosphorylation of the tyrosine residue and translocation of STATs into the nucleus, STATs are dephosphorylated by tyrosine phosphatases, and exported from the nucleus. Factors negatively regulating the JAK/STAT pathway include protein inhibitors of activated STAT (PIAS) and suppressors of cytokine signaling (SOCS) proteins [15, 19].

These cellular pathways are essential for maintaining normal functions, but their dysregulation can lead to various pathological conditions, including the transformation and metastasis of malignant cells [4, 51, 53]. Among the STAT family members, STAT3 and STAT5a have been extensively studied for their roles in physiological mammary gland development and function [2, 7, 8, 41, 57]. STAT3 and STAT5a have been implicated in breast cancer through the JAK/STAT pathway [2, 7, 26, 54, 55]. STAT3 regulates genes important for cancer progression, such as those involved in tumor development, cell proliferation, angiogenesis, metastasis, and apoptosis resistance [18, 26, 32, 55]. Conversely, dysregulation of STAT5 activation leads to excessive alveolar development, damaged breast remodeling, and breast tumorigenesis through the upregulation of Akt/PI3-kinase signaling [43]. During normal mammary development, the STAT5a regulates genes essential for mammary tissue differentiation, alveolar progenitor cell development, and pregnancy-related lactation [2, 41, 49, 51].

Although abnormalities in STAT activity have been extensively investigated in various human malignancies, veterinary research has mainly focused on investigating STAT3 expression in canine and feline mammary tumors, canine anal sac carcinoma, canine diffuse large B-cell lymphoma, feline oral squamous cell carcinoma, and feline injection-site fibrosarcoma [1, 5, 29, 35,36,37,38,39]. Given the similar features shared between feline mammary tumors and human breast cancers, it is essential to investigate the involvement of STAT3 and STAT5a in feline mammary tumors [6, 11, 58]. Limited studies have elucidated STAT3 activity based on tyrosine or serine phosphorylation in feline mammary tumors compared with their human counterparts [36, 37]. However, compared with studies on human breast cancer, studies characterizing the phosphorylation status of STAT3 in feline breast cancer are notably lacking. Furthermore, to the best of our knowledge, there have been few studies of STAT5a expression and phosphorylation status in feline mammary tumors. Therefore, in this study, we aimed to characterize STAT5a expression and phosphorylation in tumor and non-tumor lesions in the feline mammary gland using immunohistochemistry.

MATERIALS AND METHODS

Tissue samples and case selection

Overall, 102 feline mammary gland masses from 76 cats submitted for histopathological examination to the Laboratory of Veterinary Pathology at Gifu University between 2009 and 2022 were included in this study. All samples were fixed in 10% neutral buffered formalin and embedded in paraffin, sectioned into 4-μm, then dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E). All samples were histologically diagnosed by board-certified veterinary pathologists, according to the diagnostic criteria of the Surgical Pathology of Tumors of Domestic Animals, Vol. 2 [59].

The neoplastic tissue samples included 48 mammary carcinomas, 9 mammary adenomas, and non-neoplastic proliferative tissue samples with 7 fibroadenomatous changes and 23 mammary hyperplasias, 12 of which were associated with a tumor. For the normal mammary gland, 15 samples that included normal areas of the mammary gland were isolated simultaneously with the lesions. Further characteristics of the samples, including histopathological subclassifications are presented in Supplementary Table 1.

Validation of anti-human total STAT5a (tSTAT5) and phosphorylated STAT5 (pSTAT5) antibodies

The reactivity of the anti-tSTAT5 and anti-pSTAT5 antibodies used in the present study to feline tSTAT5 or pSTAT5 was validated using western blotting. Protein samples were extracted from one normal feline mammary gland and one feline mammary carcinoma using a radioimmunoprecipitation assay (Thermo Fisher Scientific, Waltham, MA, USA) lysis buffer with a proteinase and phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA). The protein samples (10 µg) were run on SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Cytiva, Tokyo, Japan) for protein detection [10]. After blocking with 3% bovine serum albumin, the membranes were incubated at room temperature (RT) for 60 min with the following primary antibodies: rabbit monoclonal antibody against tSTAT5 (1:1,000, #ab32043, Abcam, Cambridge, UK) and rabbit monoclonal antibody against pSTAT5 (Y694) (1:500, #9359, Cell Signaling Technology). Rabbit polyclonal antibody to human β-actin (1:1,000, #4967, Cell Signaling Technology) was used as a loading control. Furthermore, proteins from HeLa cells treated with sodium orthovanadate (Fujifilm-Wako Pure Chemical, Osaka, Japan), an inhibitor of cellular phosphorylation by phosphatase were used as positive controls for tSTAT5 and pSTAT5 [13, 21]. In brief, HeLa cells were maintained in high-glucose Dulbecco’s Modified Eagle Medium (Fujifilm-Wako Pure Chemical) supplemented with 10% heated-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin (Fujifilm-Wako Pure Chemical; 10% FBS/ DMEM) at 37°C under a humidified atmosphere with 5% CO2. To suppress pSTAT5 dephosphorylation, the medium was replaced with fresh 10% FBS/DMEM containing 10 mM orthovanadate when the cells reached 70% confluency. After 12 hr, the cells were harvested for subsequent western blot analysis. The cells underwent the same processing steps as the tissue samples. Proteins were visualized using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:1,000, #7074, Cell Signaling Technology), and chemiluminescence was enhanced using Immobilon Forte Western HRP Substrate (Merck Millipore, Burlington, MA, USA). Finally, a C-Digit Blot Scanner (LI-COR, Lincoln, NE, USA) was used for signal detection. For validation, immunohistochemical staining with the same tSTAT5 and pSTAT5 antibodies was performed on the same samples as those used in western blotting.

Immunohistochemistry

Four-μm-thick consecutive sections from all feline mammary samples were immunostained for tSTAT5 and pSTAT5. All sections were dewaxed and rehydrated, and antigens were retrieved in 10% ImmunoActive solution (pH 6.0) (Matsunami Glass Ind., Osaka, Japan) for 20 min at 121°C in an autoclave. Tissue sections were then blocked to neutralize endogenous peroxidase activity by soaking them in 0.3% H2O2 in methanol for 20 min at RT. Sections were then incubated with 10% normal goat serum (Nichirei Biosciences, Tokyo, Japan) for 30 min at RT to block nonspecific antibody binding. After blocking, the sections were incubated overnight at 4°C with the following primary antibodies: rabbit monoclonal antibody against tSTAT5 (1:4,000) and rabbit monoclonal antibody against pSTAT5 (Y694) (1:100). After washing with phosphate-buffered saline, the sections were incubated with Histofine® Simple Stain MAXPO(R) (Nichirei Biosciences) as secondary antibodies for 30 min at RT. Immunolabeled antigens were visualized using 3,3′-diaminobenzidine with the Dako EnVison + system (Dako, Glostrup, Denmark), and counterstained with hematoxylin.

pSTAT5 staining was quantified as the percentage of positive nuclei of neoplastic cells determined by counting at least 1,000 nuclei in 10 fields, each at a high magnification of 400×. The areas with the highest immunolabeling were selected, whereas those with necrosis were excluded. The positivity rate was determined by dividing the number of positive nuclei by the total number of nuclei scored, and the result was expressed as a percentage. The numbers of pSTAT5-positive tumor cells were determined concurrently with the confirmation of tumor cells in H&E-stained specimens prepared from serial sections.

Statistical analysis

Comparison of the mean percentages of pSTAT5-positive cells among the five groups (normal mammary gland, mammary hyperplasia, fibroadenomatous change, adenoma, and carcinoma) was performed using the Steel–Dwass test. Comparisons of the mean percentage of pSTAT5-positive cells between feline mammary carcinoma subtypes were also performed using the Steel–Dwass test. Analyses were performed using JMP Pro 16.0.0. software (JMP, Tokyo, Japan), and statistical significance was set at P<0.05. The results for pSTAT5 are expressed as mean ± standard deviation (SD).

RESULTS

Validation of anti-human tSTAT5 and pSTAT5 antibodies against feline samples

Similar to the results in HeLa cells, distinct single bands were detected in the normal feline mammary gland and feline mammary carcinoma lysates in the blots stained with anti-tSTAT5 antibody. For pSTAT5, although a distinct single band was detected in the normal feline mammary gland lysate, no band was detected in the feline mammary carcinoma lysate [20, 56]. β-actin as a loading control was detected as single bands in all lysates from normal feline mammary gland, feline mammary carcinoma, and HeLa cells. Specific bands were visible at approximately 91, 90, and 45 kDa, corresponding to the molecular weights of tSTAT5, pSTAT5, and β-actin, respectively (Supplementary Fig. 1A and 1B).

Immunohistochemical staining revealed positive staining for tSTAT5 in mammary gland luminal cells in normal feline mammary tissue and tumor cells in feline mammary carcinoma (Supplementary Fig. 1C). In addition, pSTAT5 showed a positive signal in mammary luminal cells of normal feline mammary gland tissue and a negative signal in tumor cells in feline mammary carcinoma (Supplementary Fig. 1C), employing the same specimens as those used in western blotting.

Expression of tSTAT5 in non-neoplastic and neoplastic mammary glands

In the normal and hyperplastic mammary glands, strong tSTAT5 expression was detected in the cytoplasm and nucleus of most luminal epithelial cells of the mammary ducts, including the epithelium of the mammary ductal lobules. Stromal cells, including fibrocytes, fibroblasts, and endothelial cells of the blood or lymphatic vessels, showed a positive expression of tSTAT5 in both the cytoplasm and nucleus (Fig. 1A and 1B). In the sections with fibroadenomatous changes, proliferating luminal epithelial cells in the ducts and tubes had strong tSTAT5 expression in the cytoplasm and nucleus. In addition, some of the myoepithelial cells showed cytoplasmic positivity for tSTAT5. Fibroblasts in the stroma of the sections with fibroadenomatous changes also demonstrated moderate cytoplasmic and nucleus positivity for tSTAT5 (Fig. 1C).

Fig. 1.

Fig. 1.

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Immunohistochemical features of the total-signal transducer and activator of transcription 5a (tSTAT5) in the non-neoplastic and neoplastic mammary glands. The tSTAT5 is strongly positive in the cytoplasm and nucleus of the secretory epithelial cells and ductal cells of the mammary ducts in normal (A), hyperplastic mammary gland (B), and lesion with fibroadenomatous changes (C). Moderate tSTAT5 expression is observed in the cytoplasm and strong tSTAT5 expression is observed in the nucleus in feline mammary adenoma samples (D). The expression of tSTAT5 is weak to moderate in the cytoplasm and moderate in the nucleus in feline mammary carcinomas (E and F). A myoepithelial cell with tSTAT5 expression is shown in the inset of (C) (arrowhead). The inset in each figure is the enlarged region of the non-neoplastic and neoplastic mammary lesions. (D) Simple adenoma. (E) Tubular carcinoma. (F) Comedocarcinoma. Scale=50 μm (A, C, and D). Scale=200 μm (B, E, and F).

In tumor tissues, adenomas and carcinomas showed moderate to strong tSTAT5 expression in both the cytoplasm and nucleus of tumor cells regardless of their histological type (Fig. 1D–F). The expression in the nucleus was detected in feline mammary adenomas and carcinomas, with a reduced number of nuclear-positive luminal epithelial cells in carcinomas compared with adenomas. Fibroblasts and infiltrating lymphocytes showed tSTAT5 positivity in the cytoplasm and nucleus.

Expression and percentage of pSTAT5 in non-neoplastic and neoplastic mammary glands

Weak to strong pSTAT5 expression was observed in the nuclei of luminal epithelial cells within most of the ducts, including the epithelium of the ductal lobules of both normal and hyperplastic mammary glands (Fig. 2A and 2B). Moreover, weak to strong pSTAT5 expression was observed in the nuclei of proliferating luminal epithelial cells in the ducts and tubes with fibroadenomatous changes (Fig. 2C). Furthermore, tumor cells in mammary adenomas showed moderate to strong nuclear positivity for pSTAT5 (Fig. 2D). In myoepithelial cells exhibiting fibroadenomatous changes, nuclear positivity was observed in very few cells (Fig. 2C). The percentage of pSTAT5-nuclear-positive cells in luminal epithelial cells varied among normal, hyperplastic mammary gland, and those with fibroadenomatous change, with values of 40.1 ± 25.1%, 43.2 ± 28.6%, and 18.0 ± 13.6%, respectively. Neoplastic luminal epithelial cells in mammary adenomas exhibited strong pSTAT5 expression, showing the percentage of pSTAT5-nuclear positive cells at 24.5 ± 19.2%. Conversely, only a few feline mammary carcinoma tumor cells showed pSTAT5 positivity in the nucleus (2.4 ± 5.6%) (Fig. 2E and 2F). Notably, luminal epithelial cells in the non-tumor mammary tissues adjacent to the tumor displayed nuclear positivity for pSTAT5 (data not shown). The percentage of pSTAT5-nuclear positive cells among tumor cells in feline mammary carcinomas was significantly lower than that in normal, hyperplastic mammary glands, tissues with fibroadenomatous changes, and mammary adenomas (P<0.005), as summarized in box-and-whisker plots (Fig. 3). When considering different histopathological subtypes within carcinomas, the average percentage of pSTAT5-nuclear-positive cells for tubular, tubulopapillary, solid, invasive micropapillary, and comedocarcinomas were 3.25 ± 7.88%, 2.65 ± 4.90%, 1.55 ± 1.05%, 1.98 ± 2.87%, and 0.02 ± 0.04%, respectively. No significant difference was observed among the various histopathological subtypes.

Fig. 2.

Fig. 2.

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Immunohistochemical features of phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in the non-neoplastic and neoplastic mammary glands. Weak to strong pSTAT5 expression is detected in the nuclei of secretory epithelial cells and ductal cells of normal (A), hyperplastic mammary gland (B), fibroadenomatous changes (C), and feline mammary adenoma (D). In contrast, very few tumor cells express pSTAT5 in feline mammary gland carcinoma (E and F). A myoepithelial cell with pSTAT5 expression is shown in the inset of (C) (arrowhead). The inset in each figure corresponds to the enlarged region of the non-neoplastic and neoplastic mammary lesions. (D) Simple adenoma. (E) Tubular carcinoma. (F) Comedocarcinoma. Scale=50 μm (A, C, and D). Scale=200 μm (B, E, and F).

Fig. 3.

Fig. 3.

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The box-and-whisker plot of the percentage of positive cells for phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in the non-neoplastic and neoplastic mammary glands. The comparison of the percent of the pSTAT5 nuclear positivity was statistically analyzed using the Steel–Dwass test, which revealed that the nuclear positivity rate of pSTAT5 was significantly lower in feline mammary carcinomas than in each group including normal mammary tissue, hyperplasia, fibroadenomatous change, and feline mammary adenomas. The bar crossing the box-and-whisker plot signifies the sample median, while the cross mark indicates the average (*P<0.05, P-values are indicated).

DISCUSSION

In this study, we employed immunohistochemistry to investigate the expression of tSTAT5 and pSTAT5 in the feline mammary gland. STAT5 has been studied in normal mammary glands and the development of mammary tumors in mice and humans [2, 41]. However, its association with feline mammary tumors has not yet been characterized.

The two variants of STAT5, STAT5a, and STAT5b with 94% structural homology, originate from different genes. Nonetheless, the STAT5a and STAT5b dimers bind to similar core consensus sequences, suggesting that these STAT5 isoforms regulate many of the same pathways and genes [25]. Therefore, in this study, we used the anti-STAT5a antibody used in previous studies [16, 56]. Furthermore, cross-reactivity of both STAT5a and pSTAT5 antibodies was confirmed by western blotting because cross-reactivity has not yet been reported in feline tissues. Human STAT5a and pSTAT5 have molecular weights of 91 and 90 kDa, respectively [20, 56]. A distinct band from the lysate of the normal mammary gland was observed at approximately 91 kDa and 90 kDa, suggesting that these antibodies cross-reacted and were specific for feline tSTAT5 and pSTAT5 proteins. Furthermore, a distinct band from all three samples was detected using an anti-tSTAT5 antibody. However, a single band was detected in the lysate from the normal mammary gland and orthovanadate-stimulated HeLa cells for the anti-pSTAT5 antibody, but not in the lysate from mammary carcinoma. These results indicated cross-reactivity of the antibodies used in this study with cats and supported the immunostaining findings. Immunohistochemistry showed that fibroblasts and lymphocytes reacted positively to both antibodies, as did mammary epithelial cells. This reaction in both cell types appeared to be specific because the expression of tSTAT5 was reported in both cell types [9, 14, 22].

In this study, although nuclear tSTAT5 colocalized with pSTAT5 under various conditions, feline mammary carcinoma displayed more frequent nuclear tSTAT5 positivity than pSTAT5. Because phosphorylated STAT dimerizes and translocates into the nucleus, nuclear positivity may indicate activated STATs. However, because inactivated STAT3 has been reported to dynamically shuttle between the cytoplasmic and nuclear compartments and maintain a prominent nuclear presence [24]. The mechanisms underlying nuclear translocation and STAT5 accumulation have not been elucidated in detail. However, as inactive STAT3 accumulates in the nucleus, the possibility of inactive STAT5 accumulation in the nucleus cannot be excluded.

This study revealed a strong pSTAT5 expression in normal mammary glands and non-neoplastic lesions, such as mammary gland hyperplasia. STAT5 has been reported to be persistently activated in luminal epithelial cells in the mouse and human breast tissue not only during pregnancy and lactation but also during the non-pregnancy period [30]. This study suggests that in cats, STAT5 expression and activation are similar in normal mammary glands, representing the non-pregnancy period, and hyperplastic mammary glands, representing the lactation period. Proliferating secretory mammary epithelial cells in tissue with fibroadenomatous changes and neoplastic mammary epithelial cells in mammary adenomas exhibited mild to strong pSTAT5 expression. In contrast, pSTAT5 expression was significantly decreased in mammary carcinomas, regardless of the histopathological subtype. These results suggest that STAT5 activation is less related to tumor cell growth in feline mammary carcinomas.

In human breast cancers, the expression of STAT5 and nuclear STAT5 have been reported to be lower in high-grade breast cancers than in low-grade tumors [8]. STAT5 expression is generally lower in invasive carcinomas than in the clinically favorable secretory carcinoma subtype in humans, and it also tends to be further reduced in mouse mammary tumors in metastases than in primary tumors [31, 33, 41, 48, 56]. Conversely, studies have reported that high expression levels of tSTAT5 or pSTAT5 in patients with breast cancer correlate with improved overall and disease-specific survival, suggesting an association between reduced pSTAT5 expression and breast cancer malignancy [23, 27, 31, 33, 34, 50, 57]. Although tSTAT5 showed consistent expression across various feline mammary conditions, pSTAT5 was notably absent in mammary carcinomas in this study. Consequently, this study suggests that both tSTAT5 and pSTAT5 are unlikely to serve as useful prognostic factors in feline mammary carcinomas, in contrast to their significance in human breast cancer. However, further prognostic investigations are required to confirm these results. Nevertheless, the high metastatic potential and poor prognosis of feline mammary carcinomas are well-documented and consistent with the characteristics of high-grade breast cancer in humans [6, 11]. Previous studies on STAT5a expression during carcinogenesis-induced preneoplastic lesions, including intraductal growth and ductal carcinoma in situ, suggest that STAT5a activation may play a role in the early stages of tumorigenesis in rat mammary adenocarcinomas [44]. The status of STAT5a activity in the early stages of tumorigenesis remains unknown because all cases examined in the present study were clinically identified and removed. STAT5 activity in human breast cancer has been reported to decrease with tumor progression [30]. Therefore, it is unclear from the present results whether the decrease in STAT5 activity observed in feline mammary carcinomas is significant for tumorigenesis or if it diminishes with tumor progression.

In this study, pSTAT5 was strongly expressed in various feline mammary gland conditions other than mammary carcinoma. However, we acknowledge one limitation: our reliance solely on immunohistochemistry to detect tumors in their current state. Consequently, we were unable to track the progression and development of tumors over time, which limited our understanding of the timing of tumor formation. Therefore, incorporating complementary methodologies that allow for a more dynamic and longitudinal assessment of tumor development and progression could be advantageous for future research efforts aimed at elucidating the role of STAT5 in the process of tumorigenesis. The role of pSTAT5 encompasses the regulation of proliferation, differentiation, apoptosis, angiogenesis, and immune system function [12, 40]. Notably, pSTAT5 functions as a tumor suppressor in certain contexts [23]. The observed low expression of pSTAT5 in feline mammary carcinomas suggests a potential contribution to tumorigenesis and metastasis. Further investigation into the mechanisms associated with this loss of function, including JAK, prolactin, SHPs, PIAS, and SOCS, which dephosphorylate active STATs, is warranted. This investigation attempted to elucidate the role of STAT5 in feline mammary carcinoma, using feline mammary carcinoma cell lines. Moreover, reactivating pSTAT5 activity in feline mammary carcinoma holds promise for the development of new therapeutic strategies for this condition.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare with respect to the research, authorship, and/or publication of this article.

Supplementary

Supplementary Materials
jvms-86-816-s001.pdf (1.8MB, pdf)

Acknowledgments

The authors would like to thank the veterinary clinicians who provided information on the cases examined in this study. This study was supported by Grants-in-Aid for Scientific Research (C), Grant No. 23K05534 from the Japan Society for the Promotion of Science.

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