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Article 

Evaluation of 14-3-3σ as a Prognostic Marker in Canine 
Mammary Tumors 

Ana Hîruța 1, Andrada Negoescu1*, Zoltán-Miklós Gál2, Alexandru Raul Pop2 and Cornel Cătoi 1 

1 Faculty of Veterinary Medicine, Pathology Department, University of Agricultural Sciences and Veterinary 
Medicine, Cluj-Napoca, 400372, Cluj, Romania. 

2 Faculty of Veterinary Medicine, Reproduction Department, University of Agricultural Sciences and 
Veterinary Medicine, Cluj-Napoca, 400372, Cluj, Romania;  

* Correspondence: andrada.negoescu@usamvcluj.ro; Tel.: +4 0745613960 
 

Abstract: 14-3-3σ is a regulatory protein involved in cell cycle control and has been implicated in both tumor-suppressive and 
tumor-promoting roles, depending on the biological context. While extensively studied in human cancers, limited information is 
available regarding its expression in canine mammary gland tumors. This study aimed to assess the immunohistochemical 
expression of 14-3-3σ in canine mammary tumors and evaluate its potential association with malignancy indicators such as 
histological grade and mitotic index. A total of 62 tumor samples were analyzed using immunohistochemistry, and the area of 14-3-
3σ expression was digitally quantified. Statistical comparisons were performed using non-parametric tests. An inverse trend was 
observed between 14-3-3σ expression and both tumor grade (p = 0.051) and mitotic (p = 0.0090) activity. These findings suggest a 
potential link between decreased 14-3-3σ expression and increased malignancy, supporting its relevance as a candidate prognostic 
marker in canine mammary tumors. Further studies with larger cohorts are needed to validate these observations. 

Keywords: 14-3-3 σ, immunohistochemistry, canine mammary gland tumor. 
 

1. Introduction 

 
The 14-3-3 protein family comprises highly conserved regulatory molecules present 

in all eukaryotic cells, playing essential roles in key physiological processes[1]. To date, 
over 200 target proteins have been identified, including those involved in mitogenic sig-
naling, cell survival, cell cycle regulation and apoptosis. Notably, the interaction of 14-3-
3 proteins with various oncogenes and tumor suppressor genes highlights their potential 
involvement in cancer development and progression[2]. The 14-3-3 protein family com-
prises seven isoforms (β, ε, η, γ, τ, σ, and ζ), each ranging in size from 28 to 33 kDa, which 
are highly conserved and widely expressed across various tissues[3].  

The 14-3-3 σ protein, also known as stratifin or human mammary epithelial marker 
(HME1), is a negative regulator of the cell cycle and has been closely associated with tu-
mor development. It is unique among the seven 14-3-3 isoforms for its strong link to on-
cogenesis[4]. Its expression is regulated by the tumor suppressor protein p53 in response 
to DNA damage, functioning to inhibit mitosis by sequestering the cdc2–cyclin B1 com-
plex in the cytoplasm and preventing its translocation to the nucleus. 14-3-3σ is involved 
in both G1/S and G2/M cell cycle arrest through p53-dependent transactivation in re-
sponse to DNA damage[5]. In this way, it induces G2 arrest, providing time for the repair 

of damaged DNA.  
In humans, both reduced [6,7] and elevated levels [8,9] of 14-3-3 σ expression have been implicated in 

various cancers. Furthermore, both upregulation and downregulation of 14-3-3 σ have been reported in var-
ious tumor types, suggesting a context-dependent role in tumorigenesis [3]. Notably, breast cancer cells lack-
ing 14-3-3σ expression exhibit a significantly higher frequency of G2-type chromosomal aberrations com-
pared to cells that express the protein. These findings suggest that 14-3-3σ plays a critical role in G2 check-
point control in breast epithelial cells. The loss of 14-3-3σ gene expression may contribute to breast cancer 

Received: 07.07.2025 

Accepted: 09.07.2025 

Published: 15.07.2025 

DOI: 10.52331/v30i20s71 

 

 

 

Copyright: © 2025 by the authors. 

Submitted for possible open access 

publication under the terms and con-

ditions of the Creative Commons At-

tribution (CC BY) license (http://crea-

tivecommons.org/licenses/by/4.0/). 



Cluj Vet J 2025, vol 30, issue 2 40 of 66 
 

 

development by permitting the accumulation of genetic damage that promotes malignant transformation 
[10]. 

In canine mammary tissues 14-3-3 σ has been detected in 97% of samples, localizing to both epithelial 
(ECs) and myoepithelial cells (MECs)[4]. Studies have shown that this isoform is specifically expressed in 
epithelial cells under normal conditions.  

Mammary cancer is the most frequently diagnosed malignancy in both women and female dogs[11]. 
Owing to its significant clinical impact, mammary gland tumors have been extensively investigated. Since 
ethical constraints limit experimental research in humans, animals with naturally occurring mammary tu-
mors represent valuable models for the development of in vitro systems and the advancement of cancer 
research [4]. Given the inconsistent and seemingly random pattern of 14-3-3 σ staining in epithelial cells 
(ECs) [12], the authors hypothesized that its expression might be associated with the degree of malignancy. 
To explore this possibility, histological grade and mitotic count—both established indicators of malig-
nancy—were analyzed in relation to the level of 14-3-3 σ expression. 

This study aimed to investigate the expression of 14-3-3 σ protein in neoplastic canine mammary tissue 
and to evaluate its potential as a malignancy marker for epithelial cells (ECs). 

 
2. Materials and Methods 
This study included 55 female canine patients diagnosed with mammary gland tumors. All patients 

underwent clinical examination, thoracic imaging, and either unilateral or total mastectomy. A total of 64 
tumor specimens were collected, routinely fixed in 10% neutral buffered formalin, processed, and embedded 
in paraffin wax. Tissue sections were stained with hematoxylin and eosin (H&E) and evaluated histopatho-
logically according to the Zappulli classification and grading system [13]. For each sample, the mitotic count 
was determined by evaluating ten high-power fields (40× magnification) and recording the number of mi-
totic figures observed. 

For the immunohistochemical analysis, two-micrometer-thick sections were cut from formalin-fixed, 
paraffin-embedded tissue blocks and immunolabeled using mouse monoclonal antibodies against 14-3-3σ 
(clone 5D7, 1:40; Santa Cruz Biotechnology, Heidelberg, Germany). Heat-induced epitope retrieval was car-
ried out in a pH 6 buffer (Bond ER1; Leica) for 20 minutes at 90 °C. Visualization was performed using the 
Bond Polymer Refine Detection Kit (Leica), with hematoxylin used as a counterstain. Positive labeling was 
identified by the presence of brown staining of the cytoplasm. 

The quantitative analysis was performed using the ImageJ software, version 1.54f (Wayne Rasband and 
contributors, National Institutes of Health, USA). For each case, ten microscopic fields were captured at 40X 
magnification using an Olympus UC-30 digital camera and Stream Basic software, maintaining the same 
light intensity throughout. For statistical consistency, the mean percentage value across the ten analyzed 
fields was calculated for each sample and used in subsequent analyses. 

To assess the association between the immunolabeled area and histological grade, the Kruskal–Wallis 
non-parametric test and the Jonckheere–Terpstra trend test were employed. The correlation between the 
labeled area and mitotic count was analyzed using Spearman’s rank correlation and Kendall’s tau non-par-
ametric tests. 

 

3. Results 
The mean age of the patients was 9.19 years. The majority of female dogs were mixed-breed (11/55), 

followed by German Shepherds (7/55) and Yorkshire Terriers (7/55). The database included the following 
histological subtypes of mammary carcinomas: complex carcinomas (n=19), tubular carcinomas (n=15), tu-
bulopapillary carcinomas (n=1), inflammatory carcinoma (n=1), mixed carcinomas (n=9), solid carcinomas 
(n=4), invasive micropapillary carcinoma (n=1), intraductal papillary carcinomas (n=9), and ductal carcino-
mas (n=2). Additionally, four cases were classified as special types: lipid-rich carcinoma (n=1), carcinosar-
coma (n=1), comedocarcinoma (n=1) and adenosquamous carcinoma (n=1). Immunohistochemical expres-
sion of 14-3-3σ varied across histological subtypes of canine mammary carcinomas. Positive labeling was 
observed in the vast majority of complex carcinomas (18/19), tubular carcinomas (13/15), and all cases of 
tubulopapillary carcinoma (1/1), invasive micropapillary carcinoma (1/1), intraductal papillary carcinomas 
(9/9), and ductal carcinomas (2/2). Mixed carcinomas exhibited positive labeling in 6 out of 9 cases. Among 
solid carcinomas, only 1 of 4 cases showed immunoreactivity. In contrast, none of the special carcinoma 



Cluj Vet J 2025, vol 30, issue 2 41 of 66 
 

 

subtypes—lipid-rich carcinoma (n=1), carcinosarcoma (n=1), comedocarcinoma (n=1), and adenosquamous 
carcinoma (n=1)—showed any detectable expression of 14-3-3σ. 

Across the examined samples, the cytoplasmic staining pattern of the cells was variable (Figure 1). No-
tably, in terms of immunolabeling, the cytoplasm of epithelial cells from the three most aggressive histolog-
ical subtypes—lipid-rich carcinoma, carcinosarcoma, and adenosquamous carcinoma—showed a complete 
absence of detectable staining for 14-3-3σ. 

 

Figure 1. Immunohistochemical patterns showing varying labeling intensities. 

 
The Kruskal–Wallis test did not show statistically significant differences in the area of expression 

among the three histological grades (H = 3.51, p = 0.171). However, the Jonckheere–Terpstra test revealed a 
trend toward decreasing expression with increasing tumor grade (z = –1.951, p = 0.051), suggesting a 
potential association between reduced expression area and higher tumor aggressiveness (Figure 2-left). 

 

 

Figure 2. Graphical representation of the relationship between expression area and histological grade (left) and mitotic count 

(right). 

 Left: vertical error bars- Indicate variability, blue line- connects the central values (medians), visually indicating a decreasing 

trend in expression from Grade 1 to Grade 3.  

Right: X-axis: Represents the number of mitotic figures observed per tumor, Y-axis: represents the percentage of IHC marked 

area, trend line: a linear regression line showing the general direction of the relationship. 



Cluj Vet J 2025, vol 30, issue 2 42 of 66 
 

 

A statistically significant moderate inverse correlation was found between 14-3-3σ expression and 
mitotic activity. As the mitotic count increased, the area of epithelial immunolabeling decreased. This 
relationship was demonstrated by both Spearman’s rank correlation coefficient (ρ = –0.323, p = 0.0086; 95% 
CI: –0.526 to –0.086) and Kendall’s tau (τ = –0.221, p = 0.0090; 95% CI: –0.385 to –0.018), indicating that 
higher proliferative activity is associated with reduced 14-3-3σ expression in epithelial cells (Figure 2, 
right). 

4. Discussion 
14-3-3 sigma is a regulatory protein involved in cell cycle control, particularly at the G2/M checkpoint, 

and has been shown to play dual roles in cancer biology—functioning either as a tumor suppressor or having 
oncogenic potential depending on the cellular context. It is also implicated in tumor invasion and metastasis 
pathways [14,15].  

Although traditionally classified as a tumor suppressor, a 2013 study revealed that in basal-like breast 
cancer (BLBC) in humans, 14-3-3 sigma can adopt a pro-tumorigenic role. Its expression increases 
progressively during malignant transformation, promoting tumor cell migration and invasion, independent 
of cell proliferation [16]. Additionally, in invasive ductal carcinoma, reduced 14-3-3 sigma expression is 
associated with decreased patient survival. Likewise, in ductal carcinoma in situ, higher levels of 14-3-3 
sigma expression are linked to poorer patient outcomes [17]. These features suggest the complex biological 
behaviour of this particular protein when different types of breast neoplasms are involved. 

In veterinary oncology, 14-3-3 sigma has also been explored in various species and tumor types. 
Notably, studies have documented its expression in canine mammary, gastric, and renal cell carcinomas 
and  equine penile squamous cell carcinoma [4,12,18,19] 

An intriguing finding across both squamous cell carcinoma and renal cell carcinoma is the observation 
of aberrant nuclear immunolabeling of 14-3-3 sigma. This nuclear localization is suggested to correlate with 
increased metastatic potential, serving as a potential marker of aggressive tumor behavior [19,20]. Similar 
features were observed in a recent study on canine gastric carcinoma, in which aberrant immunolabeling 
was noted in pleomorphic neoplastic cells, indicating a higher potential for metastasis [21]. Despite these 
associations, no significant correlation was found between 14-3-3 sigma expression and tumor grade or 
mitotic index in some studies [19]. In contrast, a positive correlation was observed in canine mammary 
neoplasms between 14-3-3 sigma expression and the number of mitotic figures(In contrast, a positive 
correlation was observed in canine mammary neoplasms between 14-3-3 sigma expression and the number 
of mitotic figures, degree of invasion, and presence of vascular emboli.). Authors should discuss the results 
and how they can be interpreted from the perspective of previous studies and of the working hypotheses. 
The findings and their implications should be discussed in the broadest context possible. Future research 
directions may also be highlighted. 

Our study suggests that immunohistochemical labeling of 14-3-3σ may be associated with malignancy 
markers such as tumor grade and mitotic count. Although statistical analyses did not reach conventional 
levels of significance, the results approached relevance, and the observed inverse trend between tumor 
grade, mitotic figures, and the area of positive labeling indicates a potential biological relationship. These 
findings imply that the lack of statistical significance may be attributed to the limited sample size, and 
further studies with larger cohorts are warranted to validate this association. 

 

5. Conclusions 
This study indicates a possible association between 14-3-3σ expression and malignancy features, such 

as tumor grade and mitotic activity, in canine mammary tumors. While statistical significance was not 
reached, the inverse trends observed suggest biological relevance. These findings, in line with previous 
research, underscore the need for further studies with larger cohorts to better define the prognostic value of 
14-3-3σ. 

 
Author Contributions: Conceptualization, A.H. and A.N.; methodology, A.H. and A.N.; statistical analysis, Z.-M.G.; 
resources, C.C..; data curation, A.H.; writing—original draft preparation,., A.H., A.N. and Z.-M.G.; writing—review and 
editing, A.N.; supervision, C.C. All authors have read and agreed to the published version of the manuscript. 

Funding: This research was founded by the Discipline of Veterinary Pathology at the Faculty of Veterinary Medicine Cluj-
Napoca.  



Cluj Vet J 2025, vol 30, issue 2 43 of 66 
 

 

Acknowledgments: The authors extend their gratitude to the Discipline of Animal Reproduction at the Faculty of Veterinary 
Medicine Cluj-Napoca, and the private practice reproduction referral clinic Quantas Repro Vet SRL in Cluj-Napoca for their 
help in collecting the samples. 

Conflicts of Interest: The authors declare no conflict of interest. 

References 

1.  Fu, H.; Subramanian, R.R.; Masters, S.C. 14-3-3 Proteins: Structure, Function, and Regulation. Annu. Rev. Pharmacol. Toxicol. 

2000, 40, 617–647, doi:10.1146/annurev.pharmtox.40.1.617. 

2.  Tzivion, G.; Avruch, J. 14-3-3 Proteins: Active Cofactors in Cellular Regulation by Serine/Threonine Phosphorylation. Journal of 

Biological Chemistry 2002, 277, 3061–3064, doi:10.1074/jbc.R100059200. 

3.  Tzivion, G.; Gupta, V.S.; Kaplun, L.; Balan, V. 14-3-3 Proteins as Potential Oncogenes. Seminars in Cancer Biology 2006, 16, 203–

213, doi:10.1016/j.semcancer.2006.03.004. 

4.  Suárez-Bonnet, A.; Herráez, P.; Mulas, J.M.D.L.; Rodríguez, F.; Déniz, J.M.; Monteros, A.E.D.L. Expression of 14-3-3 σ Protein 

in Normal and Neoplastic Canine Mammary Gland. The Veterinary Journal 2011, 190, 345–351, doi:10.1016/j.tvjl.2010.12.015. 

5.  Simpson, P.T.; Gale, T.; Reis-Filho, J.S.; Jones, C.; Parry, S.; Steele, D.; Cossu, A.; Budroni, M.; Palmieri, G.; Lakhani, S.R. Distri-

bution and Significance of 14-3-3σ, a Novel Myoepithelial Marker, in Normal, Benign, and Malignant Breast Tissue. The Journal 

of Pathology 2004, 202, 274–285, doi:10.1002/path.1530. 

6.  Iwata, N.; Yamamoto, H.; Sasaki, S.; Itoh, F.; Suzuki, H.; Kikuchi, T.; Kaneto, H.; Iku, S.; Ozeki, I.; Karino, Y.; et al. Frequent 

Hypermethylation of CpG Islands and Loss of Expression of the 14-3-3 σ Gene in Human Hepatocellular Carcinoma. Oncogene 

2000, 19, 5298–5302, doi:10.1038/sj.onc.1203898. 

7.  Mhawech, P.; Benz, A.; Cerato, C.; Greloz, V.; Assaly, M.; Desmond, J.C.; Koeffler, H.P.; Lodygin, D.; Hermeking, H.; Herrmann, 

F.; et al. Downregulation of 14-3-3σ in Ovary, Prostate and Endometrial Carcinomas Is Associated with CpG Island Methylation. 

Modern Pathology 2005, 18, 340–348, doi:10.1038/modpathol.3800240. 

8.  Guweidhi, A. Enhanced Expression of 14-3-3sigma in Pancreatic Cancer and Its Role in Cell Cycle Regulation and Apoptosis. 

Carcinogenesis 2004, 25, 1575–1585, doi:10.1093/carcin/bgh159. 

9.  Perathoner, A.; Pirkebner, D.; Brandacher, G.; Spizzo, G.; Stadlmann, S.; Obrist, P.; Margreiter, R.; Amberger, A. 14-3-3σ Ex-

pression Is an Independent Prognostic Parameter for Poor Survival in Colorectal Carcinoma Patients. Clinical Cancer Research 

2005, 11, 3274–3279, doi:10.1158/1078-0432.CCR-04-2207. 

10.  Ferguson, A.T.; Evron, E.; Umbricht, C.B.; Pandita, T.K.; Chan, T.A.; Hermeking, H.; Marks, J.R.; Lambers, A.R.; Futreal, P.A.; 

Stampfer, M.R.; et al. High Frequency of Hypermethylation at the 14-3-3 σ Locus Leads to Gene Silencing in Breast Cancer. Proc. 

Natl. Acad. Sci. U.S.A. 2000, 97, 6049–6054, doi:10.1073/pnas.100566997. 

11.  Ferreira, T.; Miranda, M.; Pinto-Leite, R.; Mano, J.F.; Medeiros, R.; Oliveira, P.A.; Gama, A. Integrated Study of Canine Mam-

mary Tumors Histopathology, Immunohistochemistry, and Cytogenetic Findings. Veterinary Sciences 2024, 11, 409, 

doi:10.3390/vetsci11090409. 

12.  Suárez-Bonnet, A.; De Las Mulas, J.M.; Herráez, P.; Rodríguez, F.; De Los Monteros, A.E. Immunohistochemical Localisation of 

14-3-3 σ Protein in Normal Canine Tissues. The Veterinary Journal 2010, 185, 218–221, doi:10.1016/j.tvjl.2009.05.014. 

13.  Mammary Tumors; Zappulli, V., Ed.; Surgical pathology of tumors of domestic animals / edited by M. Kiupel; Davis-Thompson 

DVM Foundation: Gurnee, Illinois, 2019; ISBN 978-1-73374-911-4. 

14.  Ko, S.; Kim, J.Y.; Jeong, J.; Lee, J.E.; Yang, W.I.; Jung, W.H. The Role and Regulatory Mechanism of 14-3-3 Sigma in Human 

Breast Cancer. J Breast Cancer 2014, 17, 207, doi:10.4048/jbc.2014.17.3.207. 

15.  Mikami, T.; Maruyama, S.; Abé, T.; Kobayashi, T.; Yamazaki, M.; Funayama, A.; Shingaki, S.; Kobayashi, T.; Jun, C.; Saku, T. 

Keratin 17 Is Co-Expressed with 14-3-3 Sigma in Oral Carcinoma in Situ and Squamous Cell Carcinoma and Modulates Cell 

Proliferation and Size but Not Cell Migration. Virchows Arch 2015, 466, 559–569, doi:10.1007/s00428-015-1735-6. 



Cluj Vet J 2025, vol 30, issue 2 44 of 66 
 

 

16.  Boudreau, A.; Tanner, K.; Wang, D.; Geyer, F.C.; Reis-Filho, J.S.; Bissell, M.J. 14-3-3σ Stabilizes a Complex of Soluble Actin and 

Intermediate Filament to Enable Breast Tumor Invasion. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, doi:10.1073/pnas.1315022110. 

17.  Yoon, N.K.; Seligson, D.B.; Chia, D.; Elshimali, Y.; Sulur, G.; Li, A.; Horvath, S.; Maresh, E.; Mah, V.; Bose, S.; et al. Higher 

Expression Levels of 14-3-3σ in Ductal Carcinoma in Situ of the Breast Predict Poorer Outcome1. CBM 2009, 5, 215–224, 

doi:10.3233/CBM-2009-0106. 

18.  Suárez-Bonnet, A.; Herráez, P.; Aguirre, M.; Suárez-Bonnet, E.; Andrada, M.; Rodríguez, F.; Espinosa De Los Monteros, A. 

Expression of Cell Cycle Regulators, 14-3-3σ and P53 Proteins, and Vimentin in Canine Transitional Cell Carcinoma of the 

Urinary Bladder. Urologic Oncology: Seminars and Original Investigations 2015, 33, 332.e1-332.e7, doi:10.1016/j.urolonc.2015.04.006. 

19.  Suárez-Bonnet, A.; Willis, C.; Pittaway, R.; Smith, K.; Mair, T.; Priestnall, S.L. Molecular Carcinogenesis in Equine Penile Cancer: 

A Potential Animal Model for Human Penile Cancer. Urologic Oncology: Seminars and Original Investigations 2018, 36, 532.e9-

532.e18, doi:10.1016/j.urolonc.2018.09.004. 

20.  Suárez-Bonnet, A.; Lara-García, A.; Stoll, A.L.; Carvalho, S.; Priestnall, S.L. 14-3-3σ Protein Expression in Canine Renal Cell 

Carcinomas. Vet Pathol 2018, 55, 233–240, doi:10.1177/0300985817738097. 

21.  Hardas, A.; Suárez-Bonnet, A.; Beck, S.; Becker, W.E.; Ramírez, G.A.; Priestnall, S.L. Canine Gastric Carcinomas: A Histopatho-

logical and Immunohistochemical Study and Similarities with the Human Counterpart. Animals 2021, 11, 1409, 

doi:10.3390/ani11051409. 
 

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