Krüppel-like factors (KLFs) are critical regulators of biological and physiological systems and have been extensively studied for their roles in cell proliferation, differentiation and survival in the context of cancer. Among the KLFs, KLF4 is highly expressed in human breast cancers and plays an oncogenic role. The present study examined the expression of KLF4 and assessed its significance in canine mammary carcinoma.
Results
Immunohistochemistry was employed to investigate the expression of KLF4 in 142 cases of canine mammary tumor. 75 of the 142 (52.8%) cases were histologically confirmed as mammary carcinoma. Quantification of immunohistochemistry was carried out using Quick score which multiply the staining intensity by the percentage of positive cells. High KLF4 expression was identified in 44 of the 75 (59%) dogs with mammary carcinoma and none in the benign cases. High KLF4 expression occurred only in the tumor cells and not the adjacent normal cells in mammary carcinoma (P < 0.001). Moreover, the high expression level of KLF4 expression was statistically associated with poor grade, late stage, histological subtypes of simple and complex carcinoma, and shorter 24-month survival. The Kaplan-Meier survival analysis also indicated that dogs with high nuclear KLF4 expression had a significantly shorter survival than those with low/moderate KLF4 expression (P = 0.011).
Conclusions
KLF4 is highly and frequently expressed in canine mammary carcinoma and correlates with a more aggressive phenotype.
Background
Canine mammary tumors are the most common tumor in female dogs. The spontaneous, naturally occurring canine mammary tumors share many features with human breast cancer such as the predominant malignant histological type being adenocarcinoma [1–3] and the expression of estrogen and progesterone receptors (ER/PR), and epidermal growth factor receptor 2 (HER2) in subsets of canine mammary carcinoma [4–7]. It has been suggested that canine mammary carcinomas may be a suitable natural model for the comparative study of human breast cancer [4, 5, 7–9].
The Krüppel-like factor (KLF) family proteins are transcription factors that play important roles in a wide range of cellular processes, including embryogenesis, proliferation, differentiation, migration, inflammation and tumorigenesis [10–13].
The KLF family consists of 17 different members in which many have been identified as potentially novel oncogenes or tumor suppressors [13, 14]. Human KLF4 was first identified using a DNA probe containing the zinc finger region of human erythroid Krüppel-like factor from human umbilical vein endothelial cell cDNA library [15]. The biological effects of KLF4 seem to depend on cancer type rather than unique. KLF4 transcription factor can function as a tumor suppressor and is down-regulated in various human cancer types such as gastric and colorectal cancer [16, 17]. On the other hand, high level and oncogenic role of KLF4 were also reported in human breast cancer and oral squamous carcinoma [18, 19]. This study investigated the presence of KLF4 and established their clinical significance in canine mammary carcinoma.
Results
One hundred forty-two dogs (43 Maltese, 11 Yorkshire terriers, 11 Shih-Tzus, 9 Pomeranians, 10 Cocker spaniels, 2 French spaniels, 2 Bichon Frisé, 7 poodles, 2 German shepherd dogs, 1 Shiba, 3 Beagles, 1 Labrador Retriever, 1 Husky, 1 Miniature Doberman, 1 Papillon, 1 Schnauzer, 1 Spitz, and 35 mongrels) were investigated in this study. Of the 142 cases, 52.8% (75/142) were histologically confirmed as carcinoma.
Analyzing the expression of KLF4 in paraffin-embedded tissues by IHC revealed up-regulated nuclear KLF4 expression in mammary carcinomas as compared to benign tumor cases (Table 1). We divided carcinoma patients into three groups, either high KLF4 expression with Quick score of 9-12, moderate KLF4 expression with Quick score of 5-8, or weak KLF4 expression with Quick score of 1-4 (Figure 1). High expression of KLF4 (as defined by a Quick score of 9 or greater) was identified in 59% (44/75) of dogs with mammary carcinoma and none in the benign tumors. Moreover, high expression level of KLF4 occurred preferentially in the tumor cells and not the adjacent non-tumor cells in mammary carcinoma (P < 0.001, Table 2 and Figure 2). Chi-square analyses for the clinicopathologic characteristics of the 75 canine mammary carcinoma cases in relation to nuclear KLF4 expression showed that high KLF4 expression correlated significantly with shorter 24-month survival (P = 0.01, Table 3). High KLF4 expression was also closely associated with poor grade, late stage, and histological subtypes of simple and complex carcinoma. The Kaplan-Meier survival curves indicated that patients with high nuclear expression of KLF4 had a significantly poor survival than those with low/moderate KLF4 expression as defined by log-rank test (P = 0.011, Figure 3).
Table 1
Immunohistochemical quantitation of nuclear KLF4 expression with the Quick score in canine mammary tumor
Quick score
Histological classification
0
1-4
5-8
9-12
total
Benign tumor
0
60
7
0
67
Carcinoma
0
12
19
44
75
Figure 1
Representative immunohistochemical staining patterns of KLF4 in canine mammary carcinoma (×400). (A) weak (B) moderate (C) strong nuclear KLF4 expression.
Table 2
Expression of KLF4 in canine mammary tumor
Pathological diagnosis
KLF4 expression
Benign tumor
Carcinoma
Total
P
Tumor part
Quick score
< 9
67(100%)
31(41.3%)
98
< 0.001
≧ 9
0
44(58.7%)
44
Non-tumor part
Quick score
< 9
67(100%)
75(100%)
142
N/A
≧ 9
0
0
0
Figure 2
Elevated KLF4 expression identified in tumor cells and not adjacent non-tumor cells in a representative canine mammary carcinoma (40X).
Table 3
Clinicopathologic characteristics of canine mammary carcinoma
KLF4 expression (Quick score)
low/moderate (< 9)
High (≧ 9)
N
P
n
%
n
%
Age
< 12 years
14
45.2%
17
38.6%
31
0.572
≧ 12 years
17
54.8%
27
61.4%
44
Ovariohysterectomy
No
26
83.9%
33
75.0%
59
0.356
Yes
5
16.1%
11
25.0%
16
Tumor Size
T1 (< 3 cm)
15
48.4%
11
25.0%
26
T2 (≧ 3 cm, < 5 cm)
7
22.6%
18
40.9%
25
0.089
T3 (> 5 cm)
9
29.0%
15
34.1%
24
Grade
I
12
38.7%
5
11.4%
17
II
14
45.2%
22
50.0%
36
0.009
III
5
16.1%
17
38.6%
22
Histological classification
Carcinoma in benign tumor
5
16.1%
0
0.0%
5
Complex carcinoma
14
45.2%
21
47.7%
35
0.020
Simple carcinoma
12
38.7%
23
52.3%
35
Location of affected gland
cranial
10
32.3%
17
38.6%
27
0.571
caudal
21
67.7%
27
61.4%
48
Stage
I
15
48.4%
5
11.4%
20
II
5
16.1%
13
29.5%
18
III
4
12.9%
7
15.9%
11
0.006
IV
4
12.9%
15
34.1%
19
V
3
9.7%
4
9.1%
7
ER
Negative
16
51.6%
23
52.3%
39
0.955
Positive
15
48.4%
21
47.7%
36
PR
Negative
6
19.4%
5
11.4%
11
0.509
Positive
25
80.6%
39
88.6%
64
Her-2/neu
Negative
26
83.9%
32
72.7%
58
0.281
Positive
5
16.1%
12
27.3%
17
Molecular phenotyping
Basal
10
32.3%
15
34.1%
25
HER-2 overexpressing
2
6.5%
5
11.4%
7
Luminal A
12
38.7%
14
31.8%
26
0.761
Luminal B
3
9.7%
7
15.9%
10
Null
4
12.9%
3
6.8%
7
Survivala
< 24 months
9
56.3%
34
89.5%
43
0.010
≧ 24 months
7
43.8%
4
10.5%
11
a Twenty-one cases lacked survival data and were excluded from the analysis.
Figure 3
The Kaplan-Meier plots for survival according to high versus low/moderate nuclear KLF4 expression. Twenty-one case lacked survival data and were excluded from the analysis.
Discussion
Studies of KLF proteins in mouse models of human diseases have revealed the normal biological roles of the KLFs as well as their involvement in the pathogenesis of a variety of diseases such as cancer [20]. Previous studies have shown that approximately 70% of human breast cancer has increased KLF4 expression and that up-regulated nuclear KLF4 expression is associated with a more aggressive phenotype [18, 21]. The oncogenic properties of KLF4 in breast cancers was also confirmed in vitro and using xenograft tumor model in which KLF4 knockdown inhibited breast cancer development [22].
The breast cancer stem cell hypotheses suggest that breast cancer is derived from a single cell with stem-like properties that is capable of tumor initiation and formation. KLF4 can inhibit differentiation and increase self-renewal in embryonic stem (ES) cells [23, 24]. Forced expression of KLF4, along with transcription factors, Oct4, c-myc, and Sox2, can reprogram or dedifferentiate somatic cells into
induced pluripotent stem cells (iPSCs) in both mice [25, 26] and human [27–29]. Taken together, these finding suggest that KLF4 is indispensable for the regulation of stem cells and contributes to tumorigenesis.
In this study, we investigated the expression and clinical relevance of KLF4 in canine mammary carcinoma. Immunihistochemistry revealed that nuclear expression of KLF4 was elevated in tumor cells of canine mammary carcinoma. Although increased KLF4 expression was not related to prognostic markers such as ER, PR or HER2. High nuclear KLF4 expression was associated significantly with a more aggressive phenotype as indicated by poor grade, late stage, histological subtypes of simple and complex carcinoma, and shorter 24-month survival in canine mammary carcinoma. Despite diffuse cytoplasmic KLF4 expression with different degree of intensity was observed among the samples. The cytoplasmic KLF4 expression was not related to any clinicopathologic parameters and survival (data not shown). The Kaplan-Meier survival analysis also indicated that dogs with high nuclear expression of KLF4 had a significantly shorter survival as compared with ones with low/moderate nuclear KLF4 expression.
We provided evidence for the first time that KLF4 is preferentially and highly expressed in canine mammary carcinoma. As in human breast cancer, KLF4 plays an oncogenic role in canine mammary carcinoma. Further studies are needed to validate whether systemic targeting of KLF4 would inhibits the oncogenic functions of KLF4 thus provides an effective strategy for the treatment of canine mammary carcinoma.
Conclusions
Nuclear expression of KLF4 is frequently elevated in canine mammary carcinoma and closely correlated with a more aggressive phenotype and shorter survival.
Methods
KLF4 Immunohistochemistry
Paraffin-embedded tissue blocks of 142 cases of canine mammary tumor diagnosed between January 2003 and April 2008 were retrieved from the archives of the School of Veterinary Medicine, National Taiwan University, Taiwan. The tumors were diagnosed according to the WHO criteria for canine mammary neoplasms [30]. Samples were first de-waxed in xylene and re-hydrated through graded alcohols, followed by a rinse using 10 mM Tris-HCl (pH 7.4) and 150 mM sodium chloride, then treated with 3% hydrogen peroxide for 5 min. Slides were incubated with 1:250 dilution of anti-KLF4 antibody (sc-20691, Santa Cruz Biotechnology, USA) for 1 hour at room temperature, then thoroughly washed three times with PBS. Bound antibodies were detected using the LSAB+ kit (Dako, USA). The slides were then counterstained with haematoxylin stain solution. Paraffin-embedded sections of human breast cancer cells of homogeneous KLF4 immunophenotype were included as positive controls. Negative controls had the primary antibody omitted and replaced by PBS. Quantification of KLF4 expression was carried out using Quick score which multiply the staining intensity by the percentage of positive cells [31–33]. The intensity of staining was scored as 0, 1, 2, and 3 standing for negative, weak, moderate, and strong staining, respectively. The percentage of tumor cells staining positively was scored as follows: 0 = 0%, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, and 4 = 76-100%, compared with the total of tumor cells. The immunohistochemical results were evaluated by two investigators scoring independently. Conflicting scores were resolved at a dual head microscope.
Molecular Phenotyping
Immunohistochemistry was performed in parallel as described above with monoclonal antibodies for ER (1:35 dilution, Dako, Denmark), PR (1:200 dilution, Thermo Scientific, USA), HER2 (1:400 dilution, Dako, Denmark), CK5 (1:100 dilution, Novacastra, UK), and P-cadherin (1:100 dilution, Novacastra, UK). ER and PR immunoreactivity was considered positive when more than 10% of the neoplastic cells expressed this marker [5]. HercepTest scoring system was applied to evaluate HER2 expression (0 = no staining or membrane staining in fewer than 10% of tumor cells; 1+ = faint, barely perceptible membrane staining in more than 10% of tumor cells; 2+ = weak to moderate complete membrane staining observed in more than 10% of tumor cells; 3+ = strong and complete membrane staining in more than 10% tumor cells) [5]. In this study, overexpression of HER2 was defined as a HercepTest score of 3+. As for CK5 and P-cadherin, cytoplasmic staining in > 50% of cells was considered positive [5]. Immunohistochemical panel which involved the evaluation of ER, HER2, CK5, and P-cadherin was used to distinguish canine mammary carcinoma subtypes [5, 34].
Statistical Analysis
Overexpression of KLF4 was defined as a Quick score of 9 or greater on the scale of 0 to 12. Patterns and correlations of KLF4 and clinicopathologic parameters of canine mammary tumor were examined by Pearson's chi-square test. Survival rate was calculated using Kaplan-Meier analysis and compared by the Cochran-Mantel-Haenszel test (log-rank test). Survival was defined as the time between date of diagnosis and date of death. Subjects still alive at the end of the study were censored at the date of last follow-up. Cases that lacked survival information were excluded from the analysis. A P value of less than 0.05 was considered to indicate statistical significance.
Declarations
Acknowledgements
This study was supported by a grant from the Department of Health, Taiwan (DOH100-TD-C-111-002).
Authors’ Affiliations
(1)
Department of Pathology, St. Martin De Porres Hospital
(2)
Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University
(3)
Graduate Institute of Medicine, Kaohsiung Medical University
(4)
Department of Pathology, Changhua Christian Hospital
(5)
Cancer Center, Kaohsiung Medical University Hospital
Moulton JE, Taylor DO, Dorn CR, Andersen AC: Canine mammary tumors. Pathol Vet. 1970, 7 (4): 289-320. 10.1177/030098587000700401.PubMedView ArticleGoogle Scholar
Prier JE, Brodey RS: Canine Neoplasia. A Prototype for Human Cancer Study. Bull World Health Organ. 1963, 29: 331-344.PubMed CentralPubMedGoogle Scholar
Ferreira E, Gobbi H, Saraiva BS, Cassali GD: Columnar cell lesions of the canine mammary gland: pathological features and immunophenotypic analysis. BMC Cancer. 2010, 10: 61-10.1186/1471-2407-10-61.PubMed CentralPubMedView ArticleGoogle Scholar
Gama A, Alves A, Schmitt F: Identification of molecular phenotypes in canine mammary carcinomas with clinical implications: application of the human classification. Virchows Arch. 2008, 453 (2): 123-132. 10.1007/s00428-008-0644-3.PubMedView ArticleGoogle Scholar
Mouser P, Miller MA, Antuofermo E, Badve SS, Mohammed SI: Prevalence and classification of spontaneous mammary intraepithelial lesions in dogs without clinical mammary disease. Vet Pathol. 2010, 47 (2): 275-284. 10.1177/0300985809358603.PubMedView ArticleGoogle Scholar
Vinothini G, Balachandran C, Nagini S: Evaluation of molecular markers in canine mammary tumors: correlation with histological grading. Oncol Res. 2009, 18 (5-6): 193-201.PubMedView ArticleGoogle Scholar
Martin PM, Cotard M, Mialot JP, Andre F, Raynaud JP: Animal models for hormone-dependent human breast cancer. Relationship between steroid receptor profiles in canine and feline mammary tumors and survival rate. Cancer Chemother Pharmacol. 1984, 12 (1): 13-17.PubMedGoogle Scholar
Strandberg JD, Goodman DG: Animal model of human disease: canine mammary neoplasia. Am J Pathol. 1974, 75 (1): 225-228.PubMed CentralPubMedGoogle Scholar
Huang CC, Liu Z, Li X, Bailey SK, Nail CD, Foster KW, Frost AR, Ruppert JM, Lobo-Ruppert SM: KLF4 and PCNA identify stages of tumor initiation in a conditional model of cutaneous squamous epithelial neoplasia. Cancer Biol Ther. 2005, 4 (12): 1401-1408. 10.4161/cbt.4.12.2355.PubMedView ArticleGoogle Scholar
Segre JA, Bauer C, Fuchs E: Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet. 1999, 22 (4): 356-360. 10.1038/11926.PubMedView ArticleGoogle Scholar
Shields JM, Christy RJ, Yang VW: Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem. 1996, 271 (33): 20009-20017. 10.1074/jbc.271.33.20009.PubMed CentralPubMedView ArticleGoogle Scholar
Turner J, Crossley M: Mammalian Kruppel-like transcription factors: more than just a pretty finger. Trends Biochem Sci. 1999, 24 (6): 236-240. 10.1016/S0968-0004(99)01406-1.PubMedView ArticleGoogle Scholar
Dang DT, Pevsner J, Yang VW: The biology of the mammalian Kruppel-like family of transcription factors. Int J Biochem Cell Biol. 2000, 32 (11-12): 1103-1121. 10.1016/S1357-2725(00)00059-5.PubMed CentralPubMedView ArticleGoogle Scholar
Yet SF, McA'Nulty MM, Folta SC, Yen HW, Yoshizumi M, Hsieh CM, Layne MD, Chin MT, Wang H, Perrella MA, Jain MK, Lee ME: Human EZF, a Kruppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains. J Biol Chem. 1998, 273 (2): 1026-1031. 10.1074/jbc.273.2.1026.PubMedView ArticleGoogle Scholar
Wei D, Gong W, Kanai M, Schlunk C, Wang L, Yao JC, Wu TT, Huang S, Xie K: Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res. 2005, 65 (7): 2746-2754. 10.1158/0008-5472.CAN-04-3619.PubMedView ArticleGoogle Scholar
Choi BJ, Cho YG, Song JW, Kim CJ, Kim SY, Nam SW, Yoo NJ, Lee JY, Park WS: Altered expression of the KLF4 in colorectal cancers. Pathol Res Pract. 2006, 202 (8): 585-589. 10.1016/j.prp.2006.05.001.PubMedView ArticleGoogle Scholar
Foster KW, Frost AR, McKie-Bell P, Lin CY, Engler JA, Grizzle WE, Ruppert JM: Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res. 2000, 60 (22): 6488-6495.PubMedGoogle Scholar
Foster KW, Ren S, Louro ID, Lobo-Ruppert SM, McKie-Bell P, Grizzle W, Hayes MR, Broker TR, Chow LT, Ruppert JM: Oncogene expression cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF. Cell Growth Differ. 1999, 10 (6): 423-434.PubMedGoogle Scholar
McConnell BB, Yang VW: Mammalian Kruppel-like factors in health and diseases. Physiol Rev. 2010, 90 (4): 1337-1381. 10.1152/physrev.00058.2009.PubMed CentralPubMedView ArticleGoogle Scholar
Pandya AY, Talley LI, Frost AR, Fitzgerald TJ, Trivedi V, Chakravarthy M, Chhieng DC, Grizzle WE, Engler JA, Krontiras H, Bland KI, LoBuglio AF, Lobo-Ruppert SM, Ruppert JM: Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clin Cancer Res. 2004, 10 (8): 2709-2719. 10.1158/1078-0432.CCR-03-0484.PubMedView ArticleGoogle Scholar
Yu F, Li J, Chen H, Fu J, Ray S, Huang S, Zheng H, Ai W: Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene. 2011, 30 (18): 2161-2172. 10.1038/onc.2010.591.PubMed CentralPubMedView ArticleGoogle Scholar
Bruce SJ, Gardiner BB, Burke LJ, Gongora MM, Grimmond SM, Perkins AC: Dynamic transcription programs during ES cell differentiation towards mesoderm in serum versus serum-freeBMP4 culture. BMC Genomics. 2007, 8: 365-10.1186/1471-2164-8-365.PubMed CentralPubMedView ArticleGoogle Scholar
Zhang P, Andrianakos R, Yang Y, Liu C, Lu W: Kruppel-like factor 4 (Klf4) prevents embryonic stem (ES) cell differentiation by regulating Nanog gene expression. J Biol Chem. 2010, 285 (12): 9180-9189. 10.1074/jbc.M109.077958.PubMed CentralPubMedView ArticleGoogle Scholar
Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006, 126 (4): 663-676. 10.1016/j.cell.2006.07.024.PubMedView ArticleGoogle Scholar
Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R: In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007, 448 (7151): 318-324. 10.1038/nature05944.PubMedView ArticleGoogle Scholar
Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ: Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008, 451 (7175): 141-146. 10.1038/nature06534.PubMedView ArticleGoogle Scholar
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007, 131 (5): 861-872. 10.1016/j.cell.2007.11.019.PubMedView ArticleGoogle Scholar
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA: Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007, 318 (5858): 1917-1920. 10.1126/science.1151526.PubMedView ArticleGoogle Scholar
Misdorp W, Else RW, Hellmén E, Lipscomb TP: Histological Classification of Mammary Tumors of the Dog and Cat. Washington, D. C.: American Registry of Pathology 1999.Google Scholar
Briffod M, Hacene K, Le Doussal V: Immunohistochemistry on cell blocks from fine-needle cytopunctures of primary breast carcinomas and lymph node metastases. Mod Pathol. 2000, 13 (8): 841-850. 10.1038/modpathol.3880149.PubMedView ArticleGoogle Scholar
Charafe-Jauffret E, Tarpin C, Bardou VJ, Bertucci F, Ginestier C, Braud AC, Puig B, Geneix J, Hassoun J, Birnbaum D, Jacquemier J, Viens P: Immunophenotypic analysis of inflammatory breast cancers: identification of an 'inflammatory signature'. J Pathol. 2004, 202 (3): 265-273. 10.1002/path.1515.PubMedView ArticleGoogle Scholar
Li J, Martinka M, Li G: Role of ING4 in human melanoma cell migration, invasion and patient survival. Carcinogenesis. 2008, 29 (7): 1373-1379. 10.1093/carcin/bgn086.PubMedView ArticleGoogle Scholar
Matos I, Dufloth R, Alvarenga M, Zeferino LC, Schmitt F: p63, cytokeratin 5, and P-cadherin: three molecular markers to distinguish basal phenotype in breast carcinomas. Virchows Arch. 2005, 447 (4): 688-694. 10.1007/s00428-005-0010-7.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
