Breast cancer is one of the most common malignancies that occur among females. The incidence of the disease has increased in recent years. The age of onset has become lower, and metastasis is the major cause of death in breast cancer. Therefore, more attention should be focused on breast cancer (Yang et al., 2008).
Breast cancer is considered to be a systemic disease, which means that most breast carcinoma metastasize before diagnosis of the primary lesion (Riordan and Auerbach, 1999). Therefore, early detection of a metastasized lesion and identification of more effective therapeutic modalities for metastatic disease are necessary if the prognosis for patients with advanced breast cancer is to improve (Schäfer and Heizmann, 1996).
Cancer cells may invade the surrounding by tissue remodeling and angiogenesis. They may spread through the bloodstream and lymphatic system to other parts of the body (Greider and Blackburn, 1996). The majority of invasive breast cancer arise from the epithelium of lobules and ducts of the glands (Ismail et al., 2008).
Research into breast cancer has highlighted the prognostic significance of a number of pathological factors (Kamby, 1990); these include the size of the primary tumor, the histological grade, and, most importantly, the involvement of the draining lymph nodes of the tumor (Rudland et al., 2000).
S100A4, a member of the S100 family of proteins, is directly involved in driving the progression of metastatic disease. S100A4 protein levels are elevated in malignant forms of breast, gallbladder, pancreatic, prostate, esophageal, gastric, lung, and thyroid tumors (Garrett et al., 2006). In humans, there are now over 20 S100 family members that are distributed tissue specifically (Zimmer et al., 1995). The first family members were discovered in the brain and given the name S100 because they are soluble in 100% saturated ammonium sulfate (Zimmer et al., 1995).
Furthermore, nonmetastatic cancer cells engineered to overexpress S100A4 produce a high incidence of tumors with a shorter latency period and higher frequency of metastasis (Grigorian et al., 1996).
More recent efforts have focused on the prognostic significance of S100A4 expression and its use as a marker for metastasis and poor patient outcome (Garrett et al., 2008).
The S100A4 gene was cloned independently by several groups under various names, including metastasin (Mts1), fibroblast-specific protein (FSP1), 18A2, pEL98, p9Ka, 42A, CAPL, and calvasculin (Watanabe et al., 1992).
Initial cloning efforts identified S100A4 as a highly expressed transcript in growth-stimulated cultured cells (Goto et al., 1988) and metastatic tumor cell lines (Ebralidze et al., 1989). In addition, S100A4 expression levels are upregulated during oncogenic transformation (Takenaga et al., 1994).
Immunohistochemical analysis of rat tissues shows that S100A4 is expressed in some absorptive and keratinized epithelia, in the parietal cells of the stomach, and in a subset of cells of the immune system in the blood, bone marrow, spleen, and lymph nodes (Gibbs et al., 1995). (Similar studies examining S100A4 expression during mouse development show that S100A4 is highly expressed in embryonic macrophages as well as in differentiating mesenchymal tissues; Klingelhöfer et al., 1997.) S100A4 expression is also observed in metastatic cancers. Immunohistochemical analyses of human cancers show significant S100A4 expression in breast, pancreatic, prostate, gallbladder, esophageal, gastric, lung, and thyroid carcinomas (Ito et al., 2004).
S100 proteins are typically homodimers that contain N-terminal and C-terminal EF-hands connected by a loop or hinge region (Garrett et al., 2006).
S100A4 specifically binds to the C-terminal end of the coiled-coil of myosin-IIA in a Ca2+-dependent manner (Li et al., 2003). Studies show that Ca2+-activated S100A4 promotes the monomeric, unassembled state of myosin-IIA by depolymerizing preassembled filaments and inhibiting the assembly of myosin-IIA monomers into filaments (Li et al., 2003).
Myosin-IIA localizes to the lamellae of migrating cells (Betapudi et al., 2006). Localization studies of S100A4 show that it is diffuse throughout the cytoplasm and enriched at sites of protrusion along the leading edge (Li and Bresnick, 2006). Recent studies show that S100A4-expressing cells become highly polarized upon chemotactic stimulation by extending enhanced forward protrusions and suppressing side protrusions (Li and Bresnick, 2006). Furthermore, the ability of S100A4 to promote directional motility is an immediate consequence of its interaction with myosin-IIA (Li and Bresnick, 2006). These observations suggest that S100A4 expression may induce a metastatic phenotype through the regulation of myosin-IIA assembly and cell motility (Garrett et al., 2008).
Germline mutations of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene located on chromosome 10q23 are the cause of Cowden disease, which is characterized by the development of hamartomas and other benign tumors, and a higher risk of developing breast and thyroid cancers later in their life (Heering et al., 2009). PTEN is a dual-specificity phosphatase active against phosphorylated peptides and phospholipids. PTEN is capable of dephosphorylating serine, threonine, and tyrosine residues as well as phosphatidylinositols phosphorylated in the D3 position (Zimmer et al., 1995; Grigorian et al., 1996).
PTEN negatively regulates AKT/PI3K (phosphatidylinositol 3-kinase) signaling, one of the most important pathways for cell survival and inhibition of apoptosis (Chu et al., 2004).
Genetic alteration of both PTEN alleles occurs in almost all types of human cancers examined, with inactivation usually because of a mutation accompanied by loss of heterozygosity. In breast tumorigenesis, loss of PTEN is associated with a poor outcome in patients with cancer (Salmena et al., 2008).
Complete loss of PTEN in mice is lethal early in development. Heterozygous mice are viable, however, and adults eventually develop loss of heterozygosity of the remaining PTEN allele, leading to the appearance of tumors in the endometrium, liver, prostate, gastrointestinal tract, thyroid, and thymus, including breast cancer. Conditional deletion of the PTEN gene in the mammary gland caused excessive ductal branching, precocious lobuloalveolar development, and delayed involution. By 10 months of age, 50% of these mice developed breast tumors (Li et al., 2002).
Currently, little is known about the relationship among expressions of PTEN and S100A4 proteins, as well as their role in tumorigenesis and outcome in breast cancer. The purpose of this study was to investigate the relationship between S100A4 and PTEN gene and their prognostic effect in infiltrating breast carcinoma.
Materials and methods
Formalin-fixed and paraffin-embedded tissue blocks from 55 cases of surgically resected breast cancers were obtained from the Pathology Department, Tanta Medical University; all specimens were re-evaluated by routine H&E stain. None of the patients had received radiotherapy, chemotherapy, or other treatments before operation. Their mean age was 50.72 (range, 40–70) years.
Immunohistochemical staining procedures
Mouse anti-human PTEN monoclonal antibody (ZM-0221, 1 : 50 dilution) was purchased from Dako (USA). S100A4 DAKO antibody (used at a dilution of 1 : 100) was used on 3–4-μm-thick sections of paraffin blocks. A secondary detection system (DAKO Envision) enhanced with a conjugated polymer was used to bind with the primary antibody. DAB chromogen was used for permanent color development and detection under a microscope.
For negative controls, sections were incubated with 0.01 mmol/l PBS (pH 7.4) instead of the primary antibodies.
Evaluation of immunostaining
Clearly brown staining restricted to the cytoplasm or the nucleus was considered as PTEN protein positive; the degree of immunostaining was graded as follows: positive rate of counted cells: 0, positive rate <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, >75%; staining intensity: 1, light brown; 2, brown; and 3, dark brown. Both integrals were multiplied: A score of 0 was defined as negative or loss of expression (−); scores of 1–6, reduced expression (+); and scores of 8–12, positive expression (++)
The S100A4 staining was mainly granular cytoplasmic.
The scoring for cytoplasmic S100A4 immunoreactivity results was subclassified as follows:
0, negative staining.
Tumors with more than 10% stained cells were defined as S100A4 positive and were scored according to the intensity of the immunostaining as follows:
1+, weak staining.
2+, moderate staining.
3+, strong staining.
SPSS 12.0 software (USA) was used to analyze all data. Statistical evaluation was carried out using the χ2-test and the Kendall test. A P value less than 0.05 was considered statistically significant.
Thirty of studied patients (54.5%) were older than 55 years, whereas 16 (29%) were between 45 and 55 years and nine (16.5%) were younger than 45 years. All were studied histopathologically and for PTEN and S100A4A immunoexpression.
Histopathologic results of cases of breast carcinoma
All the cases studied were of IDC NOS type, showing masses and cords of malignant cells surrounded by a desmoplastic reaction and lymphocytic infiltration.
According to the Nottingham grading scheme for breast carcinoma using degrees of glandular differentiation (degree of gland/tubule formation), nuclear pleomorphism, and mitotic activity, the breast cancer cases were classified as follows: five cases (9%) as grade I, 20 cases (36.5%) as grade II (Fig. 1), and 30 cases (54.5%) as grade III.
According to the size of the tumor, it was classified as follows: 10 cases (18.1%) were less than 2 cm, 40 (72.7%) were between 2 and 5 cm, and five (9.1%) were greater than 5 cm.
According to lymph node metastasis, there were 20 cases (36.4%) with no axillary metastasis, 30 cases (54.5%) with metastasis between one and three lymph nodes, and five cases (9.1%) showing more than four lymph nodes metastasis.
Results of immunohistochemical expression of PTEN in breast carcinoma cases
PTEN expression was detected as cytoplasmic staining in 35 cases (63.6%) of breast carcinoma. Weak cytoplasmic staining for PTEN (1+) was detected in 10 cases (18.2%) (Fig. 2), moderate cytoplasmic staining (2+) was detected in five cases (9%) (Fig. 3), and strong cytoplasmic staining (3+) was detected in 20 cases (36.4%) (Fig. 4). Twenty cases (36.4%) were negative to PTEN staining.
Relation between PETN expression and clinicopathologic parameters of breast carcinoma
There were significantly negative correlations between the level of PTEN expression and histological grade and axillary lymph node status (P<0.05), but PTEN expression was not correlated with patients’ age or tumor size (P>0.05). Loss of PTEN expression in histological grade III was higher than that in grades I and II (P<0.05). Loss of PTEN expression in the axillary lymph node metastatic group was higher than that in the nonmetastatic group (P<0.05). Loss of PTEN expression in tumor size greater than 5 cm was higher than that in tumor size less than 2 cm, and tumor size ranged from 2 to 5 cm, but did not reach significance (P>0.05) (Table 1).
PTEN expression showed a significant positive correlation with estrogen receptor (ER) and progesterone receptor (PR) expression of patients with breast cancer as PTEN was expressed in all cases of 3+ expressions of ER and PR.
Results of immunohistochemical expression of S100A4 in breast carcinoma cases
S100A4 expression was detected as cytoplasmic staining in 50 cases (90.9%) of breast carcinoma. Weak cytoplasmic staining for S100A4 (1+) was detected in five cases (9.1%), moderate cytoplasmic staining (2+) was detected in 10 (18.2%) (Fig. 5), and strong cytoplasmic staining (3+) was detected in 30 (54.5%). Five cases (9.1%) showed nuclear staining (Fig. 6), whereas five were negative to S100A4.
In normal breast tissue adjacent to the tumor S100A4 protein expression was detected in a variety of different cell types. The cytoplasm of smooth muscle cells of the vessel walls, lymphocytes, macrophages, and the intralobular connective tissue were consistently positive, whereas the interlobular connective tissue showed no staining for S100A4. Epithelial cells of the ducts and ductules occasionally showed positive staining, although the majority of these were not immunoreactive. The staining of normal tissue was not assessed further, although it was observed that tumors expressing high amounts of S100A4 protein also showed stronger immunoreactivity in the corresponding normal tissue.
Relation between S100A4 expression and clinicopathologic parameters of breast carcinoma
There were significantly positive correlations between the level of S100A4 expression and patients’ age and tumor size (all P<0.05), but S100A4 expression was not correlated with histologic grade or lymph node metastasis (P>0.05). S100A4 expression in all cases with tumor size greater than 5 cm or tumor size ranging between 2 and 5 cm was positive, whereas S100A4 expression was negative in all cases with tumor size less than 2 cm (P<0.05). S100A4 expression in histological grade III was higher than that in grades I and II, but did not reach significance (P>0.05). S100A4 expression in the axillary lymph node metastatic group was higher than that in the nonmetastatic group, but also did not reach significance (Table 2).
S100A4 expression showed a significant negative correlation with ER and PR expression of breast cancer as S100A4 was expressed strongly in all cases that showed negative expression of ER and PR immunostaining (P<0.05). All cases that expressed nuclear S100A4 were found to show ER and PR positivity.
Relation between PTEN expression and S100A4 proteins in breast cancer
The expression of PTEN protein in breast cancer showed a significantly negative correlation with the expression of S100A4 (P<0.05). Along with reduced or negative expression of PTEN protein, moderate or strong expression of S100A4 was shown. Nuclear expression of S100A4 was significantly correlated with PTEN expression. All cases that showed nuclear S100A4 were also found to express PTEN (Table 3).
In the current study, we examined the expression of S100A4 and PTEN and correlated their expression with other prognostic factors to determine their prognostic value.
We examined by immunohistochemistry the protein expression of S100A4, a metastasis-associated protein, in human breast cancer specimens. In addition, the expression of PTEN was investigated and the protein levels of these markers were related to clinicopathological variables.
The expression of S100A4 was detected as diffuse cytoplasmic staining in 50 cases (90.9%) and nuclear expression was detected in five (9.1%), whereas five cases (9.1%) of breast carcinoma were negative for both cytoplasmic and nuclear staining.
High levels of S100A4 were found to be significantly correlated with an increase in the tumor size and loss of both ER and PR. High levels of S100A4 were also found with an increase in tumor grade and axillary lymph node metastasis; however, this association was not statistically significant.
Our results were quite similar to those of Pedersen et al. (2002), who found a significant association between high S100A4 protein expression and histological grade III (P=0.030), as well as an inverse correlation to expression of ER (P=0.046). These findings indicate a relation between S100A4 and an aggressive phenotype, as both loss of ER and high histological grade are known indicators of aggressive disease (Pedersen et al., 2002).
The observed inverse correlation between S100A4 and ER is in agreement with previous expression studies carried out on human breast cancer specimens (Albertazzi et al., 1998).
However, Grigorian et al. (1996) examined the role of S100A4 in cancer in breast cancer models, which have shown that overexpression of S100A4 in nonmetastatic mammary tumor cells confers a metastatic phenotype.
Rudland et al. (2000) studied the correlation between the presence of S100A4 and the major pathological tumor variable. In their study they found that S100A4 expression and presence of lymph node metastasis were associated with poor prognosis. However tumor sizes and grades were not associated with bad prognosis.
The development of cancer requires a variety of different mutations to oncogenes and tumor suppressor genes, finally leading to the transformed phenotype. The importance of PTEN as a tumor suppressor in mammary carcinogenesis has been implicated by several studies describing the frequent loss of PTEN protein expression in breast tumors (Perren et al., 1999; Depowski et al., 2001).
Of the 55 breast carcinomas in our study, the expression differed widely, but positive cells for PTEN showed a uniform cytoplasmic staining pattern. Thirty-five cases (63.6%) were scored as positive and 20 (36.4%) as negative.
In the current study, we found that PTEN expression level was significantly negative correlated with histological grade and axillary lymph node metastasis. PTEN expression level was significantly positively correlated with the level of both ER and PR. However, no significant association was found between PTEN expression and tumor size.
Our results were similar to those of Yang et al. (2008), who found that PTEN expression level was significantly negatively correlated with TNM stage, histological grade, axillary lymph node status, recurrence, and metastasis (P<0.05), which shows that downregulated expression of PTEN is implicated in progression of breast cancer and can act as an objective and effective marker to reflect the pathobiological behavior of breast cancer.
Pourmand et al. (2007) reported that the prognosis of patients with positive expression of PTEN was better. On multivariate analysis, although PTEN did not act as an absolute prognostic factor, combined with related reports, mutation of the PTEN gene occurred in the advanced stage of the tumor and the low expression was related to a poor prognosis. Therefore, the expression of PTEN protein can probably act as a significant prognostic factor in breast cancer.
Shen et al. (2007) evaluated PTEN expressions in breast tumor cells by immunohistochemistry. They found a significant correlation between the loss of expression of these proteins and higher tumor grades.
Other studies have also reported reduced PTEN expression in invasive breast cancer. Perren et al. (1999) reported an incidence of 33%, whereas Depowski et al. (2001) reported loss of PTEN expression in 48% of their invasive breast cancer cases.
In addition, Zhu et al. (2007) found that the loss of PTEN expression was correlated significantly with micrometastasis detected in sentinel lymph node using both H&E and IHC methods.
Bose et al. (2002) showed that reduced expression of PTEN correlated with the highest stage.
S100A4 protein expression appears to be expressed widely in early and advanced stages. This study indicates a complex role of S100A4 in breast cancer of different stages. The difference in the expression of S100A4 protein suggests that it may be useful as an independent marker of breast cancer.
The expression of PTEN protein could probably act as a significant prognostic factor in breast cancer.
The combined detection of PTEN and S100A4 with ER and PR expression may serve as an important index to estimate the pathobiological behavior and prognosis of breast cancer.
Conflicts of interest
There are no conflicts of interest.
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