Metastasis remains the main cause of cancer mortalities , stressing the need to understand the cellular and molecular mechanisms that regulate this process. It is a complex process where cytoskeletal proteins were reported to regulate multiple cellular processes including morphological changes and motility, which are critical steps for metastasis .
Fascin is an actin-bundling motility-associated protein that plays an important role in the assembly of cell-motility structures . Therefore, it is possible that fascin expression in cancer cells may lead to a more clinically aggressive course through augmented cell motility and enhanced metastatic potential, a finding supported by in vitro observations . Most of studies have shown that fascin expression is correlated with the clinical aggressiveness of tumors and with poor patient survival. In studies of breast cancer, some researchers have shown that fascin expression increases in more advanced tumors , but the results of other studies have not agreed with those findings .
In the current study, 43.28% of invasive ductal breast carcinomas showed positive fascin expression. However, in the adjacent normal breast tissue, fascin expression was observed in myoepithelial cells and luminal cells of few ducts and acini. These results are consistent with previous studies showing striking up-regulation of fascin in a variety of malignancies including breast carcinoma [7–10, 17], while normal epithelia exhibited very low levels of expression [14, 16]. In agreement with Onodera et al. , we found positive fascin expression in DCIS of 25% of invasive ductal carcinomas with in situ component. Only few studies, investigating fascin expression in breast cancer tissues, have been conducted showing different rates of expression. Grothey et al.  found that 70% of estrogen receptor negative and 50% of progesterone receptor negative breast cancer were positive for fascin expression. Yoder et al.  observed positive fascin immunoreactivity in 16% of the invasive breast carcinomas. Rodriguez-Pinilla et al.  detected fascin expression in 25.1% of sporadic invasive breast carcinomas and in 83.3% and 16.7% BRCA1- and BRCA2-associated carcinomas, respectively. Al Alwan et al.  demonstrated fascin in the tumor cells of 40.84% of invasive ductal breast carcinoma patients.
Heterogeneity in the results was apparent within breast carcinoma studies. This could be due to difference in the commercial company supplying the primary antibody, and the method of immunohistochemical staining. Furthermore, it should be noted that each study included different histological types of breast carcinomas. Grothey et al.  investigated hormone receptor -negative breast cancer. Yoder et al.  studied primary node-positive and node-negative invasive breast carcinomas, which included infiltrating ductal carcinomas, infiltrating lobular carcinomas, mucinous carcinomas and medullary carcinomas. Rodriguez-Pinilla et al.  investigated node-negative sporadic and hereditary invasive breast carcinomas. Al-Alwan et al.  as well as our current research studied invasive ductal carcinoma only.
In our work, we found a significant relationship between fascin expression and negative prognostic factors as lymph node metastases and advanced tumor stage. Concomitant with these results, Al Alwan et al.  found a significant correlation between fascin expression and lymph nodal metastases and Yoder et al.  reported a significant association between fascin expression and advanced tumor stage. However, Yoder et al.  as well as Grothey et al.  did not find any significant relationship between fascin expression and lymph nodal status. Moreover, the later  did not find any correlation with advanced tumor stage.
The present research confirms a statistically significant inverse relationship between fascin expression and estrogen receptor and progesterone receptor status. These observations are in line with previous studies [14–17]. Hormone receptor–negative breast cancers traditionally have a worse prognosis and fewer available treatment options (ineffectiveness of hormonal therapy) compared with hormone receptor–positive tumors [34–36]. It is interesting that hormone receptor–negative breast cancers also display increased cell motility in vitro [37, 38]. In a study examining the ability of breast cancer cell lines to penetrate into a collagen-fibroblast matrix, cells expressing mRNA for estrogen receptor showed a noninvasive phenotype, whereas cells lacking estrogen receptor mRNA were shown to be highly invasive . Yoder et al.  suggested a connection between the expression of fascin and the absence of hormone receptors, increased cell motility, and decreased survival in human breast cancers. It is conceivable that fascin may serve as a downstream cytoskeletal effector contributing to the more aggressive/malignant phenotype of hormone receptor -negative breast cancer.
Interestingly, no association was identified in the present study between fascin expression and patients’ age or tumor size. These observations are in accordance with Yoder et al.  and Rodriguez et al. . However, in a recent study, fascin correlated significantly with tumor size .
There was no significant relationship between fascin immunoreactivity and tumor grade. A similar absence of correlation has also been noted by Grothey et al.  and Al- Alwan et al.  although Yoder et al.  and Rodriguez et al.  reported a significant positive association between fascin expression and tumor grade.
In vitro, studies revealed that fascin exhibits highly increased levels in breast cancer cell lines over-expressing the receptor tyrosine kinase and prognostic indicator c-erbB-2/HER-2, and that such cells exhibit dramatically increased cell dynamics and in vitro motility . The data presented in our research failed to reveal any association between fascin and HER2 status in tissue samples concomitant with Yoder et al. , Rodriguez et al.  and Al- Alwan et al. . This could be due to limited population size or an institutional bias. Alternatively, the forced overexpression of this receptor in cell cultures by transfection may represent an artificial system, which may not well reflect the biological complexity of HER2 gene amplification and protein overexpression occurring in vivo.
By using siRNA technology, Xie et al.  have successfully silenced fascin gene in EC109 cells, an esophageal squamous cell carcinoma cell line. They found that decreased level of fascin correlated with decreased formation of surface protrusions that play essential roles in cell motility. Besides the decreased formation of protrusions, Xie et al.  suggested another possible mechanism for fascin effect on cell invasiveness, as shown in the gelatin zymography, which was the decreased activity of extracellular matrix proteases as MMP-2 and MMP-9. Such proteases digest collagen type IV and other components of the basement membrane and play a key role in local invasiveness and the formation of distant metastases by malignant tumors . Xie et al.  postulated that the effects of fascin on cell invasiveness involve both changes in cell motility as well as the activity of matrix proteases. Furthermore, Onodera et al.  showed that fascin was responsible for the overproduction of MMP-9 in cholangiocarcinoma (CC), raising a possibility that fascin relates not only to increased cell motility but also to stromal degradation during the invasion of CC. In addition, the migratory effect of fascin-1 on hepatocellular carcinoma cells led to efficient invasion when assisted with secretory factors from intrinsically highly invasive cells such as MMP-9, which fascin-1 alone could not up regulate. Concomitant with these observations, our study revealed a significant strong agreement between fascin and MMP-9 expressions in breast carcinoma cases. To the best of our knowledge, this is the first study to investigate the relationship between immunohistochemical expression of fascin and MMP-9 in breast cancer.
In the current research more intense expression of fascin and MMP-9 was observed at the invasive fronts compared with other areas of tumor. Moreover, a significant moderate agreement between fascin and MMP-9 was found regarding the site of predominant intensity. These observations are in agreement with Onodera et al. . In addition, Grothey et al.  previously observed that fascin staining is often enhanced at the leading edges of infiltrating tumors, which indicates its role as a pathogenic factor for tumor cell invasion. Onodera et al.  demonstrated that overexpression of MMP-9 induced by TNF-α was also inhibited by fascin siRNA, implying that TNF-α-induced MMP-9 overexpression is mediated by fascin. Interestingly, they found that macrophages positive for TNF-α were commonly observed at the invasive front of cholangiocarcinoma compared with the central part of CC. Such locally released TNF- α from macrophages around the invasive front of CC may be responsible for such overproduction of fascin and then MMP-9. These findings may explain the correlated dense expression pattern of fascin and MMP-9 in breast carcinoma tissues at the invasive fronts, as shown in the current study.
Many previous studies have been conducted to research MMP-9 expression in human cancers, including breast cancer, but the results are still controversial. In the present study, MMP-9 expression was detected in 50.75% of breast carcinoma cases with weak expression in stromal cells. In contrast, the adjacent normal breast tissue did not express MMP-9. These results are consistent with Scorilas et al.  who observed MMP-9 staining primarily in cancer cells, and to a lesser degree in surrounding stromal cells but not in normal breast tissue. However, Pellikainen et al.  observed MMP-9 in both tumor cells and stromal fibroblasts and inflammatory cells. In addition, benign breast epithelium and vascular endothelium stained positively for MMP-9.
In the current research, 37.5% of invasive ductal carcinomas with adjacent in situ component revealed positive MMP-9 expression in the in situ carcinoma lesions. This result is in agreement with Kim et al.  who found MMP-9 mRNA expression in 50% of DCIS and 44% of invasive ductal carcinoma cases. They added that MMP mRNA expression levels suggest that an invasive potential of breast carcinoma is already obtained before morphologically overt invasive growth is observed.
In our study, there was a significant correlation between MMP-9 expression and lymph node metastases, advanced tumor stage as well as estrogen receptor negative and progesterone receptor negative hormonal status. Similar to these findings, Fan et al.  observed that MMP-9 overexpression was higher in breast cancers with lymph node metastases than those without lymph node metastases. They added that increased expression of MMP-9 protein was correlated with high TNM classification. Furthermore, Przybylowska et al.  and Slattery et al.  found a significant relationship between MMP-9 and ER-/PR- tumors. In addition, Liu et al.  observed a significant association between basal like breast cancer and MMP-9. In contrast, Zhang et al.  noted that high MMP-9 expression in tumor cells was not associated with any clinicopathological parameters or immunohistochemical expression of ER and PR. Scorilas et al.  found that MMP-9 negative tumors were obtained from patients who were diagnosed with stage III-IV disease and Grieu et al.  observed that MMP-9 21562 polymorphism was associated with ER positive tumors.
In the current research, no statistical association was detected between MMP-9 expression and patients’ age, tumor size, histological grade or HER2 status. Parallel to these results, Scorilas et al.  observed no significant association between MMP-9 and tumor grade and Zhang et al.  found no correlation between MMP-9 and c-erbB2 (HER2). In contrast, Li et al.  detected a significant relationship between positive MMP-9 immunostaining and higher tumor grade. Moreover, Fan et al.  and Przybylowska et al.  found that MMP-9 protein was positively associated with tumor size. The different results between our present study and others might be due to differences in sample size, methods of scoring criteria, and the antibodies used to evaluate expression.
Regarding breast cancer molecular subtypes, in the current research, triple negative breast cancers had the highest rate of fascin and MMP-9 expression. However, luminal breast cancer had the lowest rate of expression. It is well established in the literature that triple negative subtype is an independent prognostic factor of distant metastasis due to its strong invasive ability and metastasis ability . It represents one of the most aggressive phenotype with discrete risk factors and ominous prognostic significance . On the other hand, luminal subtype of breast cancer has better prognosis than other molecular subtypes . Fascin and MMP-9 might be markers of aggressive behaviour in breast cancer.