Hornerin Is Involved in Breast Cancer Progression

The S100 protein family consists of over 20 family members of low molecular weight calcium binding proteins [1]. These proteins share common amino acid sequence domains and a functional EF-hand motif that plays a key role in calcium binding through a helix-loop-helix topology [2, 3].

Hornerin is a member of the S100-fused protein family. It was first identified in the mouse embryo epidermis. It was also detected in the skin, tongue, esophagus, and proximal stomach of adult mouse tissues. Hornerin has a calcium binding EF-hand structure at the N-terminus followed by a spacer sequence and a large repetitive domain [4]. The hornerin protein is similar to profilaggrin (which is involved in cornification of keratinocytes) in its structural features, expression profiles, extensive posttranslational proteolytic processing, and tissue localization [5, 6]. Hornerin has been thought to be involved in pre- and post-natal mammary development in breast tissue [7]. Although the physiological role of S100 proteins has not been clearly identified, recent experimental data have suggested that they are involved in physiological and pathological processes such as gene transcription, inflammatory and immune responses, regulation of protein phosphorylation, transcription factors, antimicrobial responses, calcium homeostasis, dynamics of cytoskeleton constituents and, cell proliferation, differentiation, and death [8]. Differential expression of the S100 family members in certain cancers has been reported [1]. Experiments focusing on their role in mammary carcinogenesis have demonstrated that the expression levels of some S100 family proteins in basal-type breast cancers are higher than that in nonbasal types breast cancers [8, 9]. Upregulated expression of hornerin in less aggressive breast cancers has been reported [8, 9, 10]. S100A8 and S100A9 are known to be associated with high grade and basal-type breast cancer [8, 9, 10]. It is generally accepted that breast cancers arise by a multistep process in which normal epithelial cells transform into invasive cancer via atypical ductal hyperplasia and in situ carcinoma, though it is not necessarily a linear process [11, 12]. However, the roles of hornerin, S100A8, and S100A9 in the progression of mammary carcinogenesis have not been fully evaluated. Therefore, the objective of this study was to determine their expression levels in ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), and metastatic carcinoma in the same patient to clarify their roles in cancer progression.

Previous studies on "fused gene"-type cornified envelop precursor proteins have mainly focused on their physiological and pathologic roles in keratinization [4, 6, 18, 19]. However, recent studies have reported that "fused gene"-type S100 proteins may also be involved in the pathogenesis of neoplasia [10, 20]. The objective of this study was to determine the expression levels of hornerin, S100A8, and S100A9 in DCIS, IDC, and metastatic carcinoma in the same patient to clarify their roles in cancer progression. Our results revealed a positive correlation between hornerin expression and ER-negativity with HER2-amplification, in agreement with the results of an in vitro study that used the MCF10A cancer progression model to show that hornerin mRNA expression increased as tumorigenicity progressed [10]. However, in this study, we confirmed our results with IHC staining using formalin fixed paraffin embedded tissue and found that hornerin expression was correlated with the lobular carcinoma or node-negative group. To demonstrate the changes in expression, we specially constructed TMAs from DCIS, IDC, and metastatic lesion in lymph nodes of the same patients. IHC analyses of TMAs showed that hornerin expression differed in breast tissue according to the progression stages in the same patient, including preinvasive in situ, invasive, and metastatic. In addition, the expression of hornerin dramatically increased in a stepwise manner as breast cancer progressed from DCIS to MCN. These findings strongly suggest that hornerin is involved in breast cancer progression, specifically in the transformation from preinvasive carcinoma to invasive carcinoma. Furthermore, it was remarkable to find that the expression levels of hornerin in IDC were much higher than those in DCIS. Recently, it has been proposed that exosomes are involved in the formation of organ-specific metastatic niches [21]. Therefore, as an exosome protein, hornerin may play an essential role in the invasive process of mammary cancer progression, suggesting that it might be used as a potential marker to predict recurrence or potential metastasis. In this study, a survival analysis for hornerin in breast cancer was not performed. Therefore, further study with survival data is necessary.

S100 proteins are low molecular weight proteins ranging in size from 9 to 13 kDa. Most S100 proteins can form heteroand homo-dimers that might be essential for the generation of their active form [22]. S100A8 and S100A9 proteins usually form a heterodimer called calprotectin that is present in high levels in endothelium, macrophages, and neutrophils [23, 24, 25]. Our study showed a significant correlation between S100A8 and S100A9 expression. This finding is in accordance with a previous study [8]. Since S100A8 and S100A9 proteins have been known to exist as heterodimeric molecules [26], it would not be surprising if their expression was coordinately controlled. In the present study, a positive correlation between S100A8 expression and higher pT stage was demonstrated. However, no association between S100A9 and clinicopathologic parameters with prognostic impact was observed. Some studies have indicated that overexpression of S100A8 and S100A9 is associated with poor prognosis of IDC and lung adenocarcinoma [9, 27]. Another study has suggested an association between hornerin expression and ER-negative or high grade tumors [8].

There are some differences between our study and another study with regard to how protein expression was interpreted [10]. We interpreted it to be positive when we observed more than 10.0% of protein expressed in two TMA cores because any resultant subclone could have a specific biologic behavior due to evolutionary changes among cancer cells. As we used TMA with a unique design, including in situ, invasive, and metastatic lesions from each patient, we only enrolled a small number of cases in this study. Furthermore, we only evaluated 70 of the 94 cases recruited for this study because not all the cores fulfilled the required data.

In this study, the expression of hornerin steadily increased during breast cancer progression from preinvasive lesions to metastatic carcinoma. In addition, hornerin was expressed at higher levels in ER-negative but HER2-positive groups. These findings suggest that hornerin may be involved in mammary cancer progression. Therefore, it might be used as potential marker for poor prognosis in breast cancer management. Recently, it has been proposed that exosome proteins, such as S-100 family members, are involved in the formation premetastatic niches at metastatic sites [21]. The relationship between exosomes and S100 family members in cancer progression is starting to emerge. To clarify the roles of S-100 family members including hornerin in cancer progression, further studies with a large number of cases along with survival data are needed.