Background
Sebaceous cell carcinoma (SeCC) of the eyelid is a highly aggressive malignant tumor that arises from the meibomian glands, Zeiss glands of the eyelid, or sebaceous glands of the caruncle [
1]. Sebaceous cell carcinoma is more frequent in Asians than in Caucasians. In the West it accounts for only 0.2–4.7 % of all eyelid malignancies [
2] whereas in Asia it accounts for 11–33 %. The prevalence of SeCC is 8–23 % in Taiwan. Sebaceous cell carcinoma is notorious for masquerading as other benign and malignant lesions, often resulting in delayed diagnosis. In addition, diffuse epithelial involvement (pagetoid growth pattern) is present in about half of the cases. Therefore, it is usually associated with a high risk of recurrence and metastatic diseases. Other factors associated with a poor prognosis include vascular, lymphatic or orbital invasion, involvement of both upper and lower eyelids, poor differentiation, multi-centric origin, highly infiltrative pattern, and tumor size more than 2 cm. Etiological risk factors of SeCC include radiation exposure, advanced age, and a genetic predisposition to Muir–Torre syndrome, which includes sebaceous adenoma and sebaceous carcinoma [
2‐
5].
Unfortunately, the prognosis of SeCC is unpredictable. To date, there are no specific reliable markers available for predicting the prognosis of the patients. The retinoids have been reported to affect the growth and differentiation of epithelial tissue and to play an important role in essential biologic processes, differentiation, proliferation, and apoptosis [
6‐
10]. Furthermore, retinoids, whose biological functions are mainly mediated by retinoid receptors, have been used as a therapy for some non-melanoma skin cancers [
11]. The cellular retinoic acid-binding proteins (CRABPs) may regulate the accessibility of retinoic acid (RA) to the RA receptors and are thought to affect the prognosis of cancer. In particular, CRABP1 is expressed in hair follicles of normal skin (dermal papilla) and in the stroma of epidermal tumors. A member of the fatty acid-binding protein (FABP) family, FABP5, is found abundantly in skin epidermal cells, adipocytes, macrophages, liver, heart, sebaceous glands, and anagen follicle bulbs [
11,
12]. Previous experiments indicate that CRABP2 and FABP5 are abundantly expressed in the differentiating cells of sebaceous glands, interfollicular epidermis, and hair follicles [
11]. Therefore, combinations of RA receptors and CRABPs may be an important factor in mediating the effects of RA on transcription and cellular processes. Moreover, the dynamic patterns of expression of CRABPs also reflect cross talk of RA and Wnt/β-catenin signaling in different developmental and homeostatic situations [
11,
13‐
17]. Aberrant expression of β-catenin has been reported in different tumors such as colorectal, hepatocellular, breast carcinoma, oral squamous cell carcinoma, and non-melanoma skin tumors [
18]. The RA receptors are nuclear receptors related to the steroid and thyroid hormone receptors. So far, two classes of nuclear retinoid receptors (RARs and RXRs) have been reported, and each has three main subtypes, −α, −β, and -γ. They play a pivotal role as ligand-dependent transcription factors. RA receptors act in heterodimeric combinations with retinoid X receptors (RXRs) and facilitate DNA binding of the RAR–RXR complex [
19]. RA signaling is mediated by RA binding to RARs, which form heterodimers with RXRs, and is regulated by RA-binding proteins [
19,
20]. Retinoids, that are Vitamin A derivatives, as ligands for binding to these nuclear receptor transcription factors (RARs and RXRs), are strongly associated with the development of skin cancer and its subsequent prognosis.
Therefore, we aimed to investigate the role of RA signaling pathway in the pathogenesis of SeCC. In this study, we retrospectively analyzed the variation in the immunohistochemical expression of related signaling pathway binding proteins between SeCC cases and normal control cases, and tried to explore the role of RA pathway in the pathogenesis of SeCC.
Discussion
In this study, we present the immunohistochemical expression of β-catenin, RA signaling molecules (CRABP1, CRABP2, FABP5), and related retinoic acid receptors (RARs and RXRs) in SeCC cases. Multiple predisposing factors have been proposed, such as radiation exposure, advanced age, race, and genetic predisposition [
3‐
5]. However, the exact pathoetiology of SeCC remains unknown. As there are no available specific markers for predicting prognosis, the course of SeCC is difficult to predict. Collins and Watt proposed that dynamic patterns of CRABP expression reflect cross talk of RA and W
nt/β-catenin signaling in different developmental and homeostatic situations of skin [
11]. Zhang
, Liu and co-workers proposed that conditional expression of a murine C
tnnb1 gene gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN) [
20]. In particular, human OSSN patients exhibited nuclear translocation of β-catenin. These results indicated that β-catenin activation might have an important role in tumorigenesis, resulting in oncogenic transformation. Sen and colleagues showed that cytoplasmic overexpression of β-catenin found in the majority of cases of SeCC (66 %) of eyelid which was significantly related to tumor size [
21]. Therefore, they proposed that β-catenin overexpression in SeCC may be due to dysregulation of the W
nt/β-catenin pathway [
21]. However, its role in the pathoetiology and prognosis of sebaceous cell carcinoma was essential to be explored further. In this study, we also observed a significant increase in the level of β-catenin protein in SeCC cases (
n = 13/16, 81 %). These results indicated that the oncogenic potential of the W
nt/β-catenin transduction pathway was related to the development of SeCC. However, specific β-catenin labeling was not observed in the nuclei of SeCC cells. It suggests that the mechanism of SeCC tumorigenesis was much different from that of human OSSN.
In addition to overexpression of β-catenin in our SeCC cases, we also demonstrated the expression of CRABPs and related RA receptors in SeCC cases. CRABPs binded all-trans-RA intracellularly and might be involved in the transfer process of RA into the cell nucleus. CRABP1 played a role in presenting RA to metabolizing (CYP26) enzymes, and CRABP2 played a role in transfer of RA to nuclear RARs by direct protein–protein interactions. In our data, the CRABP1 protein was not expressed strongly in the tumor cells of SeCC cases, whereas the CRABP2 protein was overexpressed in the tumor cells of SeCC cases. These results indicated that CRABP2 protein might play an important role in the pathoetiology of sebaceous cell carcinoma.
The FABPs belonged to a group of intracellular lipid chaperones that bind fatty acids, retinoids, and hydrophobic compounds, and mediate their biological functions [
22]. In particular, FABP5 was the only one of the family to bind retinoic acid [
12]. Therefore, CRABP1, CRABP2, and FABP5 were retinoid-binding proteins expressed in mammalian skin and appendages that were known to regulate RA signaling [
11,
23]. In our data, the FABP5 protein was also aberrant expressed in some tumor cells of SeCC cases. Collins and Watt reported that CRABP1, CRABP2, and FABP5 proteins were dynamically expressed during skin development and in adult tissue [
11]. Their findings demonstrated that there was dynamic regulation of RA signaling in different regions of the skin, and provided evidence for interactions between the RA, β-catenin, and Notch pathways [
11]. Furthermore, they found that the CRABP1, CRABP2, and FABP5 proteins were overexpressed in both benign papillomas and malignant squamous cell carcinomas (SCCs) [
11]. In particular, CRABP1 was expressed in the tumor stroma, and CRABP2 and FABP5 were expressed in the sebaceous gland cells, interfollicular epidermis, and hair follicles. Our results supported those molecules were also upregulated in the tumor cells of sebaceous cell carcinoma. In this study, we observed a significant increase in the level of expression of CRABP2 and FABP5 in SeCC cases compared with controls. CRABP2 and FABP5 were expressed in hair follicles of eyelid skin in both groups, whereas CRABP2 and FABP5 were aberrantly expressed in the SeCC tumor cells. In addition, we found CRABP2 and FABP5 were aberrantly expressed in severe SeCC patients and these results may be related to distant metastasis and poor prognosis (Case 16, Fig.
1d).
In 10 cases of sebaceous cell carcinoma, Chakravarti and co-workers found that three cases had decreased RAR-α expression (absent in three cases); six cases had increased RAR-β expression; expression was absent in four cases, and two cases had decreased RAR-γ expression compared with controls [
24]. Moreover, RXR-α expression was decreased, RXR-β expression was low in seven tumors, and RXR-γ expression was absent in six tumors. They concluded that aberrant expression of retinoid receptors in sebaceous cell carcinoma of the eyelid might play a role in the pathogenesis and progression of this carcinoma [
24]. In our study, RAR-β, RXR-β, and RXR-γ were predominantly expressed in SeCC cases, whereas RAR-α, RAR-γ, RXR-α were presented at lower levels in SeCC cases with no significant difference as compared with control cases. Further studies were needed to investigate the association of dysregulation of retinoic acid receptors and the prognosis of sebaceous cell carcinoma.
Acknowledgements
We thank Ya-lan Jian and Kang-han Liao for helping experiments at the Linko Chang-Gung Memorial Hospital.
Supported in part by grants from Chang Medical Research Project G CMRPG3B0671 (YJT), CMRPG3A1293 (LKY), CMRPG3C1751 (LKY), CMRPG3E1521(LKY) and National Science Council Grants (Taiwan) 1012314B182A056MY3 (LKY), NSC grant 1042314B182A097MY3(LKY) and NSC grant 1012320B255003MY3(CYC)
Competing interests
The authors declare that they have no competing interests. No conflicting financial interest. The authors have no additional financial interests.
Authors’ contributions
Conceived and designed the experiments: LKY. Performed the experiments: LKY, YJT. Analyzed the data: HYH, DHKM, NKW, CHH, CYC. Wrote the paper: YJT, SYW. All authors read and approved the final manuscript.