Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma

Abstract

Objective: Cathepsin D (CTSD) is a pivotal orchestrator in the occurrence and development of tumors. Recently, CTSD was detected in salivary adenoid cystic carcinoma (SACC). However, its functional role in perineural invasion (PNI) of SACC remained elusive. We conducted the present study to detect the expression of CTSD in SACC, analyze the correlation between CTSD expression and prognosis of SACC patients and elucidate the role of CTSD in occurrence of PNI in SACC to lay the foundation for further studies. Methods: Immunohistochemical analysis was conducted to assess CTSD and Ki67 expression in 158 SACC samples and 20 normal salivary gland samples adjacent to carcinoma. Meanwhile, the correlation between CTSD and PNI of SACC specimens was analyzed using Wilcoxon test. QRT-PCR, immunofluorescence and western blot analysis were used to examine the levels of CTSD mRNA and protein in SACC-LM cell line. SiRNA-mediated CTSD silence was performed. Scratch wound healing assay, transwell invasion assay and DRG co-culture assay of PNI was used to detect the ability of migration, invasion and PNI. FITC-phalloidin was used to detect cytoskeletal organization. Results: Our data demonstrated that the positive expression of CTSD was observed in 74.1% (117/158) of SACC cases, and the expression of CTSD was significantly correlated with the PNI (p < 0.05). The ability of migration, invasion, and PNI could be inhibited significantly by siRNA-mediated CTSD silence (p < 0.01). Furthermore, siRNA-mediated CTSD silence inhibited cytoskeletal organization and pseudo foot formation in SACC-LM cells. Conclusion: Our results suggested that an association between PNI and expression of CTSD existed. CTSD may promote PNI of SACC accompanied by cytoskeletal organization and pseudo foot formation.

Keywords: cathepsin D (CTSD); cytoskeletal organization; invasion frontier; perineural invasion (PNI); salivary adenoid cystic carcinoma (SACC).

Figure 1

(A-D) are immunohistochemical staining of CTSD, (×200). (E-H) are immunohistochemical staining of Ki67, (200×). N represents nerve. Normal means normal salivary gland. Non-PNI means non PNI group of SACC. PNI(f) means far away nerve of SACC with PNI group. PNI(i) means nerve invasion frontier of SACC with PNI group.

Figure 2

Kaplan-Meier survival analysis on CTSD negative expression, CTSD weakly positive expression, CTSD moderately positive expression, CTSD strongly positive expression group in the patients with SACC (log-rank test, p < 0.0001).

Figure 3

Expression of CTSD in SACC tissues and SACC-LM cells. (A) immunofluorescence analysis of CTSD expression in SACC tissue and transfection efficiency of siRNA mediated CTSD knockdown in SACC-LM cells (blank control, negative control, and si-CTSD group), (200×). (B) Western blot analyses of CTSD expression in SACC tissue and transfection efficiency of siRNA mediated CTSD knockdown in SACC-LM cells (blank control, negative control, and si-CTSD group). (C) qRT-PCR analyses of CTSD expression in SACC tissue and transfection efficiency of siRNA mediated CTSD knockdown in SACC-LM cells (blank control, negative control, and si-CTSD group), (***p < 0.001).

Figure 4

Effect of CTSD on migration and invasion of SACC-LM cells. (A,B) Scratch wound healing assay showed that CTSD knockdown inhibited the migratory ability of SACC-LM cells, (200×). (C,D) Transwell invasion assay showed that CTSD knockdown inhibited the invasive ability of SACC-LM cells, (200×) (**p < 0.01).

Figure 5

Effect of CTSD on PNI of SACC-LM cells. (A,B) In vitro DRG co-culture assay of PNI showed that CTSD knockdown inhibited the ability of PNI of SACC-LM cells, (40×) (**p < 0.01). DRG represents newborn rat dorsal root ganglia (DRG).

Figure 6

Effect of CTSD on cytoskeletal organization in SACC-LM cells. siRNA-mediated CTSD silence resulted in morphological changes ranging from a migratory, fibroblastoid phenotype toward epithelial plasticity of SACC-LM, (100×). And FITC-phalloidin staining showed that siRNA-mediated CTSD silence led to a collapse in cytoskeletal organization and reduction in pseudo foot formation, (400×).