Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation.

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis-Sox9, Sox6, and Sox5-were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen-cartilage-specific ECM substances-were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.

Lymphatic vessels and related factors in adenoid cystic carcinoma of the salivary gland.

Adenoid cystic carcinoma of the salivary gland preferentially metastasizes to distant organs. It rarely metastasizes to lymph nodes. Recently, lymphangiogenesis has been associated with lymph node metastasis. Therefore, lymphangiogenesis in adenoid cystic carcinoma was evaluated from the number of lymphatic vessels and the expression of lymphangiogenic factors. Immunohistochemistry and molecular analysis were performed on clinical materials (29 cases for immunohistochemistry and 9 cases for molecular analysis). Normal submandibular gland was used as a negative control of lymphangiogenesis (10 cases for immunohistochemistry and 5 cases for molecular analysis). In adenoid cystic carcinoma, podoplanin-positive lymphatic vessels were small and often constricted, and localized to the tumor periphery. They did not have Ki67-positive endothelial cells. The lymphatic vessel density of the tumor did not exceed that of the salivary gland. By reverse transcriptase-polymerase chain reaction, adenoid cystic carcinoma and the salivary gland expressed vascular endothelial growth factor receptor-3 (VEGFR-3) similarly but VEGF-C and VEGF-D differently. Adenoid cystic carcinoma expressed VEGF-C, whereas the salivary gland expressed both VEGF-C and VEGF-D. VEGF-C was weak in adenoid cystic carcinoma and strong in the salivary gland. Real-time reverse transcriptase-polymerase chain reaction of VEGF-C showed that the ratio of the tumor to the salivary gland was 1 to 30 (P<0.01). Immunohistochemistry barely detected VEGF-C in adenoid cystic carcinoma. VEGF-C was expressed faintly by the tumor cells. VEGF-C and VEGF-D were detected in the serous acinar and duct cells and in the duct contents in the salivary gland. VEGFR-3 appeared to be expressed by lymphatic vessels in both adenoid cystic carcinoma and the salivary gland. These results indicate that lymphangiogenesis does not occur in adenoid cystic carcinoma. This condition would lead to the uncommon lymphatic metastasis.