Tumor-initiating cells (T-ICs) discovered in various tumors have been widely reported. However, T-IC populations in salivary gland tumors have yet to be elucidated. Using the established Pleomorphic Adenoma Gene-1 (Plag1) transgenic mouse model of a salivary gland tumor, we identified CD44high (CD44hi) tumor cells, characterized by high levels of CD44 cell surface expression, as the T-ICs for pleomorphic adenomas. These CD44hi tumor cells incorporated 5-bromo-2-deoxyuridine (BrdU), at a lower rate than their CD44negative (CD44neg) counterparts, and also retained BrdU for a long period of time. Cell surface maker analysis revealed that 25% of the CD44hi tumor cells co-express other cancer stem cell markers such as CD133 and CD117. As few as 500 CD44hi tumor cells were sufficient to initiate pleomorphic adenomas in one third of the wildtype mice, whereas more than 1×104 CD44neg cells were needed for the same purpose. In NIH 3T3 cells, Plag1 was capable of activating the gene transcription of Egr1, a known upregulator for CD44. Furthermore, deletion of sequence 81–96 in the Egr1 promoter region abolished the effect of Plag1 on Egr1 upregulation. Our results establish the existence of T-ICs in murine salivary gland tumors, and suggest a potential molecular mechanism for CD44 upregulation.

Over-expression of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1) plays a crucial role in the formation of pleomorphic adenoma, which is the most common type of salivary gland tumor. To understand the molecular mechanisms governing PLAG1-mediated tumorigenesis, we used a microarray-based approach to identify PLAG1 target genes. We validated the expression of several genes, including Bax, Fas, p53, p21, p16, Cyclin D1, Egfr, Trail-R/DR5, c-Fos, c-myc and Igf2, by real-time RT-PCR or western blotting. Using luciferase reporter gene assays, we determined that the promoters of Bax, Fas, p53, TRAIL-R/DR5, and c-Fos were transactivated by PLAG1. PLAG1 not only activates genes that promote cell proliferation and tumor formation but also genes that inhibit these cellular processes. Therefore, we conclude that PLAG1 may play a dual role in tumor formation.

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