In this study, we demonstrated that overexpression of stathmin promoted tumor progression and was regulated by mutp53 in OSCC. Overexpression of stathmin has been detected in several malignancies. Stathmin overexpression in hepatoma promoted local invasion, polyploidy formation, early recurrence and poor prognosis, suggesting that stathmin can be an effective therapeutic target [
11]. Proteins from pooled microdissected nasopharyngeal carcinoma and adjacent non-tumorigenic nasopharyngeal epithelial tissues were separated by 2-DE to find significant overexpression of stathmin in the tumors [
17]. Stathmin knockdown by siRNA in melanoma cells drastically repressed cell proliferation and migration, whereas ectopic expression of stathmin increased cell proliferation and migration [
18], consistent with our results. From the reports in various carcinomas, overexpression of stathmin was very consistent in several tumors and acted as a potential oncogene.
Recently, possible mechanisms that may underlie the multiple functions of stathmin in cancer have attracted much attention. In our study, cleaved PARP (the 89-kDa cleaved product) was increased after silencing stathmin, which indicated the pro-apoptotic effect of stathmin knockdown in OSCC. Furthermore, bcl-2 and cyclin D1 increased after ectopic expression of stathmin. In agreement with these results, silencing of stathmin resulted in decreased expression of bcl-2 and survivin proteins and activation of caspase-3 and caspase-9 [
38,
39].
Cdc2, also known as cyclin-dependent kinase 1, mainly regulates the progression of the G2/M phase. A conserved tyrosine (Tyr15 in humans) leads to inhibition of cdc2; its phosphorylation is thought to alter ATP orientation, preventing efficient kinase activity. Activation of cdc2 (try15) blocked cells at the G2/M phase which then induced apoptosis. In contrast, cyclin B1, the partner of cdc2, was unaltered in our study. It has also been reported that expression of p53 and p21 increased after silencing stathmin in gallbladder carcinoma [
40]. In our study, we confirmed that upregulation of p-cdc2 (try15) after stathmin silencing arrested the cell cycle and induced apoptosis in OSCC. The results suggest that the downstream signaling molecules of stathmin differ in various cancers.
Overexpression of stathmin was transcriptionally activated by mutp53 but not by wtp53 in OSCC in our study. In over 70% of cases, the TP53 mutations are missense, most frequently within the region of DNA binding. Although the spectrum of the TP53 missense mutations is vast – counting about 1800 different amino-acid changes– several hotspots in p53 mutants, particularly affecting residues R273, R248, R175 and G245, have been reported to be present with a higher frequency in head and neck cancer [
41]. Cells expressing mutp53 exhibited aggressive cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures [
42]. This was confirmed in SCC25 cells after transduction with mutp53 plasmids. Stathmin depletion caused a large percentage of the apoptosis occurring in both normal and cancer cell lines lacking p53 [
43], while significant inhibition of proliferation was also observed in OSCC cell lines with mutp53. This suggests that stathmin depletion could be used therapeutically to induce apoptosis in tumors with or without p53 expression. In our study, three p53 mutants (R175H, G245C and R282W) were constructed into one plasmid to mimic the complicated mutations of p53 in OSCC. These three mutants contributed to the mutp53 gain of oncogenic function to promote invasive growth of head and neck cancer cells via inhibition of AMPK activation [
37]. Upregulation of stathmin was also shown to be mediated by GOF mutations in p53 in human hepatoma [
21], as well as in breast cancer cell lines harboring mutp53 [
39]. The results of our study are in accord with the above reports. In type II high grade epithelial ovarian carcinomas, stathmin favored the binding and the phosphorylation of mutp53 by DNA-PKCS, eventually modulating mutp53 stability and transcriptional activity [
44]. It was speculated that a positive feedback loop between mutp53 and stathmin synergistically promoted the progression of malignant tumors. However, wtp53 has also been reported to downregulate stathmin expression [
20,
45]. This discrepancy may be related to separate molecular driving mechanisms in different tumors, but our results nonetheless provide new insight into the interaction between mutp53 and stathmin overexpression in OSCC, which extends the network driving mutp53. The transcriptional regulation of STMN1 by p53 varies in different carcinomas, which deepens our understanding of GOF p53-driven tumors. Our results confirm that stathmin is a novel target of mutp53 and jointly promotes tumorigenesis and tumor progression.