Expression and phosphorylation of stathmin correlate with cell migration in esophageal squamous cell carcinoma

Microtubules play extensive roles in cellular processes, including cell motility. Stathmin is an important protein which destabilizes microtubules. The essential function of stathmin is closely associated with its phosphorylation status. Stathmin is overexpressed in many human cancers and has a significant relationship with clinical characteristics such as grade, tumor size and prognosis. We demonstrated that stathmin was overexpressed in ESCC tissues using both 2-DE and immunohistochemistry analysis. In addition, overexpression of stathmin was significantly correlated with histological grade in ESCC. However, no correlation was found with age, gender and lymph node metastasis. Knockdown of stathmin with siRNA impaired cell migration in KYSE30 and KYSE410 cells. When EC0156 cells were treated with paclitaxel, stathmin was stably phosphorylated and migration was impaired. These observations suggest that stathmin may have a more important function in ESCC development and migration. The present study provides further understanding of the importance of stathmin in ESCC therapy or diagnosis.


Introduction
Esophageal cancer (EC) is the eighth most common cancer worldwide (1), while esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype in Asia, especially in China. It is characterized by high incidence and mortality rate (1,2). We found that stathmin is a differentially expressed protein between cancer and adjacent normal tissues in ESCC using proteomic technology.
Stathmin is an important protein which destabilizes microtubules (3,4). Microtubules are essential for many cellular processes, including mitosis, intracellular transport, supportment of cell shape and cell motility, and stathmin playes an important role in the regulation of microtubule which was involved in the construction and function of the mitotic spindle (5). Phosphorylation of stathmin led to a loss of the microtubule-destabilizing activity (6)(7)(8). Inhibition of stathmin phosphorylation produced strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (6). Stathmin phosphorylation gradient was necessary for correct spindle formation. Gradients of diffusible morphogens are known to be crucial for the supracellular self-organization of tissues and organisms (9).
Many studies had reported that stathmin was overexpressed across a broad range of human cancers, including acute leukemia, lymphoma, neuroblastoma and ovarian, prostate, breast and lung cancer (10)(11)(12).
The upregulation of stathmin in ESCC was reported and associated with differentiation degree, lymph node metastasis, invasive depth and TNM stage (13,14). Wang et al demonstrated that knockdown of stathmin by antisense oligonucleotide can inhibit the proliferation of ECa109 cells (15). The expression and exact biological function of stathmin in ESCC, especially motility, remained largely unclear.
The staining index was calculated as the multiples of staining intensity and staining area, as described (18,19) The staining index ﹤1 was considered as negative, while 1-4 as weak and >4 as strong.
Western blot analysis. Western blotting was performed as previously described (20). In briefly, protein extracted from cell lines and tissues specimens were separated using 12% SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). Membranes were blocked by 10% skim milk in 1X PBS. The membranes were incubated with the primary antibodies against stathmin (ab52630, Abcam, UK) or β-actin (Cat. No. A-5316, Sigma, MO) in suitable dilutions. Secondary antibodies were antirabbit IgG and anti-mouse IgG, respectively. Signals were detected by chemiluminescence using the ECL kit. In vitro wound-healing assay. KYSE30, KYSE410 or EC0156 cells were incubated overnight yielding confluent monolayer for wounding. Wounds were scratched using a pipette tip, photographs were taken immediately (time 0 h) and 24 h after wounding. The distance migrated by the cell monolayer to close the wounded area during this time period was measured. Photos were taken by Leica DMCI microscope (Leica, German) at x100 magnification.
Statistical analysis. Data were analyzed by SPSS 16.0 (SPSS Inc., Chicago, IL, USA). χ 2 test was used to analyze the relationship between stathmin expression with clinicopathologic characteristics. P-values ﹤0.05 were considered as statistically significant.

Results
The identification of stathmin in ESCC tissues. Firstly, we analyzed differientially expressed proteins using proteomic method between tumor and corresponding normal tissues in ESCC. Results showed stathmin was detected in all 2-DE gels of the 8 pairs of ESCC tissues (Fig. 1), as an obvious differentially expressed spot.
Stathmin is overexpressed in ESCC tissues. Subsequently, we employed immunohistochemistry to analyze the expression of stathmin in 143 ESCC tissue microarray using the antistathmin antibody. Strong staining was seen in ESCC tissues, however, the normal tissues were weaker or negatively stained (Fig. 2) (Table I). However, no correlation was found between stathmin expression and age, gender and lymph node metastasis (P﹥0.05) (Table Ⅰ).
siRNA-mediated reduction in stathmin expression resulted in impaired cell migration. We chose two ESCC cell lines (KYSE30 and KYSE410) as a model. SiRNA was employed to knockdown stathmin, and western blotting to detect the effect of siRNA. The results showed that the expression of stathmin was reduced after transfection of siRNA oligonucleotide for 72 h in KYSE30 and KYSE410 (Fig. 3A). Subsequently, wound-healing assay showed that the speed of wound recovery of siRNA stathmin was much slower than the scramble in both KYSE30 and KYSE410 (Fig. 3B). The results revealed that cell migration was impaired when deficient of stathmin.
The phosphorylation of stathmin reduced the motility of EC0156. Considering phosphorylation had been closely associtated with the function of stathmin, we wondered whether stathmin phosphorylation has influence on ESCC cell lines. We selected EC0156 for the next study. EC0156 was treated with paclitaxel, a compound extracted from taxus plants, at gradient dosage from 0 to 1.6 µg/ml. The results revealed that the 19 kDa band of stathmin was decreased after paclitaxel treatment, whereas a new band at about 21 kDa gradiently increased in a dose-dependent manner. We confirmed the new band was phosphorylated stathmin at Ser-16. The tendency increased dramatically between 0.1 and 1.6 µg/ml, reaching a peak at a dosage of 0.8 µg/ml. Conversely, KYSE30 and EC0156 without treatment had no  changes (Fig. 4A). Besides, rounded cells were observed in most of the EC0156 through microscopic analysis (Fig. 4B). The number of cells appered slightly reduced. Wound-healing assay showed that the speed of wound recovery of EC0156 cells treated with pacitaxel was much slower than the control, suggesting that cell migration was impaired after stabilized phophorylation of stathmin (Fig. 4C).

Discussion
Previously, we analyzed differientially expressed proteins using proteomic methods between tumor and nomal tissues in ESCC. We found an obviously overexpressed spot in all tumor 2-DE gels, which was identified as stathmin. Stathmin is ubiquitous, highly conserved 19 kDa cytosolic phosphoprotein that regulates microtubule dynamics (21). We employed immunohistochemistry to detect the expression of stathmin in ESCC tissue microarray. Here we demonstrated strong expression in 77.14% (108/140) of ESCC tissues. In addition, overexpression of stathmin was significantly correlated with histological grade (P﹤0.05). However, no correlation was found with age, gender and lymph node metastasis (P>0.05). Wang et al revealed that the positive rate of stathmin in 75 ESCC samples was 81.3% and the relative contents of stathmin were significantly correlated with the differentiation degree, lymph node metastasis, invasive depth and TNM stage of ESCC (13). In addition, the basic tendency of stronger stathmin staining in ESCC was consistent with our previous study, which only used 13 ESCC samples (22). So far, many studies have demonstrated stathmin to be overexpressed in many human cancers, including mesothelioma tumor (11), malignant pheochromocytomas (23,24), cervical carcinoma (25), primary nasopharyngeal carcinoma (26), gastric cancer (27), hepatocellular carcinoma (28,29), medulloblastoma (30), endometrial cancer (18) and urothelial carcinoma (31). Above all, overexpression of stathmin was significantly correlated with clinical stage, tumor grade and lymph node metastasis in cancers, including cervical carcinoma, nasopharyngeal carcinoma, gastric cancer, hepatocellular carcinoma and endometrial cancer (18,(25)(26)(27)(28)(29)(30). Moreover, stathmin may be regarded as a survival prognosis factor in ovarian cancer (32), endometrial cancer (18) and urothelial carcinoma (31). Our previous work, although the ESCC cases were only 13 for immunohistochemistry to analyze the expression of stathmin, we still found the expression of stathmin was overexpressed in ESCC tissue, but there was no correlation with tumor grades (22). In the present study, we expanded the ESCC case numbers for immunohistochemitry. Finally, we found overexpression of stathmin was significantly correlated with histological grade. However, the relationship between stathmin expression and lymph node metastasis did not replicate. There may be two reasons. The limited cases with lymph node metastasis in tissue microarray may lead to information being lost in our study. Another factor worth considered is the tiny difference of the pathologic criteria. In addition, the case number of the present study was much greater than the previous reports in ESCC.
We demonstrated that the speed of wound recovery in stathmin knockdown cells was much slower than the scramble in both KYSE30 and KYSE410 cells, suggesting cell migration was impaired when deficient of stathmin. Similar report exists in gastic cancer, in which cell invasion was significantly reduced by stathmin siRNA in the Matrigel invasion assay (27). Stathmin depletion with siRNA caused significant inhibition of lamellipodia formation, which directed by Pak1-WAVE2-kinesin complex (33). The lamellipodia formation may promote cell migration and invasion. Thus, stathmin depletion might inactivate lamellipodia formation leading to reduction of cell migration.
We demonstrated that the band at 19 kDa of stathmin was decreased when EC0156 was treated by paclitaxel. Meantime, a new band at about 21 kDa appeared in a dose-dependent manner. Subsequently, the new band was confirmed as phosphorylated stathmin at Ser-16. The basic function of stathmin was closely associated with its phosphorylated state. The stathmin-tubulin interaction was dependent on phosphorylation of stathmin (34). Rapidly switching phosphorylation of stathmin regulated microtubule assembly (21). Phosphorylation at Ser-16 and Ser-63 strongly reduced stathmin-tubulin complex formation (35). The effects of stathmin on dynamic instability were strongly attenuated by phosphorylation at Ser-16-and Ser-63 (6). Many protein kinases, including CDC2 (36), MAP (37), Auro B (38) and BGLF4 (39), can phosphorylate stathmin. Stathmin is known to undergo phosphorylation at Ser16 upon cell stimulation, such as paclitaxel at low concentration (40). Paclitaxel is an anticancer drug which interferes with microtubules (41). In the present study, we found paclitaxel may induce stathmin phosphorylation at Ser-16 and influenced the function of stathmin.
Through microscopic analysis, rounded cells were observed in most of EC0156 after the treatment with paclitaxel. The total number EC0156 appeared slightly reduced. Besides, the wound recovery speed of EC0156 treated with paclitaxel was diminished compared to the control, suggesting that cell migration may be impaired after stathmin phophorylation. It is possibly that stabilized phosphorylation of stathmin may disrupt microtubule dynamics and finally impaired the motility of EC0156, which were supported by other reports. Migration can be viewed as a repeated sequences of events that include formation of pseudopodia protrusions, attachment and translocation of the cell body in the direction of the new adhesion sites. The microtubule dynamics instability is important to generate an asymmetrical microtubules array (42). Stronger or long time stabilized phosphorylation of stathmin may impair the microtuble dynamics. Cernuda-Morollon et al found that T-cell receptor (TCR)-induced stathmin phosphorylation prevented T cell polarization on ICAM-1 by increasing Rac1 activity and reduced the migration of T cell and changed microtubule dynamics leading to loss of migratory polarity (43). Di Paolo et al revealed that phosphorylation at three sites (Ser-16, Ser-25, Ser-63) completely inhibited the tubulin binding capacity of stathmin (35). This phenomenon was also observated in cancer cells. Belletti et al revealed that Ser-16 phosphorylation of stathmin enhanced sarcoma cell adhesion and inhibited sarcoma cell motility by mutant methods (44). The present study revealed that paclitaxel may act as a stimulus factor to induce the phosphorylaiton of stathmin leading to impairment of microtubule dynamics. Our observations provide new evidence for understanding the interaction between stathmin phosphorylation and microtubules.
Our observation at cell level was not in accordance with immunohistochemistry analysis. Except for information lost or cancer diversity, different microenvironment between cultured cells and tissues may also contribute. In summary, stathmin was overexpressed in ESCC tissues and overexpression of stathmin was significantly correlated with histological grade. In addition, deficiency or stabilized phosphorylation of stathmin both impaired the migration of ESCC cells. The present study might highlight the potential of stathmin in the therapy and diagnosis of ESCC.