Clinicopathological and biological significance of mitotic centromere-associated kinesin overexpression in human gastric cancer

Abstract

Mitotic centromere-associated kinesin (MCAK) is a microtubule (MT) depolymerase necessary for ensuring proper kinetochore MT attachment during spindle formation. To determine MCAK expression status and its clinicopathological significance, real-time reverse transcriptase-polymerase chain reaction was used in 65 cases of gastric cancer. MCAK gene expression in cancer tissue was significantly higher than expression in non-malignant tissue (P<0.05). Elevated MCAK expression was significantly associated with lymphatic invasion (P=0.01) and lymph node metastasis (P=0.04). Furthermore, patients with high MCAK expression had a significantly poorer survival rate than those with low MCAK expression (P=0.008). Immunohistochemical study revealed that expression of MCAK was primarily observed in cancer cells. Additionally, a gastric cancer cell line (AZ521) that stably expressed MCAK was established and used to investigate the biological effects of the MCAK gene. In vitro results showed that cells transfected with MCAK had a high rate of proliferation (P<0.001) and increased migratory ability (P<0.001) compared to mock-transfected cells. This study demonstrated that elevated expression of MCAK may be associated with lymphatic invasion, lymph node metastasis, and poor prognosis. These characteristics may be due in part to the increased proliferative and migratory ability of cells expressing MCAK.

Figure 1

Mitotic centromere-associated kinesin mRNA expression in clinical samples. (A) RT–PCR analysis of MCAK in gastric cancer (T) and paired normal (N) samples obtained from four patients (patient numbers: MK 18, 22, 137, and 154). Mitotic centromere-associated kinesin mRNA expression was observed in cancer samples, but no expression was seen in normal samples. GAPDH was used as a control. m=marker; NC=negative control; PC=positive control. (B) Immunohistochemistry of gastric cancer surgical specimens using an antibody to MCAK. Positive staining was observed in cancer cells, but not in normal mucosal epithelium (magnification × 40 (left image)). A dotted circle indicates normal mucosa and a solid line circle indicates cancer cells. In normal mucosa (right upper image, magnification × 100), solid line arrows indicate proliferating cells of the glandular neck and dotted line arrows indicate macrophages.

Figure 2

Overall survival rate of patients with gastric cancer grouped according to MCAK mRNA expression status of the tumour. Patients with high MCAK mRNA expression (n=32) had a significantly poorer prognosis than those with low MCAK mRNA expression (n=33).

Figure 3

Experimental studies. (A) Western blotting revealed that MCAK protein was detected in transfectants but not in mock-transfected cells. β-Actin was used as a control. Right and left lanes show transfected and mock-transfected cells (cell line AZ521), respectively. (B) Growth rate of MCAK transfectants and mock-transfected cells in 10% FBS. Bar=s.d.; cell counts were greater in MCAK transfectants than in mock-transfected cells (P<0.001). (C) Cell cycle analysis of MCAK transfectants and mock-transfected cells after 24 h of serum starvation followed by 18 h serum feeding with 10% FBS. The G1-phase cell counts were unified. The S-phase fraction was greater in transfectants (44.3%) than mock-transfected cells (25.3%). (D) Migration assay. The migratory ability of transfectants was significantly stronger than that of mock-transfected cells (P<0.001). (E) Anoikis analysis. After anoikis induction for 18 h, the apoptosis rate was measured by annexin V and PI staining. Proportion of apoptotic MCAK-transfected cells (78.52%) was less than that of apoptotic mock-transfected cells (91.73%).