Abnormal cell proliferation, which results from deregulation of the cell cycle, is fundamental in tumorigenesis. The integrated mechanisms that regulate the accurate replication of DNA and correct division of cells are thus pivotal in the neoplastic process 12. Regulation of the cell cycle is complex and involves a wide variety of genes and proteins, among which the minichromosome maintenance (MCM) nuclear proteins are essential replication initiation factors. The MCM protein family consists of six major isoforms (MCM2-7), which have similar biochemical functions 3 and are equally important for continuous chromosome replication after the activation of early origins of DNA replication 4. During the cell cycle, the MCM proteins form a hexameric complex, which is a key component of the prereplication complex that assembles at replication origins during early G₁ phase 35. MCM proteins restrict DNA synthesis to only once per cell cycle 346, and regulate DNA elongation 4. These functions of MCM proteins imply that they are correlated with cell proliferation, which has been consistently supported by experimental evidence. For example, the onset of DNA synthesis was inhibited if anti-MCM2 antibody was injected into cells during G₁ 6, and depletion of MCMs after initiation irreversibly blocked the progression of replication forks 4. Moreover, MCM2 expression in developing Drosophila embryos followed a pattern corresponding to the fast dividing cells, and inactivation of MCM2 inhibited cell proliferation 7. Cellular expression of MCMs was constitutively high in proliferating cells but low or undetectable in quiescent cells 389.

Ki-67, another important cell proliferative biomarker, is a nuclear protein containing phosphorylation sites for a variety of kinases, putative nuclear targeting sequences, and a forkhead-associated domain, similar to those of cell-cycle-regulating proteins 101112. During mitosis, phosphorylation and dephosphorylation of Ki-67 occur at the breakdown and the reorganization of the nucleus, two hallmark occasions of the cell cycle. These posttranslational modifications are accompanied by the redistribution of Ki-67 from the interior of the nucleus to the periphery of the condensed chromosomes and vice versa 1213. Although recognized as a cell cycle regulating protein, the specific functions of Ki-67 remain elusive, mainly due to its lack of homology with other proteins 13. Some proposed functions of Ki-67 include organization and maintenance of the architecture of DNA, and synthesis of ribosomes during mitosis 1415. Since Ki-67 is expressed during all phases of the cell cycle except G₀, cellular expression of Ki-67 has provided a measure of tumor proliferation 1617. The prognostic value of Ki-67 has been reported in various tumors, including cancers of breast, soft tissue, lung, cervix, prostate, and brain 1718192021.

Gelsolin is the most potent actin binding and severing protein identified to date which is widely expressed in mammalian tissues 2223. It regulates the integrity and dynamics of actin cytoskeleton and maintains the proper cellular morphological structure and motility through severing, capping and nucleating actin filaments 232425. Knockout animal models have clearly demonstrated the critical role of gelsolin in facilitating cell motility 2627282930. An inverse association between gelsolin expression and patient survival time has been observed in lung cancer and breast cancer 313233, suggesting cellular motility mediated through gelsolin expression plays an important role in the progression and prognosis of malignant tumors.

Although MCM2, Ki-67, two markers of cell proliferation, and gelsolin, marker of cell motility, have been previously studied, no study has been conducted assessing the potential combined prognostic effect of these markers in non-small cell lung cancer (NSCLC).

Patient characteristics are summarized in Table 1. The majority of patients were Caucasian (93.8%). There were more male (54.7%) than female patients (45.3%). The majority of patients were former smokers (50%) or current smokers (41%). About one third of patients had a positive family history of lung cancer. The mean and median survival time among deceased patients was 29.4 and 26.5 months, respectively (range: 1.5–84.7 months). The mean and median follow-up time for living patients was 68.3 and 67.0 months, respectively (range: 42.7–128.4 months). Forty-nine patients (38.3%) were alive at the last contact on December 30^th, 2003.

The phenotypic expressions of MCM2, Ki-67, and gelsolin are shown in Figure 1, which depicts examples of negative expression (C and E), low expression (A and F), and high expression (B, D and G), respectively. Examination of MCM2, Ki-67, and gelsolin expression showed that 61.7%, 78.1% and 32.8% of tumors exhibited high expression of these three proteins, respectively (Table 1). Patient characteristics stratified by levels of MCM2, Ki-67 and gelsolin expression are shown in Table 2. None of the clinicopathological factors showed a significant association with Ki-67 expression. For MCM2, high expression was associated with sex (male patients: 64.6% vs. 38.8%), histology (squamous carcinomas: 45.6% vs. 6.1%), and grade (poorly differentiated tumors: 68.4% vs. 46.9%). For gelsolin, high expression was associated with sex (female patients: 66.7% vs. 51.2%), and histology (squamous carcinomas: 38.1% vs. 26.7%).

Univariate analysis of the prognostic significance of MCM2, Ki-67, and gelsolin showed higher levels of gelsolin were significantly associated with an elevated risk of death compared to low levels of gelsolin (RR = 1.89, 95% CI = 1.20–2.98, p = 0.006), and higher levels of MCM2 were associated with a non-significant increased risk of death compared to low levels of MCM2 (RR = 1.51, 95% CI = 0.94–2.41, p = 0.09). Ki-67 expression did not display apparent prognostic effect in this study sample (RR = 0.70, 95% CI = 0.27–1.86, p = 0.47). After controlling for age, stage, adjuvant chemotherapy, family history of lung cancer, and smoking history, the significant association remained between high levels of gelsolin and elevated risk of death (RR = 1.89, 95% CI = 1.17–3.05, p = 0.01). The prognostic effect of MCM2 was attenuated after controlling for confounders (RR = 1.36, 95% CI = 0.84–2.20, p = 0.22) (Table 3).

Composite analysis of MCM2 and gelsolin expression revealed that higher expression in MCM2 and gelsolin was significantly associated with higher risk of death compared to low expression in both biomarkers (adjusted RR = 2.32, 95% CI = 1.21–4.45, p = 0.01) (Table 3, Figure 2). Since the hazards function was not proportional for Ki-67, we did not analyze the combined prognostic effect of Ki-67 with MCM2 and gelsolin in the Cox Proportional Hazards Regression. Survival curves generated from Kaplan-Meier survival analysis are illustrated in Figure 2. The curves confirmed the results from Cox regression that patients with higher levels of MCM2 and gelsolin experienced shorter survival time than patients with low levels of MCM2 and gelsolin.

The relative balance between cell growth and cell death is altered in cancers, with the balance tipped towards uncontrolled proliferative capacity in cancer cells 143. In addition, some cancer cells acquire a markedly enhanced capacity to move, thereby predisposing them to invasion and dissemination 4445. Findings from the present study showed that tumors exhibiting higher levels of expression in MCM2, a marker of proliferation, and gelsolin, a marker of motility, conferred a higher risk of death in NSCLC.

MCM proteins are essential replication initiation factors that ensure the accurate replication of DNA in the cell cycle. Through accurate binding to chromatin during G₁ phase and detaching from chromatin during S phase, MCM proteins restrict DNA synthesis to only once per cell cycle 346. Only licensed origins containing MCM proteins can initiate a pair of replication forks, and once initiation occurs at an origin, the bound MCM proteins are displaced so that the origin cannot fire again 46. MCM proteins may also function as the helicase that unwinds DNA ahead of each replication fork 47. Evidence from different organisms and cells indicates that both permanently arrested cells and those in G₀ phase have lost expression of MCM proteins and are functionally unlicensed 484950. The replication initiation function of MCM proteins in cell cycle implies that they may be valuable markers for proliferation and prognosis in human cancers. Todorov et al. studied MCM2 expression in normal and tumor human tissues and found MCM2 was expressed significantly different in specimens examined, with expression of 97% in tumors but only 27% in normal tissues. Furthermore, the expression level in normal tissues was lower than in tumor tissues, indicating that MCM2 reflects cell proliferation 9. Dudderidge et al. studied MCM2, geminin, and Ki-67 in renal cell carcinoma and found that MCM2 expression increased dramatically with increasing grade 41. Hashimoto et al. investigated MCM2 and Ki-67 expression in lung adenocarcinomas and found that the co-expression of MCM2 and Ki-67 at higher levels were significantly associated with poorer survival 39. The prognostic value of MCM2 was also reported in bladder cancer and oligodendroglioma 4051.

Ki-67 is expressed in all stages of the cell cycle except G₀ phase, making it a valuable measurement for cell proliferation 12. In a number of cancers, elevated Ki-67 expression has been found associated with higher aggressiveness and invasiveness 525354. The prognostic value of Ki-67 has been observed in cancers of breast, prostate, cervix, and soft tissue 21. In a comprehensive review, Pugsley et al. noted that higher Ki-67 index was significantly associated with poorer survival in NSCLC in the majority of univariate analyses and in approximately half of the multivariate analyses 38. In our study, we did not observe the prognostic effect of Ki-67 expression in both the univariate and multivariate analysis, which may be partially due to the limited sample size. The small sample size also prevented additional examination of the prognostic value of MCM2, Ki-67 and gelsolin in subgroup patients. As Pugsley et al. pointed out, factors such as patient heterogeneity, retrospective nature of study design, quantification of immunohistochemistry, and cutoff value may contribute to the inconsistent findings for the prognostic value of Ki-67 NSCLC in the literature 38.

In addition to proliferation, elevated motility is essential for tumor cells to invade and disseminate 44455556. Highly metastatic cells typically exhibit enhanced locomotion capacity compared to less metastatic cells 44455758. Various studies have consistently shown that higher levels of motility are associated with higher rates of tumor metastasis or shorter survival time 31323359606162. The motility facilitating function of gelsolin has been widely observed in animal models 26272829. We and others have showed that higher levels of gelsolin are associated with poorer survival in breast and lung cancer 313233. Some studies failed to find a prognostic significance for gelsolin. However, these studies are generally subject to small sample size or inadequate follow-up time 37636465. As a generalization, the gelsolin content of the cells in most cancers is found to be down-regulated 37646566, but in a number of studies, researchers found that high gelsolin expression is inversely associated with survival time 313233. For example, we found that tumors with high and heterogeneous expression of gelsolin conferred the highest risk of death from NSCLC in patients with this disease 33. Shieh et al. found that the most potent predictor of poor prognosis was high focal gelsolin expression 31. Increased expression of gelsolin may facilitate a subset of tumor cells with increased motility, thereby enhancing its capability and probability of invading adjacent tissues and metastasis to remote organ sites 3233.

In summary, higher expression in MCM2 and gelsolin was significantly associated with poorer prognosis in patients with NSCLC, which suggests that higher tumor proliferation and motility may be important in the prognosis of NSCLC. Ki-67 did not display apparent prognostic value in NSCLC in our study sample. Composite application of proliferation and motility markers may be more valuable than use of a single marker in assessing tumor prognosis. This combinatorial approach may also prove valuable in assessing response to anticancer treatment and the subsequent clinical outcomes.

ABC: avidin-biotin-conjugate; GSN: gelsolin; MCM: minichromosome maintenance; NSCLC: non-small cell lung cancer; RPCI: Roswell Park Cancer Institute; RR: relative risk

The author(s) declare that they have no competing interests.

JY carried out and coordinated the study, conducted IHC and data analysis, and drafted up the manuscript. NR participated in the study design, interpretation of data, and revision of manuscript. KBM participated in the study design, interpretation of data, and revision of manuscript. HLA developed gelsolin scoring system, interpreted data, and revised manuscript. SJA collected the tumor and clinicopathological data. JH interpreted data and revised manuscript. JG and JSJB performed IHC examinations of tumor specimens and revised manuscript. DT participated in the study design, IHC examinations of tumor specimens, interpretation of data, and revision of manuscript. All authors read and approved the final manuscript.