Microtubule-Associated Protein 4 Is a Prognostic Factor and Promotes Tumor Progression in Lung Adenocarcinoma

Microtubule-associated protein 4 (MAP4) plays an important role in microtubule assembly and stabilization. The purpose of this study was to investigate the level of expression of MAP4 in lung adenocarcinoma (LADC) samples and to evaluate its prognostic value and the influence on cancer progression in LADC patients. The expression of MAP4 protein was analyzed using immunohistochemistry. The clinical significance and the prognostic significance of MAP4 expression were assessed by Kaplan-Meier analysis and Cox regression analysis. The roles of MAP4 in the migration and invasion of LADC cells were detected by wound-healing assays and transwell assays, respectively. We found the expression levels of MAP4 protein in LADC tissues to be significantly higher than those in noncancerous tissues. MAP4 expression was significantly correlated with differentiation, pathological T stage, and TNM stage. Kaplan-Meier survival analysis indicated that patients with high MAP4 expression had significantly poorer overall survival (OS). Cox regression analysis revealed that MAP4 expression level was an independent prognostic factor for OS. Functionally, in vitro studies showed that MAP4 knockdown efficiently suppressed the migration and invasion of LADC cells. Our data indicated that MAP4 protein may represent a novel prognostic biomarker and a potential therapeutic target for LADC.


Introduction
Cancer is an enormous public health burden worldwide. More than 14 million estimated new cancer cases and 8 million cancer deaths occurred in 2012. Lung cancer remains the leading cause of cancer mortality for humans all over the world [1], and non-small-cell lung carcinoma (NSCLC) is the predominant type of lung cancer, making up 85% of all cases [2]. Lung adenocarcinoma (LADC) is a type of NSCLC that has come to make up a growing proportion of NSCLC in recent years. Radical resection is the principal treatment for the patients with stage I-IIIa NSCLC, but the 5-year survival rate remains low. The high mortality rate is largely attributed to local recurrence and distant metastasis of NSCLC [3]. A number of randomized clinical trials demonstrated that adjuvant chemotherapy is the standard treatment for resected early-stage NSCLC patients [4][5][6], but few patients benefit from the treatment. Hence, identification of novel prognostic biomarkers relating to cancer recurrence and metastasis is critical to improving the treatment strategy for NSCLC patients.
Microtubule-associated proteins (MAPs) have many subtypes, including MAP1A, MAP1B, MAP2, MAP4, and tau proteins. MAP4 is mainly expressed in nonneuronal tissues and ubiquitously found in all cell types [7]. Heatstable MAP4 is composed of an asymmetric structure with an N-terminal projection (PJ) domain and a C-terminal microtubule-binding (MTB) domain. Studies have shown that the PJ domain takes part in the regulation of the dynamic instability and the phosphorylation of MTB domain participates in the cell cycle progression [8][9][10][11]. MAP4 has been reported to play an important role in the modulation of microtubule dynamics through interaction with septin [12]. It has been reported that MAP4 has effects on paclitaxel resistance and the rapid progression of apoptosis by the negative regulation of p53 [13][14][15][16][17]. Until now, there are still few studies of the relationship between MAP4 and human cancers. Ou and colleagues have reported that the cAMP/ PKA signaling pathway is involved with bladder cancer cell invasion by targeting MAP4-dependent microtubule dynamics [18]. Recently, overexpression of MAP4 has been demonstrated to be associated with poor prognosis and promotion of cell invasion and migration through MAP4-ERK-Jun-VEGF signaling in esophageal squamous cell carcinoma [19].
Up to now, however, no reports have been published on the relationship between MAP4 expression and clinicopathological features and prognosis of LADC patients. In this study, we demonstrated that expression of MAP4 was closely correlated with LADC progression and poor prognosis by promoting cancer cell invasion and migration.

Patients and Tissue
Samples. Fresh human LADC tissues and adjacent tissues used for this study were obtained from surgical resection specimens collected by Nanjing Medical University Affiliated Suzhou Hospital (Jiangsu, China). None of the patients received any treatment before surgery. The tissue samples were immediately snap-frozen and stored at −80°C for histological examination after surgery. Clinicopathologic parameters (Table 1) and OS data were collected. 91 LADC patients were followed up until August 2014 or until death, with a median follow-up period of 39 months (range 1-121 months). All patients signed informed consent, and the study was approved by the Institutional Ethics Committee of Nanjing Medical University.
2.2. Immunohistochemistry. Paraffin-embedded LADC tissue samples and cancer adjacent tissues were cut into 4 μm thick sections and affixed to the slides. The tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions using standard procedures. The sections were subsequently submerged in EDTA (pH 8) and autoclaved at 121°C for 5 min to retrieve the antigenicity. After washing in TBS, endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide solution in methanol for 10 min at room temperature. Then, incubation with MAP4 antibody (Proteintech, Rosemont, IL, US) diluted at 1 : 4000 in TBS containing 0.5% BSA was carried out at 4°C overnight followed by further washing with buffer to remove unbound antibody. The sections were incubated with Envision secondary antibody (DAKO, Santa Clara, CA, US) for 30 min at room temperature. Chromogen (DAB) (GeneTex, Irvine, CA, US) was added to visualize the reaction. The sample was then counterstained with commercial hematoxylin (Beyotime Biotechnology, Nantong, Jiangsu, China), dehydrated sequentially in alcohols and xylene, and mounted.
2.3. Immunohistochemical Staining Evaluation. Two experimenters who were blinded to clinicopathologic information and patient outcomes evaluated the slides independently.
MAP4 staining was mainly localized in the cytoplasm. MAP4 expression was quantified using a visual grading system based on the extent of staining and the intensity of staining. The percentage of positive-staining tumor cells was scored as follows: 0 (negative), 1 (1-30%), 2 (31-60%), and 3 (>60%). Staining intensity was scored as follows: 0 (none), 1 (weak staining), 2 (moderate staining), and 3 (strong staining) [20]. The final score was calculated by multiplying the extent of staining score by staining intensity score. Scores > 4 were considered indicative of high expression levels. Cases with discrepancies were rereviewed simultaneously, and consensus decisions were made.

Reverse
Transcription-Polymerase Chain Reaction (RT-PCR). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, US) in accordance with the manufacturer's instructions. The concentrations of RNA were determined using a NanoDrop2000 (NanoDrop, US). For reverse transcription, 1.0 μg of RNA/sample was reverse-transcribed using an oligo(dT) 12 primer and SuperScript II reverse transcriptase (Invitrogen, US) according to the manufacturer's instructions. The primers for MAP4 and β-actin were as

Association of MAP4 Protein Expression with
Clinicopathological Features of LADC. The correlations between MAP4 expression and the clinicopathological features of LADC patients are shown in Table 1. The level of MAP4 expression was significantly closely correlated with differentiation (P = 0 03), pathological T stage (P < 0 01), and TNM stage (P < 0 01). However, there were no significant differences between MAP4 expression and other clinicopathological factors, including gender (P = 0 75), age (P = 0 10), and pathological N stage (P = 0 06).

MAP4 Expression in LADC Tissues and the Paired
Adjacent Normal Lung Tissues. The expression of MAP4 protein in paraffin-embedded, archived LADC tissue samples (n = 91) and the paired adjacent normal lung tissues (n = 86) were analyzed using immunohistochemistry. MAP4 staining in LADC tissues appeared as brown particles, which were mainly localized within the cytoplasm, accompanied by a stromal reaction (Figure 1(a)). Most of the normal carcinomaadjacent tissues showed little or no staining (Figure 1(b)). The incidence of positive expression was 56.04% (51/91) in LADC tissues and 23.25% (20/86) in the normal tissues. The level of MAP4 expression was significantly higher in LADC than in normal lung tissues, as indicated by statistical analysis (P < 0 0001) (Figure 1(c)).

Survival Analysis Correlation of MAP4 Expression in LADC with Clinicopathological Characteristics.
To investigate the prognostic value of MAP4 for LADC, we evaluated the relationship between MAP4 expression and OS in all patients with Kaplan-Meier analysis. The survival curve showed that patients with high levels of MAP4 expression had a significantly poorer OS than those with low levels of expression (log-rank test, P = 0 028) (Figure 2(c)), while lymphatic metastasis and higher TNM stage also had a significantly worse OS (log-rank test, both P < 0 001) (Figures 2(a   ( Figure 5). These results suggested that MAP4 may be involved in the migration and invasion of LADC cells.

Discussion
Lung adenocarcinoma is the most common subtype of NSCLC, making up about 80% of all diagnosed lung cancers [21]. Local recurrence and distant metastasis are the principal causes of deaths in LADC patients [3]. Molecular targeted therapy has developed rapidly in the past decade. Compared with the traditional chemoradiotherapy, targeted therapy significantly improved the long-term survival rate of lung adenocarcinoma patients [22,23]. However, about half of lung cancer patients are not suitable for targeted therapy because of the absence of known driver mutations [24]. A potential biomarker that can predict responses to therapy and inform individualized treatment strategies and novel insights into the mechanism underlying LADC pathogenesis is critically needed. In this study, we demonstrated that MAP4 is an independent prognostic factor of LADC with the ability to promote cancer cell migration and invasion.
MAP4, a type of microtubule-associated proteins, was first identified in mouse neuroblastoma cells [25]. Studies have reported that MAP4 plays an important role in microtubule assembly and stabilization [8,11,12]. In the process of cancer chemotherapy, an antimicrotubule drug is administered safely at a time, and cancer cells are more sensitive to DNA-damaging agent when MAP4 is downregulated [26]. Researchers have found that the ratio of MAP4 to stathmin mRNA was higher in lung cancer tissues than in normal lung tissues, suggesting that the ratio might be a clinically relevant biomarker for NSCLCs [27]. In prostate cancer, MAP4 was also reported to be a potential biomarker for detection of prostate cancer and discrimination between prostate tumors with different malignancy and aggressiveness [28]. Downregulation of MAP4 is associated with poor differentiation and proliferation in primary oral squamous cell carcinomas [29]. The novel MALT1-MAP4 fusion protein, which is different from the known MALT1-associated chromosomal rearrangements, may be a new pathogenetic reason of diffuse large B-cell lymphoma [30]. However, few studies have focused on the relationship of MAP4 function with cancer progression or poor prognosis in human cancer.
In the present study, we hypothesized that aberrant MAP4 expression may be involved in the progression of LADC. LADC tissue samples and the adjacent morphological normal lung tissues were collected to assess alterations in MAP4 expression. We found MAP4 expression to be significantly higher in the LADC tissues than in the normal controls. This finding strongly suggested that elevated MAP4 expression may play an oncogenic role in promoting tumor    progression. Subsequently, the clinical significance of MAP4 expression in 91 LADC patients was analyzed and the MAP4 high-expression frequency was 56.04%. According to clinicopathologic characteristics, high levels of MAP4 expression was significantly closely correlated with differentiation, pathological T stage, and TNM stage but not with gender, age, or pathological N stage. Moreover, Kaplan-Meier survival curves showed that the patients with lymphatic metastasis, higher TNM stage, and elevated MAP4 expression had a significantly poorer OS. Cox regression analysis revealed that pathological N stage and MAP4 expression were independent prognostic factors of OS in LADC patients. We also found that MAP4 knockdown reduced cell migration and invasion in vitro studies. Collectively, these results strongly demonstrated that elevated MAP4 expression is associated with the increased metastatic potential of cancer cell and worse long-term survival of LADC patients. Although the underlying mechanism is unclear, the evidence seems to indicate that MAP4 is an important regulator during cancer progression. Our new findings were consistent with recent studies, which have shown a correlation between MAP4 overexpression and poorer prognostic outcome in bladder cancer and esophageal squamous cell carcinoma [18,19].
In summary, our present data demonstrated that the elevated expression of MAP4 was associated with differentiation, pathological T stage, and TNM stage in LADC tissues. Hence, MAP4 may be an independent prognostic factor for poor prognosis of LADC. The present study has some limitations, such as the small sample size and the unknown potential molecular mechanism between aberrant MAP4 expression and cancer progression in LADC. These findings remain to be confirmed by more samples and further studies. Based on these data, we propose that MAP4 may have the role of identifying the subgroup of patients with more aggressive tumors and poor prognostic outcome and it may become an attractive and promising therapeutic target for LADC patients in the future.

Conflicts of Interest
The authors declare no conflicts of interest.