Cytoplasmic Forkhead Box M1 (FoxM1) in Esophageal Squamous Cell Carcinoma Significantly Correlates with Pathological Disease Stage

Five human ESCC cell lines, HKESC-1, HKESC-2, HKESC-3, HKESC-4 and SLMT-1, and one human non-neoplastic esophageal squamous cell line, NE-1, were used in this study. These cell lines were previously established by our team [16‐20]. NE-1 cells were cultured in keratinocyte-SFM medium supplemented with bovine pituitary extract and human recombinant epidermal growth factor (Invitrogen, Carlsbad, CA). HKESC-1 and HKESC-4 cells were maintained in Minimum Essential Medium (MEM) medium (Invitrogen) with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen) and 1% penicillin and streptomycin (P/S) (Invitrogen). HKESC-2, HKESC-3, and SLMT-1 cells were cultured in MEM medium with 20% FBS and 1% P/S. All cell lines were maintained in a humidified atmosphere at 37°C containing 5% CO₂.

We recruited 64 ESCC patients who had undergone esophagectomy between 1997 and 2005 at the Department of Surgery, Queen Mary Hospital, Hong Kong. Consent to use clinical specimens for this study was obtained from the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB). There were 47 men and 17 women and the mean age was 64.6 years (range = 40–87 years). None of the patients had received neoadjuvant chemotherapy or radiotherapy before esophagectomy. Data of patient demographics, histopathology, and long-term follow-up were captured in a prospectively collected database. Specimens of primary tumor and nontumor tissues obtained from near the proximal resection margins were stored at –80°C and retained for laboratory studies and subsequent analyses. The site and size of the tumors were recorded. Standard tissue blocks were prepared by fixing the specimens in 10% formalin before embedding in paraffin wax. Five-micron sections were prepared and stained with hematoxylin and eosin for light microscopy. Stained sections were reviewed and tumors were graded according to the criteria of the World Health Organization (WHO). Cancers were staged according to TNM classification.

The expression of FoxM1 mRNA in esophageal cell lines and clinical specimens was determined using qPCR. RNA was extracted using TRIzol reagent (Invitrogen), according to manufacturer’s instructions. RT and qPCR were performed as described [21, 22]. Five hundred nanograms of RNA was reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen), following the manufacturer’s protocol. cDNA (0.3 and 0.6 μg) from cultured cells and tissues was used for qPCR using Platinum Quantitative PCR SuperMix-UDG w/ROX (Invitrogen) and FoxM1-specific primers (forward: 5′-ACC CAA ACC AGC TAT GAT GC-3′ and reverse: 5′-GAA GCC ACT GGA TGT TGG AT-3′). β-Actin was used as an internal control in a parallel experiment, using its specific primers (forward: 5′-CCA TCA TGA AGT GTG ACG TG-3′ and reverse: 5′-ATC CAC ATC TGC TGG AAG GT-3′). qPCR was performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Carlsbad, CA). Each sample was performed in at least duplicates.

The protein level of FoxM1 in esophageal cell lines was detected using immunoblot as described [23, 24]. Briefly, cultured cells were washed twice with ice-cold 1 × phosphate-buffered saline (PBS, pH 7.4). Ice-cold cell lysis buffer (Cell Signaling Technology, Danvers, MA) was added to cell monolayers to extract proteins by scraping. Cell lysates were left on ice for 15 min before being centrifuged at 13,200 rpm for 15 min at 4°C. Protein concentration was estimated using Bradford Protein Assay solution (Bio-Rad, Hercules, CA). Following the addition of 6 × sample buffer to the cell lysates, 25 μg of protein was resolved onto an 8% sodium dodecyl sulfate (SDS) polyacrylamide gel at room temperature before the transfer of proteins onto the polyvinylidene fluoride (PVDF) membrane for 2.5 h at 4°C. After blocking with 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 2 h, membranes were probed with polyclonal rabbit anti-FoxM1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1,500 overnight at 4°C. Monoclonal mouse anti-β-actin antibody (Sigma-Aldrich, St Louis, MO) at 1:10,000 was used as a loading control. After washing with TBST, membranes were incubated with either goat anti-rabbit (Zymed, Carlsbad, CA) (1:7,000 for detecting anti-FoxM1 antibody) or goat anti-mouse (Zymed) (1:20,000 for detecting anti-β-actin antibody) secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Following washing with TBST, signals were visualized using ECL Plus Western blotting reagent pack (GE Healthcare Biosciences, Piscataway, NJ). The apparent molecular weight of FoxM1 and β-actin on gels was 100 and 42 kDa, respectively.

Five-micron sections from tumors and nontumor tissues were prepared and mounted on glass slides for immunohistochemistry as previously described [23, 25, 26]. Sections were deparaffinized and rehydrated. Antigen retrieval was performed by heating sections in 0.1 M citrus buffer (pH 6.0) in a microwave for 10 min. Endogenous peroxidase activities were blocked by incubating sections with 3% hydrogen peroxide at room temperature for 20 min. After washing with TBS (pH 7.6), nonspecific binding sites were blocked with 10% normal goat serum (Dako, Glostrup, Denmark) in TBST at room temperature for 1 h. Sections were incubated with anti-FoxM1 antibody at 1:600 overnight at 4°C. After washing with TBS, sections were incubated with anti-rabbit-labeled polymer-HRP provided in EnVision + System-HRP (Dako) at room temperature for 45 min to detect the primary antibody. Signals were visualized by incubating sections with liquid DAB + (diaminobenzidine) (Dako) before counterstaining with hematoxylin. A colorectal carcinoma section from the tissue microarray was used as a positive control (Fig. 1a), while normal goat serum as a substitution for anti-FoxM1 antibody was used as a negative control (Fig. 1b). Stained sections were examined by Dr. Chan under light microscope. The expression of cytoplasmic FoxM1 was evaluated according to the methodology of Liu et al. [10] with modifications. The expression of FoxM1 was categorized according to the percentage of cancer cells stained positive with anti-FoxM1 antibody (score from 0 to 3, where 0 ≤ 5%, 1 = 6–25%, 2 = 26–50%, and 3 ≥ 51%) and the intensities of the immunostain (1 + , 2 + , 3 +). A weighted index score (0–9) was calculated for each section by multiplying the values of these two categories. For example, the weighted index score should be 6 if 30% of cancer cells were stained (score = 2) with strong signals (score = 3). For cytoplasmic FoxM1 expression, tumors were further grouped into a low-expression group (weighted index score = 0–4) and a high-expression group (weighted index score = 5–9). For nuclear FoxM1, its expression segregated the tumors into an “absence” group or a “presence” group.

Data are expressed as mean ± SEM. Pearson’s χ² test was used to assess the correlations between immunohistochemical data and categorical clinicopathological characteristics. The Kaplan-Meier method was used for survival analysis, and the differences in survival were estimated using the log-rank test. A P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 12.0 for Windows (SPSS, Inc., Chicago, IL).

A high level of FoxM1 mRNA was found in ESCC cell lines compared to that in the non-neoplastic squamous cell line NE-1 using qPCR. An increase of 24.34-, 57.45-, 103.72-, 25.81-, and 13.92-fold in the mRNA level of FoxM1 was observed in HKESC-1, HKESC-2, HKESC-3, HKESC-4, and SLMT-1 cells, respectively, compared to that in NE-1 cells (Fig. 2). All of ten ESCC tissues studied showed an increase in FoxM1 mRNA when compared to their corresponding nontumor counterparts. The upregulation of FoxM1 ranged from about 3- (patient 9) to 500-fold (patient 6) (Fig. 3).

The protein level of FoxM1 in cultured cells was analyzed using immunoblot. Non-neoplastic NE-1 cells expressed an undetectable protein level of FoxM1. Except HKESC-3 cells, ESCC cells, including HKESC-1, HKESC-2, HKESC-4, and SLMT-1, showed an elevated protein level of FoxM1 compared to NE-1 cells (Fig. 4).

Immunohistochemistry was performed to investigate the expression and localization of FoxM1 in 64 ESCC tissues and 10 randomly selected nontumor tissues from the same patient cohort. Cytoplasmic expression of FoxM1 was found in 98.44% (63/64) of ESCC tissues (Fig. 5), while its nuclear expression was observed in 25% (16/64) of ESCC tissues (Fig. 6). All nontumor tissues were positive for cytoplasmic FoxM1, whereas 20% (2/10) of them expressed nuclear FoxM1. Specifically, FoxM1 expression was found mainly in the proliferating layer of the non-neoplastic epithelium and the expression was relatively low in the more differentiated layer (Fig. 7).

Patients with lower expression of cytoplasmic FoxM1 were more likely in the early stage of ESCC (P = 0.018) than those with higher expression (19/37, 51.4% vs. 6/27, 22.2%, respectively) (Table 1). No significant difference in survival after surgery could be found between patients with low expression of cytoplasmic FoxM1 and those with high expression (14.75 vs. 23.28 months, respectively, P = 0.977) (Fig. 8). Patients with nuclear expression of FoxM1 were younger than those without nuclear FoxM1 (57 vs. 67.13 years, P < 0.001) (Table 2). The median postoperative survival for patients with nuclear FoxM1 was similar to those without (14.75 vs. 25.64 months, P = 0.697) (Fig. 9).