NS1-binding protein radiosensitizes esophageal squamous cell carcinoma by transcriptionally suppressing c-Myc

Acquisition of tissue specimens

The training cohort consisted of 98 patients with advanced ESCC with paraffin-embedded tissue archived at Sun Yat-sen University Cancer Center (Guangzhou, China) between 2002 and 2008. Thirty healthy esophageal mucosa tissue blocks were retrieved as the control. The validation cohort consisted of 46 patients with advanced ESCC receiving treatment at the Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). All tissue specimens were obtained as diagnostic biopsies via esophagoscopy and pathologically confirmed before initiation of any antitumor therapy. All patients received cisplatin-based chemotherapy and concurrent radiotherapy (daily dose of 1.8–2.0 Gy to a total dose of 60–70 Gy over 6–7 weeks). In addition, 10 paired fresh ESCC tissues and adjacent non-neoplastic esophageal mucosa tissues were collected at Tianjin Medical University Cancer Institute and Hospital. ESCC was staged according to the 6th edition of the International Union against Cancer (UICC 2002).

The study protocol was approved by the Ethics Committees at Sun Yat-sen University Cancer Center and Tianjin Medical University Cancer Institute and Hospital. Written informed consent was obtained from all patients. Patient data were anonymized.

Patient evaluation

Starting from 4 weeks after chemoradiotherapy, patients were evaluated every 3 months for the 1st year and then every 6 months for the next 2 years, and thereafter annually according to the World Health Organization (WHO) criteria. The diagnostic examinations consisted of esophagography, computed tomography (CT), chest X-ray, abdominal ultrasonography and bone scan, when necessary, to detect tumor recurrence and/or metastasis. Complete response (CR) was defined as no evidence of disease on imaging and complete resolution of all assessable lesions by endoscopic biopsy. Partial response (PR) was defined as a 30% or greater reduction in tumor maximum dimension and no progression of assessable lesions. Stable disease (SD) was defined by a reduction by < 50% or increase < 25% in tumor size. All these conditions had to last for at least 4 weeks and there was no appearance of new lesions. Progressive disease (PD) was defined as an increase ≥ 25% in tumor size or the appearance of new lesions.

Cells

Human ESCC cell lines KYSE30, KYSE510, KYSE410, and KYSE140 (South China State Key Laboratory of Oncology, Sun Yat-sen University), and TE-1 (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and primary cultured esophageal squamous epithelial cells (South China State Key Laboratory of Oncology) were used in the current study. KYSE30, KYSE150, KYSE410, and KYSE140 were maintained in RPMI-1640 (Gibco, Buffalo, Grand Island, NY, USA) and TE-1 in DMEM, supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ incubator. KYSE30 and TE-1 were authenticated by short tandem repeat fingerprinting at China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China). Radiation was delivered using 320 kV X-ray machine (Precision X Ray Inc.) at a dose rate of 2.3 Gy/min.

Immunohistochemistry

Paraffin-embedded tissue blocks were cut into 4-μm-thick sections, and dewaxed using xylene, followed by rehydration through gradient ethanol. Antigen retrieval was carried out by heating in a microwave. Tissue slides were immersed in 3% hydrogen peroxide for 15 min. Nonspecific binding was blocked by normal goat serum. Tissue slides were incubated overnight with anti-NS1-BP antibody (1:500, Abcam) at 4 °C, and then rinsed with phosphate buffered saline (PBS). Subsequently, the sections were incubated with a secondary antibody for 40 min at 37 °C, washed with PBS, and then stained with diaminobenzidine, followed by counter staining with hematoxylin.

NS1-BP expression was assessed by two independent pathologists who were blinded to patient data. Upon disagreement, the two pathologists reassessed the results until a consensus was reached. A staining index obtained as the intensity of positive staining (moderate low, 1; moderate high, 2; strong, 3) and the proportion of immune-positive cells of interest (0%–25%, 1; 25%–50%, 2; 50%–75%, 3; 75%–100%, 4) was calculated. The two scores were multiplied to yield the total score (1, 2, 3, 4, 6, 8, 9, and 12). Expression level was classified into either low (score 1–4) or high (score 6–12).

Western blotting assays

Antibodies against the following proteins were used for immunoblotting assays: NS1-BP, survivin and P27 (Abcam, Cambridge, MA, USA), c-Myc, cleaved-caspase3, cleaved-poly(ADP-ribose) polymerase (PARP), cyclin-dependent kinase (CDK) 4, phospho-ATM (Ser1981), phospho-Chk1 (Ser345) and phospho-Chk2 (Thr68) (Cell Signalling Technology, Danvers, MA, USA), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). GADPH was used as the loading control. Protein concentration was detected using a BCA kit.

MTT assays

Cell viability was measured by a commercially available 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma, St. Louis, MO, USA). Briefly, cells were seeded in 96-well plates, and then cultured for 4 h after addition of MTT. Optical density (OD) was measured at 490 nm using a microplate reader. The test was repeated 3 times independently.

Flow cytometry

For analysis of apoptosis, ESCC cells were stained by annexin V-FITC and propidium iodide to determine the percentage of apoptotic cells using a kit from BD Biosciences (Bedford, MA, USA). Each sample was then analyzed with flow cytometry (BD FACSCanto II Flow Cytometer; BD Biosciences).

For cell cycle analysis, cells were washed twice with PBS, and fixed in 70% ethanol overnight at 4 °C. The fixed cells were pelleted by centrifugation at 2000×g for 10 min, re-suspended in PBS, stained with 10 mg/mL propidium iodide for 5 min, and incubated in the dark at 4 °C for 30 min. The DNA content was determined using a flow cytometer (Model 100, Beckman Coulter, Kraeme, CA, USA) with excitation and emission settings of 488 and 610 nm, respectively. The proportion of G₂/M cells was calculated based on the relative DNA content, as measured by the flow cytometer using LYSIS software (Becton–Dickinson).

Clonogenic assays

Survival following ionizing irradiation (IR) was assessed for the ability of cells to maintain their clonogenicity. Briefly, after IR, KYSE30 and TE-1 ESCC cells were trypsinized, counted, and seeded for colony formation in 6-well plates with 50–5000 cells per well. After incubation intervals of 14–21 days, colonies were stained with crystal violet and manually counted. An aggregate consisting of 50 cells or more was considered a colony and scored, and 5 replicate wells containing 10–150 colonies per well were counted for each treatment. Experiments were done in triplicate.

Plasmid construction, lentivirus production and transduction

NS1-BP cDNA was cloned into a pCDH cDNA expression lentivector (System Biosciences; Mountain View, CA, USA). For loss-of-function study, plasmid containing a validated short hairpin RNA (shRNA) targeting NS1-BP was cloned into the vector pLLU2G, which is derived from pLL3.7 and contains separate GFP and shRNA expression elements as well as elements required for lentiviral packaging [20]. The target sequence of NS1-BP for constructing lentiviral shRNA was 5′-CCAGCAATGGCAAATTATA-3′ (shNS1-BP). The cDNA of NS1-BP construct and shRNA were purchased from Genechem (Shanghai, China). The lentiviral expression construct and packaging plasmid mix were co-transfected into 293 cells to generate the recombinant lentiviruses.

Immunofluorescence

Cells grown on coverslips were fixed in 4% paraformaldehyde. Fixed cells were permeabilized with 1% Triton-100 and blocked with 10% normal goat serum. Cells were then incubated with a primary antibody against γ-H2AX (Cell Signalling) for 2 h at 37 °C in a humidified chamber, followed by incubation with a secondary antibody for 1 h. Immunofluorescence images were captured using with FV10-ASW viewer software (Olympus, Tokyo, Japan).

Luciferase assays

For dual luciferase reporter assays, 5 × 10⁴ cells were seeded into each well of 24-well plates. A firefly luciferase reporter construct under the control of the c-Myc promoter (1 μg) and a Renilla luciferase reporter construct under the control of the TK promoter for normalization of transfection efficiency (10 ng) were co-transfected into cells in triplicate using FuGENE6 (Roche Diagnostics, Basle, Switzerland) at a ratio of 1 μg of plasmid to 3 μL of FuGENE6. Luciferase activity was examined at 24 h post transfection using the Dual-Luciferase Assay kit (Promega, Beijing, China) and normalized to firefly luciferase activity. Experiments were performed at least three times independently and each combination was tested in triplicate.

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assays were performed by using a kit from Merck Millipore (Darmstadt, Germany), with minor modifications [21]. The NS1-BP antibody (Abcam) and protein A/G PLUS-Agarose (Santa Cruz Biotechnology) were used. In human cells, four promoters, P0, P1, P2, and P3, of c-Myc have been documented, with P2 being the maximally used promoter [15, 22]. NS1-BP is one of the alpha-enolase/MBP-1 partners, and binds to the P2 promoter of c-Myc specifically [14, 23]. Herein, the P2 promoter was used in the ChIP assays, and the P0 promoter was used as a non-related c-Myc promoter negative control. The primer sequences used for the ChIP assays were as follows: P2, forward, 5′-AGGCGCGCGTAGTTAATTCAT-3′ and reverse, 5′-CGCCCTCTGCTTTGGGA-3′; P0, forward, 5′-CCCAACAAATGCAATGGGAGTTTATTCA-3′ and reverse, 5′-TCAAGAGTCCCAGGGAGAGTGGAGG-3′.

Xenograft assays

Four-week-old male severe combined immunodeficient (SCID-Beige) mice were injected subcutaneously with 2 × 10⁵ TE-1-NS1-BP or TE-1-NS1-BP-control cells. When the tumor mass became palpable (about 120 mm³), mice were randomly divided into a control group and an IR group (n = 6/group). Effects of IR (6 Gy, 2 Gy/fraction every other day for 3 days) were monitored. Tumor volume (V) was determined by measuring the length (L) and width (W) of the tumor with a caliper and calculated using the formula V = (L × W²) × 0.5. All the procedures were carried out in accordance with the guidelines of the laboratory animal ethics committee of Tianjin Medical University Cancer Institute and Hospital.

Statistical analysis

Statistical analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as mean ± standard error (SE) and analyzed with two-sided Student’s t test. χ² test was used to analyze the association between NS1-BP expression and clinicopathologic features of ESCC patients. Survival data were analyzed using the Kaplan–Meier analysis followed by the log-rank test. Univariate and multivariate survival analysis were done using the Cox-regression model. Statistical significance was set at P < 0.05.

NS1-BP is downregulated in ESCC tissues

Protein expression was first examined by Western blotting assays in 10 paired fresh ESCC and adjacent normal esophageal tissues. NS1-BP levels were lower in ESCC tissues than adjacent normal tissues in 8 cases (Fig. 1a). Immunostaining revealed lower NS1-BP expression in the primary lesions in the training cohort that consisted of 144 patients with advanced ESCC versus 30 normal esophageal mucosa tissues (Fig. 1b–f). Based on the immunohistochemistry evaluation, 59% (85/144) of the ESCC tumor tissues had low NS1-BP expression. In contrast, only 30% (9/30) of normal esophageal mucosa tissues had low NS1-BP expression (P = 0.012). NS1-BP levels were significantly lower in tumor tissues than normal esophageal mucosa tissues (Fig. 1f, P < 0.01).

Downregulated NS1-BP predicts chemoradiotherapeutic resistance

NS1-BP downregulation predicts a shorter DSS

NS1-BP suppresses growth and promotes apoptosis of ESCC cells

All five ESCC cell lines expressed lower NS1-BP levels than the control cells (Fig. 2a). TE-1 and KYSE-30 cells with stable NS1-BP transfection had moderate NS1-BP expression levels (Fig. 2b). The MTT assays showed that ectopic overexpression of NS1-BP inhibited growth of both TE-1 and KYSE-30 cells compared with empty vector control (Fig. 2c). Flow cytometry indicated that NS1-BP significantly promoted apoptosis of TE-1 and KYSE-30 cells (P < 0.05, Fig. 2d).

NS1-BP modulates ESCC radiosensitivity in vitro

Clonogenic assays showed that NS1-BP-overexpressing ESCC cells had lower clonogenicity than the control cells after IR (Fig. 3a). Compared with the negative controls, over-expression of NS1-BP significantly increased the apoptotic rate of cells upon treatment with IR (Fig. 3b).

The number of γ-H2AX foci, a hallmark of DNA damages, increased in NS1-BP-overexpressing TE-1 and KYSE-30 cells compared with the corresponding controls 12 h after IR (Fig. 3c). In contrast, depletion of NS1-BP enhanced cellular radioresistance, and decreased the number of apoptotic cells and the amount of unrepaired DNA damages induced by IR (Fig. 4a–e). Over-expression of NS1-BP significantly decreased IR-induced G₂/M cell cycle arrest after IR (4 Gy) in both ESCC cell lines (Fig. 3d). Western blot analysis showed dramatic increases in cleaved caspase-3 and PARP in NS1-BP-overexpressing ESCC cells compared with the control (Fig. 3d).

NS1-BP suppresses c-Myc at the transcriptional level

Overexpression of NS1-BP inhibited c-Myc expression in both TE-1 and KYSE-30 cells (Fig. 5a). Western blot analysis confirmed that the key proteins required for cell cycle checkpoint control also showed corresponding changes (Fig. 5a). In particular, survivin, a regulator of the mitotic spindle checkpoint, and CDK4 levels were decreased, whereas the inhibitory protein of cell cycle progression p27 increased in NS1-BP-overexpressing ESCC cells (Fig. 5a). The infection of NS1-BP-overexpressing ESCC cells with c-Myc lentivirus to achieve ectopic overexpression of c-Myc restored the levels of survivin, CDK4, and p27 (Fig. 5b).

Western blot analysis showed that IR activated both ATM/Chk1 and ATM/Chk2 signalling (Fig. 5c). Overexpression of NS1-BP significantly inhibited IR-induced ATM/Chk1 phosphorylation, but did not affect Chk2 phosphorylation (Fig. 5c). To further evaluate the mechanism whereby NS1-BP suppresses c-Myc expression, we tested the effect of NS1-BP overexpression using a reporter plasmid containing the c-Myc promoter upstream of the firefly luciferase gene. Compared with the control cells, luciferase activity was reduced by over 50% in NS1-BP-overexpressing cells (Fig. 5d). ChIP assays indicated that NS1-BP binds to the promoter regions of c-Myc in both NS1-BP-overexpressing cells and the control cells (Fig. 5e).

NS1-BP radiosensitizes ESCC cells in vivo

We next investigated whether NS1-BP could affect ESCC cellular response to IR in vivo. NS1-BP-overexpressing TE-1 cells and the control cells were inoculated into female athymic nude mice. When tumors reached a size of at least 180 mm³, the xenografts were irradiated at a dose of 6 Gy (2 Gy × 3 fraction). Consistent with the in vitro results, NS1-BP inhibited growth of TE-1 xenografts (Fig. 6a). NS1-BP also significantly delayed tumor xenograft growth treated with IR (Fig. 6a). Immunohistochemistry showed lower c-Myc expression in NS1-BP-overexpressing cells than the TE-1 vector control cells (Fig. 6b).

c-Myc levels negatively correlate with NS1-BP expression

NS1-BP expression was inversely correlated with c-Myc levels (Pearson r = − 0.7989, P < 0.01, Fig. 7a, b). Higher c-Myc levels were also associated with a shorter DSS (Fig. 7c, d).