Pituitary tumor-transforming 1 increases cell motility and promotes lymph node metastasis in esophageal squamous cell carcinoma

Abstract

Human pituitary tumor-transforming 1 (PTTG1)/securin is a putative oncoprotein that is overexpressed in various tumor types. However, the involvement of PTTG1 in gastrointestinal cancer development and progression remains unclear. In this study, we investigated the clinical significance and biological effects of PTTG1 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical studies performed on 113 primary ESCC specimens revealed a high prevalence of PTTG1 overexpression (60.2%), which was significantly associated with lymph node metastasis (regional, P = 0.042; distant, P = 0.005), advanced tumor stage (P = 0.028), and poorer overall survival (P = 0.017, log-rank test; P = 0.044, Cox proportional hazard model). Eleven ESCC cell lines expressed PTTG1 protein at levels 2.4 to 6.6 times higher than those in normal esophageal epithelial cells (HEEpiC). PTTG1 protein expression was confined to the nucleus in HEEpiC cells but present in both the cytoplasm and nucleus in ESCC cells. Two small interfering RNAs (siRNA) inhibited PTTG1 mRNA and protein expression in three ESCC cell lines by 77% to 97%. In addition, PTTG1 down-regulation by these siRNAs significantly reduced cell motility in all three ESCC cell lines (P < 0.01) in vitro, as well as popliteal lymph node metastases of ESCC cells in nude mice (P = 0.020). Global gene expression profiling suggested that several members of the Ras and Rho gene families, including RRAS, RHOG, ARHGAP1, and ARHGADIA, represented potential downstream genes in the PTTG1 pathway. Taken together, these findings suggest that PTTG1 overexpression promotes cell motility and lymph node metastasis in ESCC patients, leading to poorer survival. Thus, PTTG1 constitutes a potential biomarker and therapeutic target in ESCCs with lymph node metastases.

Figure 1

PTTG1 protein expression in ESCC tumor tissues and cell lines. A, PTTG1 protein expression level in primary ESCC tumors was classified according to cytoplasmic staining of PTTG1. Top left, normal esophageal epithelium with negative PTTG1 expression; top right, ESCC with weak PTTG1 expression (1+, 0–10% of cells stained); bottom left, ESCC with moderate PTTG1 expression (2+, 10–30%); bottom right, ESCC with strong PTTG1 expression (3+, >30%). B, overall survival analysis by the Kaplan-Meier method. Primary ESCCs with moderate to strong PTTG1 expression (2+ and 3+) were classified as PTTG1 positive. Patients with PTTG1-positive tumors (n = 68) had significantly worse survival than did those with PTTG1-negative tumors (n = 45; P = 0.017, log-rank test). C, PTTG1 protein expression in 11 ESCC cell lines and in primary-cultured normal esophageal epithelial cells (HEEpiC). Equal amounts of whole cellular protein (15 μg) were loaded in all lanes. Immunoblot membranes were probed with anti-PTTG1 or anti–β-actin antibodies. The KYSE series and HSA/c composed the human ESCC cell lines. Bottom, PTTG1 expression fold change in ESCC cell lines relative to HEEpiC, normalized to β-actin expression. D, subcellular localization of PTTG1 in ESCC cell lines (HSA/c, KYSE140, and KYSE410) and HEEpiC. Equal amounts of the cytoplasmic or nuclear fractionated protein (20 μg) were loaded in all lanes. Purity of fractions was verified with anti-GAPDH (cytoplasm) and anti–lamin A/C (nucleus) antibodies. C, cytoplasmic fraction; N, nuclear fraction.

Figure 1

PTTG1 protein expression in ESCC tumor tissues and cell lines. A, PTTG1 protein expression level in primary ESCC tumors was classified according to cytoplasmic staining of PTTG1. Top left, normal esophageal epithelium with negative PTTG1 expression; top right, ESCC with weak PTTG1 expression (1+, 0–10% of cells stained); bottom left, ESCC with moderate PTTG1 expression (2+, 10–30%); bottom right, ESCC with strong PTTG1 expression (3+, >30%). B, overall survival analysis by the Kaplan-Meier method. Primary ESCCs with moderate to strong PTTG1 expression (2+ and 3+) were classified as PTTG1 positive. Patients with PTTG1-positive tumors (n = 68) had significantly worse survival than did those with PTTG1-negative tumors (n = 45; P = 0.017, log-rank test). C, PTTG1 protein expression in 11 ESCC cell lines and in primary-cultured normal esophageal epithelial cells (HEEpiC). Equal amounts of whole cellular protein (15 μg) were loaded in all lanes. Immunoblot membranes were probed with anti-PTTG1 or anti–β-actin antibodies. The KYSE series and HSA/c composed the human ESCC cell lines. Bottom, PTTG1 expression fold change in ESCC cell lines relative to HEEpiC, normalized to β-actin expression. D, subcellular localization of PTTG1 in ESCC cell lines (HSA/c, KYSE140, and KYSE410) and HEEpiC. Equal amounts of the cytoplasmic or nuclear fractionated protein (20 μg) were loaded in all lanes. Purity of fractions was verified with anti-GAPDH (cytoplasm) and anti–lamin A/C (nucleus) antibodies. C, cytoplasmic fraction; N, nuclear fraction.

Figure 2

Knockdown of PTTG1 expression in ESCC cells by siRNAs. HSA/c, KYSE140, and KYSE410 cells were transfected using the following conditions: LF−, untransfected control; LF+, transfection with Lipofectamine RNAiMAX alone; NTC, nontargeting control siRNA; P1, siRNA 1 directed against PTTG1; P2, siRNA 2 directed against PTTG1. A, PTTG1 mRNA expression levels in siRNA-treated cells. Each value represents the ratio of PTTG1 level versus HSA/c LF−, normalized to β-actin. Numbers at the bottom represent the ratio of PTTG1 expression for each condition relative to NTC-transfected cells. B, PTTG1 protein expression in siRNA-treated cells. Equal amounts of whole cellular protein (9 μg) were loaded in all lanes. Numbers at the bottom represent the ratio of PTTG1 expression for each condition relative to PTTG1 expression in NTC-transfected cells. C, subcellular localization of PTTG1 in siRNA-treated HSA/c cells. Equal amounts of cytoplasmic or nuclear fractionated protein (20 μg) were loaded in all lanes. Purity of fractions was verified with anti-GAPDH (cytoplasm) and anti–lamin A/C (nucleus) antibodies. D, immunofluorescence staining for PTTG1. Cells were stained with PTTG1, which was labeled with Alexa Fluor 568 (red), and nuclei were counterstained with DAPI (blue). Left, cells transfected with NTC stained without PTTG1 primary antibody and only with secondary antibody-Alexa Fluor 568; middle, cells transfected with NTC; right, cells transfected with P1. Photographs were taken at identical exposure intervals (18.2 ms; magnification, ×100).

Figure 3

Effects of PTTG1 knockdown on cell migration in vitro and lymph node metastasis in vivo. A, cell migration/invasion assays in vitro. Columns, mean of triplicate experiments; bars, SD. Left, Transwell migration assay; numbers of cells migrating through Transwell membranes are shown. Middle, Matrigel invasion assay; numbers of cells migrating through Matrigel inserts are shown. Right, invasion index (%); ratios of cells migrating through Matrigel inserts relative to mean numbers of cells migrating through Transwell membranes are shown. **, P < 0.01, versus NTC for each group (Tukey-Kramer test). B, lymph node metastasis assay in nude mice. Top left, a leg of a nude mouse at 28 d after inoculation of HSA/c cells into the footpad. The leg skin was stripped to show a swollen popliteal lymph node. The distance between the footpad tumor and the popliteal lymph node was ~1 cm. Bottom left, H&E staining of a footpad tumor (magnification, ×200). Note the “cancer pearl” in the center. Middle, PTTG1 immunostaining of a footpad tumor treated with NTC (top) and P1 (bottom; magnification, ×100). Right, representative H&E staining of popliteal lymph nodes treated with NTC (top) and P1 (bottom; magnification, ×100).