Human tissue samples were obtained through surgical resection at Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University from June, 2006 to July, 2009. All tissue specimens were collected, frozen and stored in liquid nitrogen until assayed. All tissue samples were obtained with informed consent, and all procedures were performed in accordance with the Internal Review and the Ethics Board of the Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University. All clinical information was obtained from patients during follow-up. Moreover, overall survival (OS) was calculated as the time from the date of diagnosis to the date of death or the date of the last follow-up (if death did not occur). Disease-free survival (DFS) was calculated as the time from the date of surgery to the date of the first recurrence or metastasis after surgery (in patients with recurrence or metastasis) or to the date of the last follow-up (in patients without recurrence and metastasis). Lymph node recurrence-free survival (LNRFS) was defined as the time from the date of surgery to the date of lymph node relapse, and distant recurrence-free survival (DRFS) was defined as the time from the date of surgery to the date of distant recurrence. Clinical information of the samples is shown in detail in Table I.

SCCA protein expression was analyzed by immunohistochemistry (IHC) on paraffin-embedded PTC tissue samples as previously described in our study (35). Briefly, the specimens were cut into 5-µm sections and baked at 65°C for 30 min. The sections were deparaffinized and antigenic retrieval was carried out. The sections were treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity followed by incubation with 1% bovine serum albumin to block the nonspecific binding. The SCCA antibody (cat. no. ab154971, 1:100 dilution; Abcam, Cambridge, MA, USA) was incubated with the sections overnight at 4°C. After washing, the tissue sections were treated with biotinylated secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 60 min at room temperature. After rinsing with PBS, the slides were immersed for 2–5 min in DAB (3,3′-diaminobenzidine) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution, then monitored under a microscope. The reaction was terminated with distilled water. Slides were then counterstained with hematoxylin, dehydrated and coverslipped.

The quantitative analysis of IHC staining was performed for SCCA protein expression levels in tissue specimens. Two experienced investigators scored independently all the slides using a method previously described (35). Scores considered both the proportion of positive-staining tumor cells and the staining intensity. The proportion of positively stained tumor cells was graded as follows: 0 (no positive tumor cells), 1 (10% positive tumor cells), 2 (>10-50% positive tumor cells) and 3 (>50% positive tumor cells). The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown) and 3 (strong staining, brown). The staining index (SI) was calculated as follows: SI = staining intensity × proportion of positively stained tumor cells. Using this method of assessment, we evaluated SCCA expression in tumor tissues by SI (scored as 0, 1, 2, 3, 4, 6 or 9). Cutoff values to define the high and low expression of SCCA were chosen based on a measure of heterogeneity with the log-rank test statistics with respect to OS. SCCA staining was quantified using a two-level grade system as follows: 0–3 indicates low expression and 4–9 indicates high expression. Mean optical density (MOD) represents the strength of staining signals by measuring per positive pixels.

K1 and 293T cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplementing with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂ according to the manufacturer's instructions. Ectopic SCCA or Ras expression in the K1 cell line was achieved using retroviral vectors. Briefly, SCCA or Ras cDNA was cloned into retroviral transfer plasmid pMSCv to generate the pMSCv-SCCA or pMSCv-Ras expression vector, which was co-transfected in 293T cells (ATCC) using standard calcium phosphate transfection method. Thirty-six hours after the co-transfection, supernatants were collected and incubated with the K1 cells for 24 h for the following assays. Knockdown SCCA or Ras cells were transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) using siRNA (Shanghai GenePharma Co., Ltd., Shanghai, China) according to the manufacturer's instructions. siRNA sequences of the sense strands were as follows: siSCCA-1, 5′-GCACAACAGATTAAGAAGGTT-3′ and siSCCA-2, 5′-CCGCTGTAGTAGGGATTCGGAT-3′; siRas-1, 5′-GGACGAAUAUGAUCCAACATT-3′ and siRas-2, 5′-UGUUGGAUCAUAUUCGUCCTT-3′.

RNA was extracted using TRIzol reagent and a reverse transcription kit following the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). PCR reactions were performed under the following conditions: Pre-denaturation at 94°C for 7 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, elongation at 72°C for 1 min and elongation at 72°C for 10 min. The primers for quantitative real-time PCR assay were: SCCA, 5′-CATTTGTTTGCTGAAGCCACTAC-3′ and 5′-CATGTTCGAAATCCAGTGATTCC-3′; GAPDH, 5′-AAGGTCGGAGTCAACGGATTTG-3′ and 5′-CCATGGGTGGAATCATATTGGAA-3. All primers were synthesized by Shanghai General Biotech, Co., Ltd. (Shanghai, China). GAPDH was used as an endogenous control. The 2^−ΔΔCq method was used to quantify the relative mRNA amount (36).

The samples were lysed in RIPA buffer consisting of 20 mM Tris-HCl (pH 7.9), 137 mM NaCl, 5 mM EDTA, 1 mM EGTA, and 1% Triton X-100 that contained protease inhibitors for 20 min at 4°C, and total protein concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific, Inc.). Twenty micrograms of proteins was separated on a 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics, Indianapolis, IN, USA). Then, the membranes were blocked in 5% skim milk in TBS-Tween buffer at room temperature for 2 h and incubated with a primary antibody against SCCA (dilution 1:1,000; cat. no. ab154971; Abcam), Ras (dilution 1:1,000; cat. no. 3339; Cell Signaling Technology, Inc.) or GAPDH (dilution 1:2,000; cat. no. ab9485; Abcam) at 4°C overnight. Then, the membranes were incubated with HRP-conjugated secondary antibodies (dilution 1:1,000; cat. no. 3056; Epitomics, Burlingame, CA, USA) at 4°C for 2 h. The peroxidase activity was detected using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA).

In brief, cell viability was evaluated in accordance with the MTT method. K1 cells were seeded in 96-well plates at 3×10⁴ ectopic expressed cells/ml and cultured for pre-selected time-points. Then, the cells were incubated with 10 µl of 0.5% MTT solution for 4 h at 37°C. The supernatant was discarded, and 150 µl of dimethyl sulfoxide (DMSO) was added to each well. The 96-well plates were shaken for 10 min until the crystals dissolved completely. The absorbance was measured at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Cells (1×10⁶) were suspended in 200 µl serum-free medium and seeded in the top chambers of 24-well Transwell plates (Corning Inc., Corning, NY, USA) coated with 30 µl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The bottom chambers of the Transwell plates were filled with 600 µl medium containing 10% FBS. Cells were allowed to migrate for 48 h at 37°C. After migration, cells in the top chambers were removed using a cotton swab, and the cells which migrated to the bottom chambers were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA). The fixed and stained cells were counted in three independent fields under a using an Olympus BX51 microscope at ×100 magnification (Olympus Corp., Tokyo, Japan). At least, three chambers were counted for each experiment. For the migration assay, a similar protocol was followed except for the replacement of the top chamber of the Transwell plate with an uncoated chamber. The bottom chamber was filled with 700 µl medium containing 10% FBS, and cells were allowed to migrate for 24 h.

K1 cells (1×10⁷) with ectopic SSCA expression were injected into the mammary fat pads of 6- to 8-week-old athymic female nude mice purchased from the Shanghai Experimental Animal Center (Shanghai, China). Nine mice weighing 23–25 g were used, and they were maintained under SPF conditions at 20–26°C, relative humidity 40–70% and a 12-h light-dark cycle. All food was treated with high temperature steam disinfection (60 min, 120°C). All water was acidified by hydrochloric acid and adjusted to a pH between 2.5 and 2.8. All efforts were made to minimize animal suffering. Tumor growth and progression were monitored for more than 9 weeks. Tumor size was measured once a week by calipers, and tumor volume was calculated as: Volume (mm³) = length × width² × 0.5. At the end of the experiment, animals were euthanized by CO₂ asphyxiation, and tumors were carefully resected, weighed and stored for analysis.

Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The Chi-squared test was used to analyze the associations between SCCA expression and clinicopathological factors. Kaplan-Meier analysis was used for overall survival (OS), disease-free survival (DFS), lymph node recurrence-free survival (LNRFS) and distant recurrence-free survival (DRFS) calculations. Cox regression analysis determined the prognostic significance and pathologic characteristics. The correlation between SCCA and Ras mRNA expression was determined using the Pearson's correlation test. The comparison of two independent groups was analyzed using Student's t-tests. Multiple group comparisons were analyzed with one-way ANOVA and Tukey's post hoc test. All statistical tests were two-tailed, and P-values <0.05 were considered statistically significant.

A total of 258 postoperative samples obtained from patients diagnosed with different stages of PTC, based on the TNM staging system (37), were analyzed: Stage I (n=52), stage II (n=68), stage III (n=79) and stage IV (n=59). The clinical features of the patients with high and low levels of SCCA expression are summarized in Table I. Low SCCA expression was observed in 47.29% (122/258) of the samples, while 52.71% (136/258) showed high SCCA expression levels. In tumors >4 cm, 67.65% (69/102) of the samples showed high SCCA expression, while only 32.35% (33/102) of tumors showed low SCCA expression. Moreover, more than 50% of patients (131/258) showed extra thyroid invasion. Moreover, 39.15% of patients (101/258) presented with lymph node metastasis (LNM), and 27.52% of patients (71/258) had distant organ metastasis (DOM). Based on the TNM staging system, 46.51% of patients (120/258) presented with a stage I+II disease, and 53.49% of patients (138/258) with a stage III+IV disease.

As summarized in Table I, there was a significant correlation between high SCCA expression and tumor size (P=0.0001), extrathyroid invasion (P=0.0029), lymph node metastasis (P=0.0011), distant organ metastasis (P=0.0167), and TNM stage (P=0.0022). These results indicate that SCCA expression is significantly correlated with aggressive metastasis and progression in PTC.

Next, we explored whether SCCA expression levels are correlated with tumor histologic features, by examining 28 non-cancerous tissues and 258 PTC tissues, including 52 stage I, 68 stage II, 79 stage III and 59 stage IV tissues, by IHC. SCCA expression in non-cancerous tissues was low or non-detected. In contrast, SCCA protein was highly expressed in higher stages of PTC, compared with non-cancerous tissues (Fig. 1A). In addition, SCCA expression increased as the stage of PTC increased. The mean optical density (MOD) of SCCA staining among non-cancerous tissues and PTC specimens of different stages is documented in Fig. 1B. Stronger SCCA staining was observed as the PTC malignant stage degree increased (P<0.05). In agreement with the IHC results, SCCA mRNA levels, as determined by qRT-PCR assays, also increased and were higher as the malignant degree increased (P<0.05; Fig. 1C). To validate these findings, we examined SCCA protein expression levels in PTC stages I to IV by immunoblotting. Compared with lower stage PTC, SCCA protein expression was markedly strong in higher stage tumors (Fig. 1D). Noteworthy, there was a significant correlation between high SCCA expression and lymph node metastasis, and in most cases a strong SCCA staining was correlated with tumors with lymph node metastasis (Fig. 1E). Moreover, the primary PTC with LNM showed high SCCA mRNA level, compared with the tumors without LNM (P<0.001). Collectively, our findings indicate that higher SCCA expression levels are associated with PTC stage and metastasis.

We next investigated whether a correlation exists between SCCA levels in PTC and patient survival, using Kaplan-Meier analysis. During the 8-year patient follow-up, the overall survival (OS) rate was (119/136) 87.5% in the high SCCA expression group, but it was (117/122) 95.9% in the low SCCA expression group (P=0.0092; Fig. 2A). Our results also indicate that SCCA overexpression was significantly correlated with low disease-free survival (DFS) (P=0.0007; Fig. 2B). The prognostic value of SCCA expression was also estimated in the various subgroups of PTC patients, based on the TNM stage. In agreement with our previous observations, DFS in PTC patients with high SCCA expression was significantly reduced, compared to those with low SCCA expression in either stages I+II subgroup (P=0.0185; Fig. 2C) or stages III+IV subgroup (P=0.0133; Fig. 2D).

Since tumor metastasis plays an important role in poor prognosis and reduced survival, the correlation was assessed between SCCA expression and lymph node recurrence-free survival (LNRFS) and distant recurrence-free survival (DRFS), by the Kaplan-Meier method. We observed that SCCA expression was significantly associated with LNRFS (P=0.0376; Fig. 2E) and DRFS (P=0.0122; Fig. 2F).

To determine whether SCCA expression could be regarded as an independent prognostic factor, a Cox regression analysis for DFS was performed (Table II). Moreover, univariate analysis of the clinical features revealed significant correlation between DFS and tumor size (P=0.003), lymph node metastasis (LNM) (P=0.001), distant organ metastasis (DOM) (P=0.012), extrathyroid invasion (P=0.018), SCCA expression (P<0.001) and TNM stage (P=0.002). Additionally, using a multivariate analysis, we observed significant relationships between DFS and tumor size (P=0.015), LNM (P=0.001), DOM (P=0.026), extrathyroid invasion (P=0.021), SCCA expression (P<0.001) and TNM stage (P=0.003; Table III), which were regarded as significant prognostic factors for PTC patients. Taken together, our findings indicate that SCCA protein may be considered as a predictive prognostic factor in PTC patients.

To evaluate whether SCCA plays an important role in tumor growth, we performed SCCA overexpression and knockdown assays using plasmid and specific siRNAs (Fig. 3A). SCCA overexpression significantly promoted K1 thyroid cancer cell proliferation. In contrast, SCCA knockdown significantly inhibited cell proliferation as demonstrated using MTT assays in vitro (P<0.05; Fig. 3B and C). To assess whether SCCA was involved in PTC growth in vivo, we performed a tumor xenograft model using K1 cells, expressing different levels of SCCA protein. Representative images of the xenograft tumors at 9 weeks are shown (Fig. 3D). Nine weeks following implantation, tumors in which SCCA was knocked-down showed a reduced xenograft volume, while tumors with overexpressed SCCA had accelerated growth, compared to the control (P<0.05; Fig. 3E). In agreement, SCCA knockdown decreased tumor weight, while SCCA overexpression significantly increased tumor weight (P<0.01; Fig. 3F). Taken together, our data indicate that SCCA knockdown inhibits tumor growth, suggesting that SCCA protein could represent a target for PTC therapy.

Tumor invasiveness is significantly correlated with aggressive metastasis (6). To determine whether the SCCA protein plays an important role in tumor invasion, we performed migration and invasion assays in K1 cells with overexpressed or silenced SCCA. Overexpression of SCCA significantly increased K1 thyroid cancer cell migration, while SCCA knockdown significantly reduced the migratory capability of the K1 cells (P<0.05; Fig. 4A and B). In addition, SCCA overexpression significantly promoted the invasive capability of K1 thyroid cancer cells, while SCCA knockdown significantly inhibited the invasion of K1 cells (P<0.05; Fig. 4C and D). Collectively, these data suggest that SCCA protein may be associated with tumor metastasis.

Genetic alterations in the mitogen-associated protein kinase (MAPK) pathway play an important role in the progression of PTC (29). In addition, BRAF point mutations, RET/PTC oncogene rearrangements, and RAS point mutations are frequent in advanced PTC and are associated with poor prognosis (6,29,38). Previously, it was shown that Ras activation upregulated SCCA protein levels in colon cancer cell lines (34). However, whether Ras upregulates SCCA protein in PTC remains unclear. For this reason, to determine whether Ras upregulates SCCA protein to promote PTC progression, we analyzed SCCA expression using an RAS overexpression plasmid and specific siRas in vitro. Ras overexpression significantly increased SCCA protein level, while Ras silencing markedly decreased the SCCA protein level (Fig. 5A and B). In concordance, knockdown of Ras markedly decreased SCCA mRNA levels, while Ras overexpression increased the mRNA level of SCCA (P<0.01; Fig. 5C and D). Moreover, Ras overexpression significantly enhanced K1 thyroid cancer cell proliferation and invasion in vitro, while knockdown of Ras significantly suppressed cell proliferation and invasion (P<0.01; Fig. 5E and F). Noteworthy, Ras and SCCA mRNA expression levels increased from stage I to IV with similar style in PTC tissues by qRT-PCR assay (Fig. 5G). Moreover, SCCA mRNA levels were significantly correlated with Ras levels in PTC tissues (Fig. 5H, r=0.9449, P<0.0001). Taken together, these results indicate that Ras could upregulate SCCA expression to promote PTC proliferation and invasion.