Expression of Stanniocalcin-1 and Stanniocalcin-2 in Laryngeal Squamous Cell Carcinoma and Correlations with Clinical and Pathological Parameters

Background Stanniocalcin-1 (STC1) and stanniocalcin-2 (STC2) are secreted glycoprotein hormones involved in various types of human malignancies. The roles of STC1 and STC2 in laryngeal squamous cell carcinoma (LSCC) remain unknown. We investigated correlations between STC1 and STC2 expression and clinicopathological or prognostic factors in LSCC. Methods Pre-surgical peripheral blood samples were collected between 2012 and 2013 from 62 patients with LSCC. Quantitative RT-PCR analysis was performed to examine mRNA levels of STC1 and STC2. Immunohistochemistry was performed to retrospectively analyze 90 paraffin-embedded LSCC tissue samples, which were obtained from patients who received surgery between 2006 and 2009. These patients did not have histories of treatment or malignancies. Univariate analysis of patient survival was performed by the Kaplan–Meier method. Multivariate analyses were performed with the Cox proportional hazards model. Results The relative mRNA levels of STC1 and STC2 in peripheral blood were significantly greater in LSCC patients than those of healthy volunteers (both P<0.05). STC2 protein expression in tumor tissues was associated with invasion into the thyroid cartilage, T-Stage, lymphatic metastasis, clinical stage, and pathological differentiation (all P<0.05). In addition, STC2 protein expression was an independent prognostic factor for overall survival in patients with LSCC (P = 0.025). In contrast, STC1 expression only correlated with clinical stage (P = 0.026) and was not an independent or significant prognostic factor. Conclusions Circulating STC1 and STC2 mRNA are potentially useful blood markers for LSCC. Our results strongly suggest that the STC2 protein, but not STC1, may be a valuable biomarker for LSCC malignancies and a prognostic marker for poor outcome following surgery. Future studies should examine STC2 as a novel molecular target for the treatment of LSCC.


Introduction
Laryngeal squamous cell carcinoma (LSCC) is one of the most common upper aerodigestive tract epithelial malignancies in the world [1]. LSCC can develop in any part of the larynx, including the glottis, supraglottic, and subglottic areas. In China, the incidence of LSCC has gradually increased over the past several decades. Currently, LSCC is the second most common malignant tumor of the head and neck in China [2]. Although patients with LSCC benefit from advanced diagnostic and therapeutic management, survival remains poor and has not improved during the past 30 years [3]. Current tumor-node-metastasis (TNM) staging criteria and differentiation grade are the main factors used to predict outcome in patients with LSCC [4,5]. However, these parameters do not accurately predict the future course of earlystage LSCC. Moreover, the molecular mechanisms by which LSCC initiates and progresses remain unclear. Therefore, the identification of sensitive and specific molecular markers of LSCC would facilitate early prevention, diagnosis, and treatment. Identification of relevant biomarkers is essential for understanding the pathogenesis of LSCC and for developing new targeted treatment strategies for this tumor type.
In order to investigate the expression profiles of STC1 and STC2 in LSCC, we assessed their expression levels in formalinfixed, paraffin-embedded tissue samples obtained from 152 patients. STC1 and STC2 mRNA expression levels were also assessed in blood specimens from 62 patients with LSCC and 30 healthy volunteers. Furthermore, possible correlations with clinicopathological and prognostic parameters were analyzed.

Ethics Statement
This study was conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent for the collection of samples and subsequent analysis. The study was approved by the Institutional Review Board of the First Affiliated Hospital of Nanjing Medical University (2012-NT-027).

Clinical Samples and Patient Population
Sixty-two patients who were diagnosed with LSCC and treated between January 2012 and April 2013 at the First Affiliated Hospital, Nanjing Medical University, were enrolled in this study. The median age of the patients was 60 years (range, 36 to 87 years), and the study included 58 males and 4 females. Thirty volunteers who visited the hospital for health examinations and appeared to have normal laryngeal mucosae according to laryngoscopic examinations were also enrolled. The healthy controls consisted of 27 males and 3 females with a median age of 59 years (range, 35 to 86 years). Five milliliters of peripheral blood (PB) were collected from each patient before surgery. Mononuclear cells were isolated from PB with lymphocyteseparation media (Sigma, St. Louis, USA). Total RNA was isolated with the RNeasy kit according to the manufacturer's protocol (Qiagen, Hilden, Germany) and stored at 280uC until further processing.
We also examined data for 90 eligible patients with LSCC (84 males, 6 females) who had detailed clinical records and follow-up data. These patients received surgery in our department from 2006 to 2009 and were followed for at least 2 years or died within two years of surgery. Clinical follow-up data were obtained by telephone or from outpatient records.
All of the patients with LSCC in our study had the following inclusion criteria: no history of radiotherapy or chemotherapy and a diagnosis of primary squamous cell carcinoma of the larynx without other malignancies. Clinical and pathological data were collected for the 152 patients with LSCC (62 patients from 2012-2013 and 90 patients from 2006-2009). Data included age, anatomical site, thyroid-cartilage invasion, differentiation grade, lymph node metastases, treatment, and recurrence. Tumor stage (T-stage) was classified according to the 2002 TNM staging system of the Union for International Cancer Control (UICC). Paraffinembedded LSCC samples were longitudinally sliced into 4-mmthick sections for immunohistochemical (IHC) analysis.  controlled by using five dilutions of cDNA. STC1 or STC2 mRNA levels were expressed as fold-increased or fold-decreased relative to GAPDH mRNA expression. The mean values of the replicates for each sample were calculated and expressed as cycle threshold (Ct). Gene expression levels were expressed as 2 2DCt , in which DCt was the difference between the Ct value of STC1 or STC2 and the Ct value of GAPDH.

Immunohistochemical Analysis
IHC staining for STC1 and STC2 was performed with Ultrasensitive immunohistochemistry S-P kits (Maixin Biology Corporation, Fuzhou, China) according to the manufacturer's instructions. Sections (4-mm thick) were deparaffinized, washed in Tris-buffered saline (TBS), and incubated with 3% H 2 O 2 in methanol for 20 minutes to block endogenous peroxidases. Sections were then washed with TBS and incubated with 5% skim milk in TBS for 20 minutes. Blocked sections were incubated with antibodies against STC1 (rabbit polyclonal anti-STC1 antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or STC2 (rabbit polyclonal anti-STC2 antibody; Novus Biologicals, Littleton, CO, USA) for 90 minutes at 37uC. A secondary antibody was applied to each section for 10 minutes at room temperature, and the slides were rinsed three times in phosphatebuffered saline (pH 7.4) after each incubation step. The slides were counterstained with hematoxylin, mounted, and observed by light microscopy.

Evaluation and Scoring of Immunohistochemistry
All IHC slides were independently and blindly assessed and scored by two pathologists with agreement. The final score was an average of the two scores. The staining score for STC1 or STC2 was assessed semiquantitatively according to the percentage of positive staining and the staining intensity. An unequivocal positive reaction was defined as a brown signal in the cytoplasm or on the cell membrane. The staining intensity was scored as 0 (no staining), 1 (weak staining exhibited as light yellow), 2 (moderate staining exhibited as yellow-brown), 3 (strong staining exhibited as brown). The percentage of positive staining was scored as 1, #10%; 2, 11-50%; 3, 51-80%; and 4, $81%. The final staining score (1 to 7) was calculated as the sum of the staining intensity and percentage score. For statistical evaluation, the expression of STC1 or STC2 was classified as negative (#10% of the area stained positive regardless of intensity), low (staining score was 3-4 and .10% of area stained positive), or high (staining score $5 and .10% of area stained positive) [15].

Statistical Analysis
Data were analyzed with SPSS19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 5.0 (GraphPad Inc., San Diego, CA, USA). Differences in STC1 or STC2 mRNA expression levels in the PB samples of patients with LSCC and that of healthy volunteers were assessed by the Mann-Whitney test. The associations of STC1 or STC2 expression in PB samples or tumors with clinicopathological variables were also evaluated by the Mann-Whitney test. The correlation between STC1 or STC2 expression in tumors and the circulating STC1 or STC2 mRNA level was analyzed by the Spearman's rank correlation coefficient. Survival times were evaluated by the Kaplan-Meier method with the log-rank test. Multivariate analyses were performed with the Cox proportional hazards model. P-value less than or equal to 5 percent was considered to be statistically significant.  Figure 1B). The levels of STC1 and STC2 mRNA expression were significantly higher in the PB samples from LSCC patients than those of healthy volunteers (both P, 0.05).

Correlation between Circulating STC1 or STC2 mRNA Level and Clinicopathological Findings in LSCC Patients
The data on the correlation between circulating STC1 or STC2 mRNA level and clinicopathological findings in LSCC patients is listed in Table 1. Peripheral STC1 expression significantly correlated with clinical stage. A possible relationship with thyroid-cartilage invasion was also found (P = 0.072). However, circulating STC1 mRNA levels were not significantly associated with other clinicopathological characteristics. STC2 expression was significantly correlated with thyroid-cartilage invasion, lymphatic metastasis, and clinical stage. Furthermore, a possible relationship between STC2 expression and T-Stage was found (P = 0.061). There was no significant correlation between STC2 expression and age, smoking index, drinking, type of laryngeal carcinoma, or pathological differentiation ( Table 1).

Expression of STC1 and STC2 in LSCC Tissues
IHC analysis showed that positive staining was observed in most of the tumor tissues and in a small portion of the adjacent normal tissues. Moreover, STC1 and STC2 staining was stronger in LSCC tissues than that of the corresponding normal laryngeal

Correlation of STC1 and STC2 Expression with Clinicopathological Findings in LSCC Patients
The data on the correlation of STC1 and STC2 expression with clinicopathological findings in LSCC patients is listed in Table 2. STC1 expression in tumor tissues significantly correlated with clinical stage. Potential relationships between STC1 expression and thyroid-cartilage invasion or T-Stage (P = 0.086, P = 0.083, respectively) were also identified. There was no significant correlation between STC1 expression and age, type of laryngeal carcinoma, lymphatic metastasis, or pathological differentiation. STC2 expression in tumor tissues significantly correlated with thyroid-cartilage invasion, T-Stage, lymphatic metastasis, clinical stage, and pathological differentiation. No significant correlation was found between STC2 expression and age or type of laryngeal carcinoma ( Table 2). In addition, the expression of STC2 in tumor tissues positively correlated with circulating STC2 mRNA levels in LSCC patients. However, there was no significant correlation between the levels of STC1 in tumor tissues and circulating STC1 mRNA levels ( Figure 4).

Correlation of STC1 or STC2 Expression with LSCC Prognosis
There were no significant differences in overall survival (OS) rates among LSCC patients with negative, low, or high STC1 expression levels in tumor tissues. In contrast, patients with high levels of STC2 expression in tumor tissues had significantly poorer OS rates than those with negative or low STC2 expression. Similarly, OS rates were lower in LSCC patients with low STC2 than those of patients with negative STC2 expression ( Figure 5).
Univariate Cox regression analyses determined that thyroidcartilage invasion, T-Stage, lymphatic metastasis, clinical stage, pathological differentiation, and STC2 expression in tumors were significantly associated with poor OS (all P,0.05). These six parameters were included in multivariate analyses. Multivariate Cox analyses demonstrated that lymphatic metastasis, pathological differentiation, and STC2 expression were independent factors with prognostic value for OS (P = 0.033, P = 0.018, and P = 0.025, respectively) in patients with LSCC ( Table 3).

Discussion
Aberrant expression of STC1 or STC2 correlates with poor prognosis in multiple types of cancer [8][9][10][11][12][13][14]. We found significantly greater levels of STC1 and STC2 mRNA in the PB samples of LSCC patients than those of healthy volunteers. These data are consistent with observations reported for gastric cancer [16,17], esophageal squamous cell carcinoma [18], and non-small cell lung cancer [12]. Furthermore, we investigated whether STC1 or STC2 levels in LSCC tumors were potential molecular markers of prognosis. We found that overexpression of the STC2 protein in surgically-resected LSCC tissues was associated with features of tumor progression and was an independent prognostic factor for OS. In contrast, STC1 protein expression correlated with fewer features of tumor progression and was not an independent or significant prognostic factor for OS in LSCC.
STC1 and STC2 are involved in tumor progression and metastasis [8]. However, the functional relationships between  STCs and human cancers have not been elucidated. Animal and cell culture studies have provided some mechanistic data, which reveal possible functions for STCs in cell growth and apoptosis. The growth-related functions of STCs may be associated with the pro-and anti-apoptotic effects of these proteins. Anti-apoptotic effects have been reported for STC1 in some types of cancer [19,20], whereas pro-apoptotic effects have been observed in other tumor types [21,22]. These inconsistencies suggest that STC1 may not be the key regulator of apoptosis in these systems [8]. Instead, the anti-or pro-apoptotic roles of STC1 may vary depending on the source, stage, or subset of cancer cells. Thus, the functions of STC1 in carcinogenesis are diverse and complex. In contrast to STC1, STC2 has been consistently found to have an antiapoptotic role in cancer cells [23,24]. The mechanism by which STC2 blocks apoptosis may be related to inhibition of plasma membrane store-operated Ca 2+ entry (SOCE), which protects cells from apoptosis [25]. The progression of solid tumors is usually associated with hypoxia. STC2 is a target gene of hypoxiainducible factor-1, which stimulates the proliferation of cancer cells during hypoxic conditions [26]. Some mechanistic and functional studies have showed that STC2 expression is induced by hypoxia and contributes to the suppression of apoptosis and  survival of cancer cells [23,26,27]. These findings suggest that STC2 is a positive regulator of tumor progression.
In some malignant neoplasms, including esophageal squamous cell cancers [13], gastric cancers [11], colorectal cancers [28], and renal cell carcinomas [29], high expression of STC2 correlates with tumor progression and poor prognosis, which is consistent with our findings in LSCC. Current evidence supports the notion that STC2 expression is associated with suppression of apoptosis, and cancer development, and dormancy with later relapse [8,26,27,30]. Similar mechanisms may explain why the cytoplasmic overexpression of STC2 in LSCC cells leads to a more clinically aggressive disease course. These findings suggest that assessing STC2 expression in combination with established clinicopathological features may be valuable for making informed prognostic or therapeutic decisions in patients with LSCC.
A considerable number of studies demonstrate that STC1 promotes tumor migration and invasion [9,18,20,31], with the exception of two studies in breast and ovarian cancer [32,33]. These inconsistent results may be due to the dynamic and complicated regulatory functions of STC1 in cell growth and apoptosis [8]. Furthermore, the expression of STC1 varies among different tissues and may also vary throughout a given tissue section [7]. Therefore, the functions of STC1 are likely to vary among human tumors. Due to some limitations of this study, the relationship between STC1 expression and LSCC progression and malignancy may not be evaluated precisely. However, our results indicate that the role of STC2 in the development of LSCC is more important than that of STC1.
Tumor cells can disseminate from the primary tumor through the blood or lymphatic circulation during early stages of disease. These cells can survive without causing clinical symptoms and eventually promote recurrence of disease [34][35][36]. Thus, it is imperative to monitor circulating tumor cells as an approach to improve diagnosis or prognosis and develop more effective treatments. Consequently, it is important to identify sensitive and specific blood markers of circulating tumor cells. STC1 and STC2 are involved in various biological mechanisms of tumor progression [8,[11][12][13][14]. Both STC1 and STC2 are considered to be promising biomarkers, because their mRNA levels are elevated in the PB of cancer patients [12,[16][17][18]. However, little is known regarding the clinical significance of STC1 and STC2 expression levels in PB from patients with LSCC.
Our results showed that circulating STC1 or STC2 mRNA levels were significantly correlated with one or more features indicative of worse tumor biology. In comparison with STC1, circulating STC2 mRNA was up-regulated more significantly in LSCC patients than that of healthy volunteers. In addition, circulating STC2 mRNA was significantly associated with more characteristics of tumor migration and invasion. Therefore, we can conclude that STC2 mRNA expression is a more promising and reliable blood marker for predicting biological tumor aggressiveness in patients with LSCC. In addition, circulating STC2 mRNA levels correlated with the expressions of STC2 in tumor tissues in LSCC patients. However, expression levels of the mRNA and protein of STC1 did not correlate. This might partly explain why STC2 mRNA expression correlates more closely with biological tumor aggressiveness in LSCC.
In combination with clinicopathological features and other biomarkers of LSCC, STC1 and STC2 expression may be useful for stratifying patients for individual treatments, such as adjuvant chemotherapy or radiotherapy. Some patients demonstrate disease recurrence or metastasis at an early stage after receiving the initial operation. In contrast, others demonstrate long-term survival despite diagnosis at a late stage of disease. This discrepancy may be due to differences in the molecular biology of individual tumors [37]. STC1 or STC2 status may play important roles in tumor biology. Thus, if a patient is found to express high levels of STC1 or STC2, comprehensive treatments, including adjuvant radiochemotherapy, should be recommended. This approach may improve patient survival by eliminating circulating tumor cells and suppressing micrometastases. Moreover, a recent study shows that STC1 and STC2 promote angiogenic sprouting and stimulate vascular endothelial growth factor (VEGF) signaling [38]. Therapies targeted against mediators of angiogenesis, such as VEGF inhibitors, have been shown to prolong the lives of numerous cancer patients. However, primary or secondary resistance to anti-VEGF therapies and multiple side effects remain important challenges to this treatment approach [39]. Hence, alternative, less toxic therapies are needed. STCs represent novel targets against which anti-angiogenesis cancer treatments can be developed. In the present study, STC1 and STC2 expression did not show similar correlations with the development and progression of LSCC. STC2 expression has more clinical significance for the diagnostic and prognostic assessment of LSCC, whereas STC2 may be more likely to serve as a new molecular therapeutic target. The limitations of this study included the retrospective design and small sample size. In addition, clinical data were not available for existing physical illnesses, occupational information, post-surgical radiotherapy, and other factors that may have influenced the findings of this study. Further investigations in other patient populations or large-scale, well-characterized patient samples may be required to confirm our findings.
In summary, we evaluated the expression status of STCs in tumors and PB of patients with LSCC. Our data indicate that circulating STC1 and STC2 mRNA are potentially useful blood markers for LSCC. Most importantly, these results strongly suggest that the STC2 protein may be a valuable biomarker of malignancy in patients with LSCC and an effective prognostic marker of poor post-surgical outcome. The STC1 protein did not show significant correlations with prognosis of patients with LSCC. Further studies are needed to clarify the mechanisms by which STC1 and STC2 are involved in the development and progression of LSCC. These studies will also help clarify the regulatory mechanisms and roles of STCs in tumor angiogenesis, proliferation, and apoptosis.