Abstract
Natriuretic peptide receptor A (NPRA) is one of the natriuretic peptide receptors. NPRA has been reported to play a role in the carcinogenesis of various tumours, as well as functional roles in renal, cardiovascular, endocrine, and skeletal homeostasis. The clinicopathological significance of NPRA in tongue squamous cell carcinoma (TSCC) was examined in this study. The overexpression of NPRA was more frequent in TSCC (21/58, 36.2%) than in the normal oral epithelium (0/10, 0%) ( P < 0.05). It was also more frequently observed in cancers with higher grades according to the pattern of invasion (grades 1–2 vs. grades 3–4, P < 0.01). Additionally, there was a tendency towards an association between the N classification and NPRA expression (N0 vs. N1–2, P = 0.06). Significant correlations were also observed between the expression of NPRA and that of VEGF-A ( P < 0.001) and VEGF-C ( P < 0.001). The high-NPRA expression group had a significantly poorer prognosis, with a 5-year disease-specific survival rate of 39.7%, compared to 97.0% in the low-expression group ( P < 0.001). Multivariate analysis suggested that the overexpression of NPRA may also be an independent prognostic factor ( P < 0.05). In conclusion, NPRA is associated with VEGF expression levels, invasion, and metastasis, and may be a prognostic factor in TSCC patients.
Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the head and neck region and accounts for more than 90% of cancers in the oral cavity . The oral tongue is the most common site of OSCC. The primary therapeutic modality for OSCC is surgery.
Although recent advances in surgical techniques and anticancer agents have improved tumour regression and survival for patients with OSCC, the wide surgical resection of OSCC inevitably causes various oral dysfunctions. Therefore, new treatment strategies are urgently needed.
The presence of neck lymph node metastasis is strongly related to a poor prognosis in squamous cell carcinoma of the head and neck . Moreover, previous studies have reported that an alteration in the expression of adhesion-related molecules is associated with a poor prognosis in OSCC patients . Several tissue and biological markers have been identified as possible indicators of tumour aggressiveness and metastatic capability .
Angiogenesis and lymphangiogenesis are also crucial for tumour progression and nodal metastasis in OSCC . Some of the main angiogenic and lymphangiogenic factors identified belong to the vascular endothelial growth factor (VEGF) family of ligands and receptors, and include the angiogenic factors VEGF-A and VEGF receptor 2 (VEGFR2), as well as the lymphangiogenic factors VEGF-C/VEGF-D and VEGFR3 .
Natriuretic peptide receptor A (NPRA) is one of the natriuretic peptide receptors; it is a membrane-bound guanylate cyclase that serves as the receptor for both atrial and brain natriuretic peptides (ANP and BNP, respectively) . NPRA synthesizes the intracellular second-messenger cyclic guanosine monophosphate (cGMP) and activates cGMP-dependent protein kinase (PKG) in response to ANP binding . The expression of NPRA in cells of inflamed and injured tissues and in tumours has been reported . NPRA has also been shown to have effects on the cardiovascular system, including natriuretic, diuretic, vasorelaxant, and anti-proliferative responses altering the intracellular levels of cGMP . Furthermore, it affects cell growth, proliferation, apoptosis, and inflammation through cGMP-regulated transcription factors, ion channels, phosphodiesterases, and possibly other effector proteins . Increases in blood pressure and hypertensive heart disease have been shown in NPRA-gene knockout mice . More recently, NPRA has been reported to play a role in the carcinogenesis of various tumours, as well as functional roles in renal, cardiovascular, endocrine, and skeletal homeostasis . Moreover, the expression of VEGF was found to be down-regulated in the lungs of NPRA-deficient mice when compared to wild-type mice . However, the relationships between the expression of NPRA and clinicopathological features, as well as between the expression of NPRA and VEGF, have not yet been investigated in tongue squamous cell carcinoma (TSCC).
The purpose of this study was to determine the clinicopathological significance of NPRA in TSCC and clarify its correlation with VEGF expression in TSCC. An immunohistochemical analysis was performed to determine the relationships between the expression of NPRA and clinicopathological features in clinical TSCC samples.
Materials and methods
Patients
The study protocol was approved by the Ethics Committee of Nagasaki University Graduate School of Biomedical Sciences. Paraffin-embedded sections were obtained from the biopsy specimens of 58 patients with TSCC who had undergone radical surgery in Nagasaki University Hospital. The tumour stage was classified according to the TNM classification of the Union for International Cancer Control, and the histological differentiation was defined according to the World Health Organization classification. The pattern of invasion was determined according to the classification of Yamamoto et al. . As controls, 10 samples of the normal oral epithelium were obtained from 10 patients undergoing the routine surgical removal of third molars; informed consent was obtained from these patients.
Immunohistochemical staining and evaluations
Serial 4-μm-thick specimens were taken from tissue blocks. The sections were deparaffinized in xylene, soaked in target retrieval solution buffer (Dako, Glostrup, Denmark), and placed in an autoclave at 121 °C for 5 min for antigen retrieval. Endogenous peroxidase was blocked by incubating sections with 0.3% H 2 O 2 in methanol for 30 min. Immunohistochemical staining was performed using the Envision system (Envision+; Dako, Carpinteria, CA, USA). The primary antibodies used were directed against NPRA (ab70848; Abcam, Cambridge, UK), VEGF-A, and VEGF-C (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Sections were incubated with the primary antibody overnight at 4 °C. Reaction products were visualized by immersing the sections in diaminobenzidine (DAB) solution, and the samples were counterstained with Meyer’s haematoxylin and then mounted. Negative controls were prepared by replacing the primary antibody with phosphate-buffered saline.
The immunoreactivity of NPRA was scored based on the staining intensity and immunoreactive cell percentage as follows . The percentage of immunoreactive cells was graded on a scale of 0 to 4: score 0 for ≤5% positive tumour cells, score 1 for 6–25% positive tumour cells, score 2 for 26–49% positive tumour cells, score 3 for 50–75% positive tumour cells, and score 4 for ≥76% positive tumour cells. The staining intensity was graded from 0 to 3: 0 for no staining, 1 for weak staining (light yellow), 2 for moderate staining (yellow–brown), and 3 for strong staining (brown). The final score was obtained by multiplying the quality and intensity scores. A final score of 0 was considered negative, of 1–3 was regarded as weakly positive, and of 4–8 was regarded as strongly positive. In this study, strongly positive staining of NPRA was defined as the overexpression of this molecule.
In accordance with a previous study on VEGF expression , proportional scores described the estimated fraction of positively stained tumour cells as follows: staining index 0 = no staining, 1 = <10% of tumour cells, 2 = 10–50% of tumour cells, 3 = 50–80% of tumour cells, and 4 = >80% of tumour cells. The intensity score represented the estimated staining intensity as follows: staining index 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The immunohistochemical overexpression of VEGF-A and VEGF-C was defined as a total score greater than 4 . Total score is defined as the sum of scores of staining index and intensity scores.
Statistical analysis
Statistical analyses were performed using StatMate III (ATMS Co., Tokyo, Japan). The relationships between the expression of NPRA and clinicopathological features were assessed using Fisher’s exact test. Continuous data are presented as the mean ± standard deviation. Datasets were examined by one-way analysis of variance (ANOVA) followed by Scheffé’s post-hoc test. A survival analysis was performed with Kaplan–Meier curves and related log-rank tests. Prognostic factors were assessed using the Cox proportional hazards model. P -values of less than 0.05 were considered significant.
Results
Relationships between NPRA expression and clinicopathological features
Immunohistochemistry with an anti-NPRA polyclonal antibody was performed on samples obtained from 58 patients with TSCC. Representative immunohistochemical staining results are shown in Fig. 1 A and B. The overexpression of NPRA was undetectable in the normal epithelium. NPRA staining was mainly detected in the cytoplasm of squamous cell carcinoma cells ( Fig. 1 B). The nuclei of tumours were also partially stained. The overexpression of NPRA was more frequent in TSCC (21/58, 36.2%) than in the normal oral epithelium (0/10, 0%) ( P < 0.05). It was also more frequently observed in cancers of higher grades according to the pattern of invasion grades 1–3 vs. grade 4C/4D, P < 0.01; Table 1 ). Additionally, there was a tendency towards an association between the N classification and NPRA expression (N0 vs. N1–2, P = 0.06). These results strongly suggest that the overexpression of NPRA might be a strong predictor of survival through invasive potential in TSCC patients.
| Characteristics | Number of samples | NPRA overexpression (−) | NPRA overexpression (+) | P -value |
|---|---|---|---|---|
| Normal epithelia | 10 | 10 | 0 | <0.05 |
| Squamous cell carcinoma | 58 | 37 | 21 | |
| Sex | ||||
| Male | 40 | 26 | 14 | NS |
| Female | 18 | 11 | 7 | 0.776 |
| Age (years) | ||||
| ≤63 | 29 | 19 | 10 | NS |
| >63 | 29 | 18 | 11 | 0.785 |
| T classification | ||||
| T1 + T2 | 51 | 34 | 17 | NS |
| T3 + T4 | 7 | 3 | 4 | 0.219 |
| N classification | ||||
| N0 | 44 | 31 | 13 | NS |
| N1 + N2 | 14 | 6 | 8 | 0.061 |
| Stage | ||||
| Stage I–II | 43 | 30 | 13 | NS |
| Stage III–IV | 15 | 7 | 8 | 0.109 |
| Differentiation | ||||
| Well-differentiated | 52 | 35 | 17 | NS |
| Moderately/poorly differentiated | 6 | 2 | 4 | 0.101 |
| Pattern of invasion | ||||
| Grades 1–3 | 48 | 35 | 13 | <0.01 |
| Grades 4C/4D | 10 | 2 | 8 | |
| Local recurrence | ||||
| Negative | 50 | 34 | 16 | NS |
| Positive | 8 | 3 | 5 | 0.095 |
| Secondary metastasis | ||||
| Negative | 43 | 30 | 13 | NS |
| Positive | 15 | 7 | 8 | 0.109 |
Correlation between the expression of NPRA and VEGFs in TSCC
Angiogenesis and lymphangiogenesis have been shown to play crucial roles in tumour progression and nodal metastasis in OSCC . The family of VEGFs, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor, and VEGF-F, has previously been reported as crucially involved in angiogenesis and lymphangiogenesis . Of these VEGFs, VEGF-A and VEGF-C expression levels have previously been correlated with lymph node metastasis in oesophageal squamous cell carcinoma . In the present study, the relationships between the expression of NPRA and the expression of VEGF-A and VEGF-C were examined. Immunohistochemical staining of VEGF-A and VEGF-C was detected in the cytoplasm of both normal tissue and tumour cells ( Fig. 1 C and D). These proteins were found to be strongly expressed at the invasion front of the tumour. The overexpression of VEGF-A was more frequent in TSCC (28/58, 48.3%) than in the normal oral epithelium (0/10, 0%) ( P < 0.01). In addition, the overexpression of VEGF-C was more frequent in TSCC (21/58, 36.2%) than in the normal oral epithelium (0/10, 0%) ( P < 0.01). Correlations were also observed between the expression of NPRA and that of VEGF-A and VEGF-C (VEGF-A, P < 0.001; VEGF-C, P < 0.001; Table 2 ). These results also strongly suggest that the overexpression of NPRA might be a strong predictor of survival.
| Characteristics | Number of samples | NPRA overexpression negative | NPRA overexpression positive | P -value |
|---|---|---|---|---|
| VEGF-A expression | ||||
| Negative | 30 | 26 | 4 | <0.001 |
| Positive | 28 | 11 | 17 | |
| VEGF-C expression | ||||
| Negative | 37 | 32 | 5 | <0.001 |
| Positive | 21 | 5 | 16 | |
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