Role of endothelin receptor signalling in squamous cell carcinoma

Endothelin plays important roles in various physiological functions including vascular constriction. Recent studies reported that the endothelin receptors ETA and ETB are highly expressed in lung and skin tumor tissues. In contrast, there are few reports on endothelin signalling in the proliferation of head and neck cancer. We found that both ETA and ETB endothelin receptors were overexpressed in tumor cells of tongue cancer samples by immunohistochemistry. ETA and ETB were expressed in cultured lingual and esophageal squamous cell carcinoma (SCCs) cell lines. When both cultured cell lines were treated with an ETA selective antagonist (BQ123) or an ETB selective antagonist (BQ788), inhibition of cell growth was observed. Similar results were observed when SCCs were treated with specific siRNA for the suppression of ETA or ETB. Furthermore, inhibition of the mitogen-activated protein (MAP) kinase pathway by the treatments with ET receptor antagonists and siRNA was also observed. These results indicate that endothelin signalling may, in part, play important roles in cell growth in SCCs through the MAP kinase pathway.


Introduction
Endothelin (ET) plays important roles on various physiological functions including vascular constriction (1)(2)(3)(4). ET family comprises three isoforms, ET-1, ET-2, and ET-3, that bind to two receptor subtypes, endothelin A (ET A ) and endothelin B (ET B ) receptors (1)(2)(3)(4). Recent studies reported that ET A and ET B were highly expressed on lung, colon and skin cancers (5)(6)(7). In addition, several reports suggested that ET-1 plays important roles in tumorigenesis, tumor progression, and metastasis (8)(9)(10). Thus, the ET receptors and their signalling pathways may be a therapeutic target in cancer therapy (11). However, little is known about the role of ET signalling on tumor cell proliferation of oral squamous cell carcinoma (SCC).
Human SCC is major neoplasm in esophagus or oral cavity and the incidence has recently been increasing (12)(13)(14). The optimal treatment for early carcinoma of oral cavity is surgical operation. However, overall survival remains largely unchanged (12)(13)(14). In addition, the decrease in quality of life (QOL) after wide excision of tongue is also important issue for patients. Therefore, different therapies are required. In our previous studies, we investigated the whole genome analysis using DNA microarray to find the potential target genes involved in tumor cell growth, and reported the critical role of several important molecules on the cell growth of SCCs (15)(16)(17)(18)(19). According to the results of DNA microarray, we found increased expression of ET receptor mRNA in cell lines of oral SCCs and the alteration of expression level on SCC growth (15,18,19). Therefore, we have examined whether ET receptors may be expressed in primary oral SCC tissues, and whether ET receptor-signalling may play a critical role of SCC growth. Our results imply a potentially important and novel role of ET function on SCC growth, and suggest that ET receptor-signalling might be useful target in the therapy of SCCs.

Materials and methods
Tissue samples. All of clinical studies were approved by the Ethics Committee of Osaka University Dental Hospital. Twentythree samples of squamous cell carcinoma (SCC) located in the tongue were obtained from surgical resection tissue specimens at Osaka University Dental Hospital after informed consent was obtained. The patients, who received no preoperative therapy including chemotherapy and irradiation therapy, were randomly selected ( Table I). The age range was 33-92 years (average: 62.0±13.9 years, mean ± SD).

Immunohistochemical staining of ET A and ET B .
The expression of ET A or ET B in tissues was detected by anti-ET A or ET B specific polyclonal antibody using standard immunohistochemical techniques on formalin-fixed and paraffin-embedded continuous sections. Incubation with anti-ET A or ET B polyclonal antibody was performed at 4˚C for 16 h, then the sections were washed out. After the application with secondary antibody, the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used with a 3,3'-diaminobenzidine (DAB) substrate kit, according to the manufacturer's instructions. The staining endpoint was determined when the standard tissue sections were constantly stained to the intensity as described previously (18,19).
The intensity of the immunohistochemical staining with anti-ET A or ET B antibody was evaluated by scoring the staining reaction in four groups: (-), none/weak; (+), weak/moderate; (++), moderate/strong, and (+++), very strong cytoplasmic staining intensity, respectively (18,19). To check the reproducibility of the evaluation system concerning the immunohistochemical staining for the ET A and ET B proteins, another oral surgeon and pathologist who were unaware of the original assessment re-evaluated the results of staining according to the system above. Tumor areas were confirmed by both of the pathologist and surgeon under the microscopy. Non-tumor areas were selected, the comparatively normal areas were separated away from the tumor areas, and confirmed by the pathologist.
Cell survival assay using ET receptor antagonists. SCC cells were treated with ET receptor antagonists, BQ123 for ET A and   Table I) by immunohistochemical observations. The brown color represents positive staining of ET A and the blue represents counterstaining. Scale bar represents 100 µm. (B) Comparison of the expression of ET A between tumor area and non-tumor area. Averaged score of strengthen of ET A expression are expressed from the data in Table I. Each column represents mean ± SEM from 9-23 cases in tumor area or non-tumor area, respectively. ** P<0.01. (C) ET B expression on tumor area (right panel) of primary lingual squamous cell carcinoma and non-tumor area (left panel) in the same tissue section (case 13 in Table I) by immunohistochemical observations. The brown color represents positive staining of ET B and the blue represents counterstaining. Scale bar represents 100 µm. (D) Comparison of the expression of ET B between tumor area and non-tumor area. Averaged score of strengthen of ET B expression are expressed from the data in Table I. Each column represents mean ± SEM from 9-23 cases in tumor area or non-tumor area, respectively. **

P<0.01. (E) Correlation between ET A and ET B expression. Each point represents each individual.
Western blot analysis. Adherent or suspended cells were washed in PBS, and cell extracts were prepared by lysing cells in lysis buffer. The proteins were separated by electrophoresis using 10% SDS-PAGE, and transferred to nitrocellulose membrane (Millipore, Bedford, MA). Detection of proteins were performed by each polyclonal antibody and visualized by using the ECL detection kit (Amersham, London, UK) following the manufacturer's suggested procedure.

Lingual SCCs in tumor tissues express ET A and ET B .
Lingual SCC primary tissues were stained using anti-ET A or anti-ET B specific antibody, respectively. Positive staining of ET A was observed in tumor area (Fig. 1A, right). In contrast, none of staining of ET A was observed in non-tumor area in the same tissue section (Fig. 1A, left). Similar staining pattern was also observed in other tumor tissue sections (Table I). Statistically significance of the ET A expression between tumor and nontumor area was observed (Fig. 1B). In addition, positive staining of ET B in tumor area, but not non-tumor area, was also observed in the same tissue section (Fig. 1C). Statistical significance of the ET B expression between tumor and nontumor areas was also observed ( Fig. 1D and Table I). These results are similar to that of ET A . Good correlation between ET A and ET B expression was observed (Fig. 1E).
ET receptor antagonists suppress cell growth of lingual and esophageal SCC. According to the data of ET receptor expression in SCCs, we hypothesized that ET receptor-signalling might play an important role on the cell growth of SCCs. To investigate the hypothesis, we used ET receptor antagonists, BQ123 for ET A and BQ788 for ET B . As shown in Fig. 2A and B, ET receptor antagonists, BQ123 and BQ788 suppressed the cell growth of In addition to the results of growth suppression of lingual SCC by the inhibition of ET receptors, both antagonists also suppressed the cell growth of esophageal SCC cell line, KYSE70 ( Fig. 2C and D). These results indicate that ET receptor-signalling is required for the growth of SCCs.

ET A and ET B -siRNA suppress cell growth of lingual and esophageal SCC.
To clarify the exact function of ET receptors on the growth of SCCs, we used small interfering RNA (siRNA) for ET A and ET B . ET A and ET B -siRNA effectively decreased the ET receptor protein levels in SCCs. The inhibition of cell growth on SAS was clearly observed when ET A or ET B was knocked down by the treatment with siRNA ( Fig. 3A and B). Similar suppression of cell growth by the knockdown of ET A or ET B was also observed when esophageal SCC cell line, KYSE70 was treated with siRNA for ET A or ET B (Fig. 3C and D). These results clearly indicate that ET receptor-signalling is required for the growth of SCCs.
Investigation of potential mechanisms. We next investigated the mechanisms of inhibition of cell growth induced by the suppression of ET receptor-signalling. Western blot analysis showed the expression of ET A and ET B proteins on the lingual SCC cell line SAS (Fig. 4A). Although the specific antagonists blocked the ET A or ET B signalling, no alterations of receptor protein expression levels were observed (Fig. 4A). In contrast, blockade of ET receptor-signalling by the treatment with antagonists caused the suppression of phosphorylation of MEK and Erk (mitogen-activated protein kinase), the important members of MAPK pathway (Fig. 4B). In addition, similar suppression of MAPK pathway by knockdown of ET receptors was observed when SAS and KYSE70 were treated with ET A or ET B -siRNA ( Fig. 4C and D). These results indicate the involvement of MAPK pathway on the ET receptor-signalling mediated cell growth of SCCs.
In contrast, no inhibition of phosphorylation of focal adhesion kinase (FAK), a 125 kDa non-receptor tyrosine kinase (20,21), by the suppression of ET receptor signalling was observed (data not shown).
We also investigated the effect of blockade of ET receptorsignalling on expression of integrins such as integrin α5 and β1 (22,23). However, no alterations of integrin α5 and β1 expressions were observed (data not shown). These results suggest that the cell growth suppression of SCCs by the knockdown or blockade of ET receptors is mediated through the direct inhibition of MAPK signalling pathway.

Combination therapy of ET A or ET B -siRNA and anti-tumor drugs.
Reduction of dosage of anti-tumor drugs for cancer chemotherapy is clinically important to minimize the side effects, although the complete tumor cell death is required. Combined treatment of ET B -siRNA (20 nM) with anti-tumor drug, cisplatin (2.5 µM), drastically inhibited the cell growth of SAS in comparison to that in each single treatment (Fig. 5A). Similar results were also observed in the combined treatment of ET A -siRNA (20 nM) with cisplatin (data not shown). These results indicate that combination therapy of ET A or ET B -siRNA and ordinal anti-tumor drugs may be a novel and useful therapy for SCCs.

Discussion
There have been several reports on the expression of ET receptors in various human cancers (5)(6)(7), and it is considered to be the relationship between ET receptor-signalling and tumor cell growth. There are, however, few reports on the evaluation and investigation of the exact role of ET receptor-signalling using human SCC tissues and cultured cell lines of oral and esophageal carcinomas.
In the present study, using an immunohistochemical method, we demonstrated significantly higher levels of expression of ET A and ET B protein in human lingual cancer tissues than in non-tumor areas in the same tissue samples. Similar results were also observed on the cultured SCC cell lines such as SAS, lingual SCC, and KYSE70, esophageal SCC. These results indicate the involvement of ET receptor-signalling on SCC growth. Furthermore, we showed that the suppression of ET receptor protein by siRNA or the blockade by antagonists caused the inhibition of SCC growth. In our experimental conditions, both the treatment with ET A and ET B antagonists and siRNA strongly inhibited the cell growth of SCCs. These results strongly suggest the important role for ET receptor-signalling in SCC cell survival. In fact, recent reports strongly indicated the involvement of ET and its receptor on oral cancer (24,25). In addition, it was also reported that suppression of endothelinconverting enzyme-1 caused the inhibition of SCC proliferation (26). Our results, together with those reports, strongly suggest the importance of ET synthesis and its receptor-signalling pathway on oral SCC proliferation.
It is reported that phosphorylation of FAK is involved in the inhibition of apoptosis and promote cell growth in SCC cell lines (15,18). FAK is a 125 kDa non-receptor tyrosine kinase and an important regulator of cell survival, invasion, migration, and cell cycle progression (15,18,20,21). FAK is functionally important in transducing intracellular messages that are associated with growth factor signalling (15,18,20,21,27). The intracellular messages link p-FAK at Tyr 925 to signalling pathways that activate MAPK cascades. In our present study, however, the inhibition of phosphorylation of FAK in SCCs treated with ET antagonists and siRNAs was not observed. In contrast, the inhibition of the phosphorylation of MEK and Erk by the treatment with ET antagonists and siRNAs was clearly observed. These results indicate that the inhibition of MAPK pathway by the suppression of ET receptor-signalling is due to the direct inhibition of MAPK pathway, but not through FAK pathway (Fig.  5B). Several reports have indicated the coupling of ET receptorsignalling and MAPK pathway (28,29). Our results agree with those reports and indicate that the mechanisms of the inhibition of cell growth by ET receptor-siRNAs and antagonists are, in part, due to the inhibition of MAPK pathway.
Reduction of dosage of anti-tumor drugs for cancer chemotherapy is clinically important to minimize the side effects, although the complete tumor cell death is required. Combined treatment of low concentration of ET receptor-siRNA (20 nM) with low concentration of anti-tumor drug, cisplatin (2.5 µM), drastically inhibited the cell growth of SAS in comparison to that in each single treatment. Cisplatin is extensively characterized as DNA damaging agent and the cytotoxicity of cisplatin is attributed to the ability to form inter and intra-strand nuclear DNA crosslinks (30,31). In contrast, inhibition of cell growth by ET receptor-siRNAs presented in our study was mainly due to the direct inhibition of MAPK pathway. Therefore, those two pathways on growth inhibition are different. This difference of mechanisms between ET receptor-siRNA and cisplatin may lead to show synergistic effect on the inhibition of tumor cell growth ( Fig. 5A and B). Our results indicate that the decrease in ET receptor levels in SCCs that strongly express ET receptors increases the sensitivity against chemotherapy, and that the siRNA for ET receptors combined with anti-tumor drugs might be a useful therapy to reduce the dosage of anti-tumor drugs.
In summary, we showed the overexpression of ET A and ET B in tumor cells of human primary lingual SCC tissues and cultured SCC cell lines, and suggest a potentially important role for ET receptor-signalling on the cell growth of human SCCs.