Modulation of semaphorin 3C & 4D expression in cancerous tissues from individuals with laryngeal squamous cell carcinoma

Semaphorins are mostly known for their importance in repulsive axon guidance but are now recognized as crucial regulators of morphogenesis and homeostasis in various tissues1. These molecules and their receptors are associated with sensitivity to cytosolic signalling in various diseases, such as schizophrenia, neurodegenerative diseases and especially cancer1. Various semaphorins are reported to enhance or inhibit tumour progression by promoting or inhibiting tumour angiogenesis, metastasis and tumour cell survival2. Squamous cell carcinoma (SCC) is the most common type of head-and-neck cancer, and the fifth-most common cancer in the world3. This malignancy is multifactorial and common treatment strategies consist of surgery, chemotherapy and/or radiotherapy4,5. In more than half of the individuals with locally advanced head-and-neck cancer there is recurrence, and the prognosis of these individuals is poor3.

Abnormal angiogenesis is one of the pivotal mechanisms responsible for tumour development and progression through different mediators6. Vascular endothelial growth factor (VEGF) has been identified as a major enhancer of angiogenesis and its signals are transmitted by two tyrosine kinase receptors (VEGFR-1 and VEGFR-2) binding to all forms of VEGFs7. Such receptors were first identified in endothelial cells and were called neuropilin-1 and neuropilin-2. Since neuropilins were formerly known as class 3 semaphorin receptors, these could modulate angiogenesis induced by VEGF. Class 3 semaphorins (Sema3A, Sema3B, Sema3C, Sema3D and Sema3E) can act as potent inhibitors of angiogenesis8. Furthermore, there are reports suggesting that, long-term stimulation of endothelial cells by Sema3A and Sema3F may lead to apoptosis being a part of the anti-angiogenic effect of semaphorins9,10. There are reports concerning the role as well as expression of different semaphorin family members in progression of various cancer types. Hence, studying the properties of this protein family may be beneficial to the development of anti-angiogenic therapies and the prognosis of cancer11. Most class 3 semaphorins (Sema3A, 3B, 3D, 3F and 3G) prevent cell migration. However, Sema3C reportedly enhances tumour cell migration and is highly expressed in metastatic tumour cells12,13. Upregulation of the Sema4D has been indicated in individuals with chronic lymphocytic leukaemia and is thought to lead to cell growth and survival in B cell acute lymphocytic leukaemia14,15. Sema4D can potentiate the invasive ability of pancreatic cancer cells and its high expression is associated with a higher amount of lymph angiogenesis, the lymphatic vessel invasion and the occurrence of lymph node metastases16,17. This semaphorin was significantly associated with an unfavourable outcome in cervical cancer patients17. Sema6D was associated with more invasive behaviour and enhanced metastatic potential in breast cancer18. Upregulation of Sema6D was also indicated in gastric carcinoma representing that this semaphorin plays an important role in the development of gastric carcinoma19,20.

Semaphorins can influence tumour progression through influencing VEGF-induced angiogenesis. For instance, SEMA4D contributed to the tumour progression and poor prognosis in cervical cancer through increasing VEGF expression21. Sema4D has been shown to cooperate with VEGF to enhance tumour progression and angiogenesis in oral squamous cell carcinoma, ovarian cancer, colorectal cancer and head-and-neck SCC21.

In order to assess the role of semaphorins in tumour progression or suppression, this study undertook to investigate the expression of Sema3A, 3C, 4D, 6D and VEGF gene transcripts as well as the serum level of Sema3A in individuals with laryngeal SCC (LSCC).

Material & Methods

Study participants: In this study, 60 individuals with LSCC who underwent surgery at Khalili Hospital of Shiraz University of Medical Sciences, Iran were selected for enzyme-linked immunosorbent assay (ELISA) (group 1) to determine serum Sema3A levels. The LSCC diagnosis was made according to the laryngoscopy analysis and pathological findings on biopsy samples collected and viewed under a microscope by a pathologist22. Tumour tissues and the surrounding healthy tissue samples (as controls), were collected from 30 of the participants (group 2) during surgery (before starting treatment) to determine the mRNA expression of semaphorins using quantitative real-time PCR (qPCR). The corresponding adjacent normal tissues were 3 mm apart from tumour tissues. Individuals with pathology except SCC and those undergoing radiotherapy and chemotherapy were excluded from the study.

All participants provided written informed consent before enrolling in the study. This study was approved by the Ethics Committee of Shiraz University of Medical Sciences, Iran. All procedures were set up and done at the Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences.

The sample size was determined by G^*Power SSC software 3.1 (designed by Erdfelder, Faul, & Buchner, 2007) based on the previous studies (α=0.05, power=80% and standard deviation=6).

RNA extraction and cDNA synthesis: Tissue samples were cut into small pieces and total RNA was extracted using RNX plus (Cinagene, Iran). cDNA was synthesized from the extracted RNA using the cDNA synthesis kit according to the manufacturer’s instructions (Fermentas, Finland).

Quantitative real-time PCR (qPCR): Human Sema3A, 3C, 4D, 6D and VEGF gene transcripts were amplified by thermal cycler (ABI, USA) and using appropriate primers. Each PCR reaction was carried out in a final volume of 20 μl containing 5 μl cDNA, 10 μl of 2X SYBR Green Master Mix (ABI, USA), 0.3 μl of each forward and reverse primers (Supplementary Table) and 4.4 μl DEPC-treated water. Thermal cycling for all the genes was initiated with a denaturation step at 95°C for 10 min, followed by 50 cycles with the subsequent programme for each cycle: 95°C for 15 s, 57°C for 30 s and 60°C for 1 min. Quantitative values were obtained from the threshold cycle (Ct), where the increase in the fluorescent signal is associated with an exponential increase in the product of qPCR. The expressions of Sema3A, 3C, 4D, 6D and VEGF mRNA were determined using the 2^-ΔΔCt method and defined as fold change22. The β-actin housekeeping gene was used for target gene expression normalization23.

Enzyme-linked immunosorbent assay (ELISA): The protein expression of SEMA3A in the serum of 60 individuals with LSCC and 30 normal age- and sex-matched individuals was evaluated using an ELISA kit (Crystal Day Biotech, China) as per the manufacturer’s protocol. At first, 100 μl of standard (3, 6, 12, 24, 48 and 96 ng/ml) and 40 μl of serum samples were added to the wells then 10 μl of biotinylated anti-Sema3A was added to each well. A volume of 50 μl of streptavidin-conjugated horseradish peroxidase (HRP) enzyme was then added to each well and incubated at 37°C for 90 min and then washed five times. Next, 50 μl of each chromogen A and B solution were added and incubated at 37°C for 10 min, then the enzymatic reaction was stopped using 50 μl of stop solution, and the absorbance was measured at 450-620 nm in a microplate spectrophotometer.

Statistical analysis: The intergroup differences in variables were estimated using Mann–Whitney and Kruskal–Wallis H nonparametric tests. The data of ELISA assessment were analyzed using Student’s t test. All data analyses were carried out using the Statistical Package for the Social Science (SPSS) version 21 software (IBM Corp, Armonk, USA), and relative expression was plotted and assessed by Prism 6 software (Inc., USA). Finally, P<0.05 was considered statistically significant.

Results

Demographic information of participants: Demographic information of the cases is presented in Table. The mean age was 60.5±18.6 yr (range: 40-79 yr) for group 1 (ELISA test) and 60±19.5 years (40-79 yr) for group 2 (qPCR). Regarding tumour size, the higher percentage was referred to T2 (2-4 cm) (48.4% in group 1 and 43.3% in group 2). The majority of the participants in either groups emerged with advanced clinical stage (stages III and IV) as it was 86.7 per cent in group 1 and 80 per cent in group 2 (Table).

Relative gene expression of semaphorins: The mRNA expression of Sema3A and Sema6D was lower in tumour tissues compared to controls, although their differences were not significant. Sema4D was expressed with a 6-fold significant decrease in tumour samples compared to healthy tissues (P=0.0011). On the contrary, the mRNA expression of Sema3C showed 6.3-fold significant increase in tumour samples (P=0.001). The mRNA expression of VEGF was higher in tumour tissues compared to controls, although the difference was not significant (Fig. 1).

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Fig. 1

Transcripts level of semaphorins in LSCC individuals. Data are presented as median. Data are presented as fold change and β-actin housekeeping gene was used for gene expression normalization. LSCC, laryngeal squamous cell carcinoma; sema, semaphorins; VEGF, vascular endothelial growth factor (P^**<0.01 vs. non-cancerous tissue).

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Semaphorins gene expression and tumour stage of laryngeal squamous cell carcinoma (LSCC): The expression of the Sema3C gene was found to be associated with the stage of LSCC tumour. The expression of this gene was amplified by increasing the stage of the disease as this difference was significant for tumours with stage IV (P=0.0038). Sema4D showed a significant negative association with the stage as it showed the less expression in individuals with stage IV (P=0.0187) compared with stage II and III and non-tumoural tissues. There were no significant differences between different stages of LSCC regarding the VEGF expression; however, the intra-tumoural expression of VEGF showed 2.4-fold higher expression in tumour tissues with pathological stage IV compared with adjacent normal tissues (Fig. 2).

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Fig. 2

Semaphorins gene expression and tumour stage of LSCC. Data are presented as median. (P^*<0.05, ^**<0.01 vs. non-cancerous).

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Serum concentration of Sema3A: The mean serum concentrations of Sema3A were lower in the cases (19.05±2.1 pg/ml) in comparison with healthy controls (25.62±4.0 pg/ml) though not significant (P>0.05) (Fig. 3).

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Fig. 3

Sema3A serum concentration in cases and healthy participants. Data are shown as mean ± SEM. SEM, standard error of the mean.

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In addition, with increasing age, serum concentration of Sema3A increased, as the highest concentration was seen in participants in the age range of 70-79 yr. No relationship was observed between the clinicopathological features of the study participants and serum concentration of Sema3A (P>0.05).

Discussion

Semaphorins are known as both proangiogenic and anti-angiogenic mediators depending on the expression of their various receptors, neuropilins and plexins, in different disease conditions8. As various semaphorin members are important in the regulation of tumour growth and progression, we assessed the expression of different semaphorins as well as the relationship between their expression and the degree of tumour progression in individuals with LSCC. The anti-angiogenic activity of Sema3A justifies its inhibitory effect on the development of both haematological and solid malignancies such as multiple myeloma and prostate and breast cancers, respectively8. Sema3A is capable of directly affecting the behaviour of tumour cells and inhibits tumour progression, for instance, through regulating the phosphorylation and nuclear translocation of phosphatase and tensin homolog and the activation of the forkhead transcription factor FOXO-3a24. On the other hand, there are reports showing tumour-promoting effects of Sema3A in hepatocellular carcinoma, glioblastoma multiforme and pancreatic cancer through upregulation of various molecules such as epithelial cell adhesion molecule and galectin-325-28. Based on these reports, it seems that the dual role of Sema3A depends mostly on the type of the cancer, and in LSCC, it may have anti-tumorigenic effects as its concentration in the sera of the participants of the present study was lower than those from normal individuals.

Literature predominantly introduces Sema3C as a mediator orchestrating angiogenesis, tumour progression and thus of poor prognosis in various types of cancers27. However, it has been reported that Sema3C promotes the activation of multiple receptor tyrosine kinase pathways in prostate cancer cells, which is a key mechanism for mediating cancer growth, survival and treatment resistance26. Similarly, it has been found that aberrant Sema3C expression is associated with poor survival in individuals with pancreatic cancer27. Sema3C knocking down attenuates the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) in pancreatic cancer cell lines28,29. Sema3C might be a new therapeutic target for preventing cervical cancer as it is associated with poor prognosis of cervical cancer and promotes tumour growth through the activation of the pERK pathway30. In line with these reports, the results of the present study showed higher expression of the Sema3C gene in tumoural tissue as compared to non-tumoural tissues. There was also an association between Sema3C and higher stages of LSCC. Consequently, the pro-tumorigenic role of Sema3C in the development of LSCC is predictable, probably through its promotive effects on cell proliferation, colony formation, angiogenesis and orchestrating EMT. On the other hand, like other members of Sema3 family, Sema3C has been reported to have contradictory roles. Furthermore, U87 glioblastoma cells overexpressing Sema3C have been shown not to encourage new vessels formation in chick chorioallantoic membranes demonstrating anti-angiogenic nature31. Similarly, Salimi-Sotoodeh et al identified Sema3C as well as Sema3A as appropriate inhibitors of pathological angiogenesis in human meningioma32. Therefore, we speculated that the effects of this semaphorin may be dependent on the environment and tumour type.

Sema4D has been reported to be a regulator of tumour progression by promoting angiogenesis33. Furthermore, an association was observed between Sema4D upregulation and poor prognosis in cervical cancer34-36. Alternatively, Sema4D is an immune semaphorin that induces the antibody production by B-lymphocytes and antigen presentation, activation and maturation in dendritic cells. This semaphorin is able to promote the production of cytokines such as interleukin-12 and tumour necrosis factor-α by monocytes34. Based on the present data reporting a decreased expression of Sema4D, and considering the immune-promoting effects of this protein, it can be concluded that downregulation of Sema4D may partly impair the activity and function of distinct immune cells such as B- and T-lymphocyte, DCs and monocytes in individuals with LSCC35.

As a limitation this study did not assess individuals with benign tumours, which is needed to be clarified in future studies. Furthermore, the expression data from the study may not be sufficient to interpret pertinent mechanisms and hence more studies are required.

Overall, based on the results of this study demonstrating an increased expression of Sema3C in LSCC tissues compared with non-tumour surrounding tissues as well as its increase in stage IV, this protein may be considered as an angiogenic or tumourogenic factor in individuals with LSCC. In contrast, since Sema4D showed decreased expression and considering its role as an immune mediator, it may cause the downregulation of the activity of immune cells in this malignancy.