Effect of 3q oncogenes SEC62 and SOX2 on lymphatic metastasis and clinical outcome of head and neck squamous cell carcinomas

Abstract

Chromosome 3q26 amplification represents a frequent alteration in head and neck squamous cell carcinomas (HNSCCs). Overexpression of 3q26 encoded genes SEC62 and SOX2 was detected in various cancers, including HNSCCs, indicating their potential function as oncogenes. In our study, we elucidated the function of SEC62 and SOX2 in HNSCC patients, with a main focus on their effect on lymphatic metastasis and patient survival. We analyzed SEC62 and SOX2 expression in tissue specimens from 65 HNSCC patients and 29 patients with cervical cancer of unknown primary (CUP); a higher SEC62 and lower SOX2 expression was observed in the lymph node metastases from HNSCC patients compared with the respective primary tumor. Lymph node metastases from CUP patients showed higher SEC62 and lower SOX2 expression compared with lymph node metastases from HNSCC patients. When proceeding from the N1 to the N3 stage, SEC62 expression in the lymph node metastases showed an increase and SOX2 expression showed a decrease. Moreover, both genes showed a highly significant relevance as prognostic biomarkers, with the worst prognosis for patients with high SEC62 and low SOX2 expression levels. In functional analyses, knockdown of SEC62 resulted in an inhibition of HNSCC cell migration while, conversely, SEC62 and SOX2 overexpression stimulated cell migration. Taken together, our study showed that the expression of the 3q oncogenes SEC62 and SOX2 affects lymphatic metastasis and cell migration in HNSCC and CUP patients and has a high prognostic relevance in these diseases.

Keywords: 3q amplification; SEC62; SOX2; head and neck cancer; prognostic biomarkers.

Figure 1. Overall survival of CUP and HNSCC patients

In the Kaplan-Meier analysis, the overall survival of CUP and HNSCC patients was compared revealing a significantly worse prognosis for CUP patients (p=0,026; log-rank-test). Black dots on the survival curves represent censored data.

Figure 2. SEC62 and SOX2 expression in the primary tumor and lymph node metastases of HNSCC patients

SEC62 and SOX2 expression was evaluated using immunohistochemistry in tissue specimens from lymph node metastases and the respective primary tumor of 65 HNSCC patients and compared with each other. (A) Strong (left picture) and weak expression (right picture) of SEC62 (cytoplasmic staining signal). (B) Strong (left picture) and weak expression (right picture) of SOX2 (nuclear staining signal). (C) SEC62-mIRS and (D) SOX2-mIRS for the primary tumor (left) and the metastases (right) of the HNSCC patients are shown using box and whisker blots. Each box represents the range from the first quartile to the third quartile. The median is indicated by a line. The whiskers outside the boxes represent the ranges from the minimum to the maximum value of each group. In (C) and (D), the mIRS values of the tumor and the respective metastasis are connected by a line. A green line indicates an increase in mIRS, a black line indicates an unchanged mIRS and a red line indicates a decrease in mIRS in the metastasis compared with the primary tumor.

Figure 3

Comparison of SEC62 and SOX2 expression between lymph node metastases of HNSCC and CUP patients (A and C) and between different N-stages (B and D). The SEC62-mIRS (A) and SOX2-mIRS (C) for the lymph node metastases of HNSCC patients compared with the lymph node metastases of CUP patients and for all included patients grouped according to their N-stages (B and D) are shown using box and whisker blots. Each box represents the range from the first quartile to the third quartile. The median is indicated by a line. The whiskers outside the boxes represent the ranges from the minimum to the maximum value of each group.

Figure 4. Analysis of copy number variations and expression level of SEC62 and SOX2 in UM-SCC1 cells

(A) CGH analysis showed gains on the long arm of chromosome 3 (3q), including the 3q26 region. (B) FISH analysis showed amplifications of the SEC62 gene (green signals, left picture) and the SOX2 gene (green signals, right picture). A probe directed against the centromere region of chromosome 10 (#10cen) was used as an internal hybridization control (red signals). Nuclei (60× magnification) were counterstained with DAPI. At the protein level, we observed a strong SEC62 expression (C) but no SOX2 expression using immunocytochemistry (D).

Figure 5. Effect of SEC62 and SOX2 expression on the proliferation and migration of UM-SCC1 cells

The expression level of SEC62 and SOX2 was modified in UM-SCC1 cells using siRNA- and plasmid-transfection. (A) The cell index was measured as an indicator for cell proliferation of UM-SCC1 cells transfected with either two different SEC62-siRNAs, a SEC62-plasmid or SOX2-plasmid and compared with cells transfected with control siRNA or a control plasmid. (B) The cells that migrated through the 8-μm pores of the insert system were fixed and marked with DAPI (white dots). Representative images are shown for the transfected cells. (C) The number of migrated cells was quantified and is presented as a percentage of the respective control cells (= 100%) using box and whisker blots. Each box represents the range from the first quartile to the third quartile. The median is indicated by a line. The whiskers outside the boxes represent the ranges from the minimum to the maximum value of each group. (D) SEC62 and SOX2 protein levels of the cells were measured using western blot analyses at the end (72 h) of the migration experiments. Rough microsomes (RM) served as positive control for Sec62. The relative expression of SEC62 per β-actin is indicated at the bottom as mean value of three identically performed experiments (n = 3) with the respective standard error.

Figure 6. Prognostic relevance of SEC62 and SOX2 expression levels in cervical lymph node metastases of HNSCC and CUP patients

In the Kaplan-Meier analysis, high SEC62 expression and low SOX2 expression were predictors of worse prognosis (A, B). A combination of both biomarkers showed an even higher prognostic relevance with the worst prognosis for patients who had a high SEC62 and low SOX2 expression level (C).