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- Material and Methods
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Metadherin (MTDH) is involved in tumourigenesis and cancer progression in multiple human malignancies. However, the MTDH protein has rarely been reported in laryngeal squamous cell carcinoma (LSCC). The expression pattern of the MTDH protein in 176 primary archival LSCC and 27 corresponding adjacent noncarcinoma specimens was detected by immunohistochemistry and further correlated with clinicopathological parameters. The results demonstrated that 161 (91.48%) primary LSCC samples stained positive for MTDH; however, staining was barely detectable in all adjacent noncarcinoma samples. Moreover, the expression of the MTDH protein was significantly associated with the primary tumour site (p = 0.021), T classification (p = 0.002), clinical stage (I + II/III + IV; p < 0.001), lymph node metastasis (p < 0.001) and postoperational recurrence (p < 0.001). Kaplan-Meier analysis revealed that MTDH expression was significantly associated with worse disease-free survival (DFS) and overall survival (OS) rates in patients with LSCC (both p < 0.001). When lymph node metastasis and MTDH expression were considered together, patients with lymph node metastasis and high MTDH expression had both poorer DFS and OS rates than others (both p < 0.001). Finally, multivariate analysis demonstrated that MTDH expression was an independent prognostic factor for both DFS and OS rates in patients with LSCC. Strong MTDH expression was negatively correlated with a canonical epithelial–mesenchymal transition molecule E-cadherin (p < 0.001) and positively associated with proangiogenic protein vascular endothelial growth factor (p < 0.001). MTDH overexpression was tightly associated with more aggressive tumour behaviour and a poor prognosis, indicating that MTDH is a valuable molecular biomarker for LSCC progression.
Laryngeal squamous cell carcinoma (LSCC), accounting for ∼1.5% of all cancers, is the most common malignancy arising in the upper airway in adults.[1, 2] Although patients with LSCC benefit from advanced diagnostic and therapeutic management, including routine surgery, chemotherapy, radiotherapy and biological therapy, outcomes for these patients have remained unsatisfactory during the last few decades. Cervical lymph node metastasis and postoperational recurrence have been reported to be the most decisive factors causing the dismal prognosis in patients with LSCC after surgical resection.[3, 4] However, the molecular mechanism of cancer initiation and progression remains unclear. Therefore, identifying sensitive and specific molecular markers that can predict patients with biologically aggressive neoplasms is of great significance for developing prospective strategies for the prevention and management of patients with LSCC.
Metadherin (MTDH, also identified as astrocyte-elevated gene-1/AEG-1 and lysine-rich CEACAM-1-associated protein/Lyric) was originally identified as a protein induced in primary human fetal astrocytes infected with HIV-1 or treated with HIV envelope glycoprotein (gp120) or tumour necrosis factor-alpha.[5-8] MTDH functions by regulating the NF-κB signalling pathway. MTDH is also a downstream target molecule of Ha-ras and c-myc and mediates their pro-tumour effects, suggesting its potential roles in tumour initiation and development.[10, 11] In normal immortal cloned rat embryo fibroblast cells, the ectopic overexpression of MTDH has induced morphological transformation and enhanced invasion and anchorage-independent growth in soft agar and has formed aggressive tumours in nude mice, demonstrating that MTDH alone can function as an oncogene for rodent cells. MTDH protein has been reported to be directly regulated by miR-375 (Ref. ) or miR-136 (Ref. ), and MTDH itself is present in the RNA-induced silencing complex and regulates the mRNA expression of various tumour suppressors. Aberrant MTDH expression is prevalent in various human solid neoplasms, including those of breast cancer, prostate, glioma, oesophageal squamous cell carcinoma, hepatocellular carcinoma and gastric cancer. Moreover, high MTDH expression is associated with aggressive cancer phenotypes, such as advanced clinical stage, increased metastatic ability and decreased patient survival, revealing MTDH as a potential prognostic biomarker in a panel of human malignancies. Recent in vitro studies revealed that MTDH is significantly increased in nasopharyngeal carcinoma (NPC) samples. LSCC is distinct from NPC by its epidemiology, histopathology, clinical characteristics, methods of treatment and patterns of failure. The expression pattern of MTDH protein in LSCC and especially the prognostic significance of MTDH protein in LSCC thus remain to be elucidated.
To clarify the clinical implication of MTDH in LSCC, we investigated the expression of MTDH protein in 176 LSCC tissue samples and further assessed whether the expression of MTDH protein was associated with clinicopathological variables and prognosis in patients with LSCC. Finally, considering that both epithelial–mesenchymal transition (EMT) and angiogenesis are well-known processes involved in tumour metastasis, we assessed the expression of E-cadherin (a typical molecular biomarker associated with EMT) and vascular endothelial growth factor (VEGF, a potent protein promoting angiogenesis) in the same patient cohort to potentially provide mechanistic insight into the roles of MTDH in LSCC.
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- Material and Methods
- Supporting Information
The aberrant expression of MTDH has been previously reported in diverse human malignancies, including breast, glioma, prostate, melanoma and oesophageal cancers.[16-21] MTDH is a versatile molecule, implicated in multiple cancer malignant processes including proliferation, chemoresistance,[33, 34] protective autophagy and metastasis.[5, 34] To the best of our knowledge, this is the first study to explore the prognostic value of MTDH protein in LSCC and to report that the upregulated expression of MTDH protein is a predictor of different aggressive tumour behaviours such as advanced clinical stage and metastasis and post-operational recurrence in patients with LSCC. The significant association between MTDH protein expression and tumour origin sites has never been reported previously, which was not the case in our study. A plausible explanation for this result is that superaglottic and subglottic carcinoma tends to be much more susceptible to cervical lymph node metastasis, which is consistent with the positive correlation between MTDH expression and metastasis.
Although close links between the expression of MTDH and lymph node metastasis in patients with LSCC has been established in current investigation, determining possible molecular mechanisms will require deeper investigation. Our data clearly revealed that MTDH expression was found to be negatively associated with E-cadherin expression (a typical molecular biomarker associated with EMT) and positively correlated with the expression of VEGF (a potent protein promoting angiogenesis). The result was consistent with recent publications in breast and hepatocellular carcinoma, in which ectopic MTDH overexpression could significantly enhance the invasion and migration of tumour cells by inducing EMT. Additionally, MTDH has been reported to be involved in the process of tumour angiogenesis in both in vitro and in vivo conditions.[12, 39, 40]
In our investigation, MTDH is predominantly cytoplasmic in tumour cells except for that a minority of LSCC specimens (12/176, 6.82%) presented with nuclear and perinuclear staining of MTDH protein. The function of MTDH in different cellular locales remains unclear. Recent report demonstrates that cytoplasmic MTDH can act as an RNA-binding protein to regulate the expression of drug resistance-associated proteins. As to nuclear MTDH, we have to note that all samples with nuclear and perinuclear staining in our study were metastatic. This observation coincides with previous publications in breast cancer, in which advanced clinical stage sections display noticeably increased MTDH nuclear translocation and a large proportion of cancer cells in liver metastases reveal MTDH translocation to the nucleus. Similar positive correlations between MTDH nuclear staining and both advanced stages and poor prognosis were also reported for hepatocellular carcinoma and melanoma. MTDH was found to directly interact with p65 subunit of NF-κB complex and to modulate the function of NF-κB in nucleus, indicating that MTDH protein is decisively involved in the regulating process of gene expression mediated by the NF-κB signalling pathway.[9, 43] Paradoxically, the fact that nuclear staining of MTDH correlates with a favourable prognosis in prostate cancer was also reported. The authors of the previous study hypothesised that nuclear MTDH might have a function in normal prostate epithelial cells that is lost during tumourigenesis. These controversial results reflect the complexity of regulation function of nuclear MTDH protein. It is possible that nuclear MTDH protein performs context-specific functions depending on the microenvironment of cells and the type of the tumour. These hypotheses need to be elucidated in future.
Currently, outcome prediction is still based on critical clinical parameters, such as the clinical phase, tumour sites and histopathologic study. Recent advances in molecular biology of human malignancies provide a hint that the discovery molecular abnormalities may facilitate early diagnosis and prognosis prediction. Our study revealed that MTDH protein level was significantly negatively associated with the postoperational prognosis in patients with LSCC. When stratified according to tumour T classification and clinical stage, MTDH protein expression also had good prognostic capability in both early- and late-stage patients with LSCC.
Our current data, together with previous reports,[3, 4] suggested that lymph node metastasis was inversely correlated with the prognosis for patients with LSCC. Survival analysis via combining cervical lymph node metastasis and MTDH protein status showed that patients with LSCC with both high MTDH expression and positive lymph node metastasis had worse prognosis than that of patients with LSCC with other phenotypes. These results suggested that much more powerful prognosis predictions for patients with LSCC can be obtained by considering lymph node metastasis and MTDH protein together, which may contribute to appropriate decisions about management strategies.
In summary, our data demonstrated that aberrant MTDH protein expression was tightly associated with aggressive clinical behaviours in human LSCC, indicating MTDH as a useful biomarker for prognosis in LSCC. Additionally, based on in vitro and in vivo studies and numerous expression analysis using patient specimens, emerging evidence demonstrates that MTDH plays diverse roles in the process of tumourigenesis in multiple organs, indicating that MTDH presents as an attractive therapeutic target. Therefore, further intensive research in both in vitro and in vivo conditions is being conducted in our laboratory to identify the exact functional mechanisms of MTDH in the process of malignant transformation in LSCC.