In this study, mRNA levels of EMT-related genes ECAD, VIM, ZEB1, SLUG was evaluated in Skip N2 metastasis, which is a common entity in the lymphatic spread of the tumor in lung cancer patients and the molecular mechanism has not yet been elucidated. The findings of the study showed that tumors with Skip N2 features were associated with better prognosis, especially in the histological subtype of Adenocarcinoma, at 1-year survival compared to tumors with sequential N1 + N2 metastasis. These findings were also supported by examining mRNAs expressions isolated from paraffin blocks of patients’ lymph node by qPCR method. According to these analyzes, the epithelial marker E-cadherin, which is a better differentiation marker, was found in high rates in Skip N2 tissues, while SLUG and parallel to this, Vimentin mRNA expressions were suppressed compared to Non-Skip N2 tissues. A moderate increase in ZEB1 levels, which is also known as an EMT factor, in Skip N2 tissues in contrast to SLUG was noted. Overall summary of the findings of this study is illustrated in Fig. 6.
Slug is a protein that belong to the SNAIL family transcription factors, and it contributes significantly to the EMT process and therefore, plays a role in the metastasis of cancer cells, by reducing the expression of the E-cadherin protein, which provides intercellular connectivity . Slug expression is normally at very low levels in epithelial cells and therefore its increase is associated with poor differentiation and thus poor prognosis. In this study, the non-Skip N2 group with high Slug expression was found to have a worse 1-year survival compared to the Skip-N2 group with significantly suppressed Slug levels. The disappearance of this difference in 5-year survival may be related to the selective tumoral cloning or intra-tumoral heterogeneity, frequently encountered in lung cancer, in particularly adenocarcinoma subtype .
There are similar studies in the literature that find that Skip N2 metastasis is associated with overall survival in lung cancer [16-18]. One of these studies reported that Skip N2 is a good prognostic indicator when it is involved in the upper lobes, especially in Adenocarcinomas , even though many other studies have found it predominantly in the upper lobe, it has indicated that no statistical significance was found in this difference . According to our findings, while neither tumor size, histological subtype, nor other demographic factors were associated with overall survival, 1-year survival was found to be a good prognostic indicator, especially in adenocarcinomas. According to the International Association for the Study of Lung Cancer Staging Project, it has been recommended that the physicians should clearly record the pathological N2 presence and detailed anatomical features of the lymph node metastasis status of the patients. They have pointed out that it might be a strong possibility to be included in the classification as an indicator [19-21]. Moreover, a recently published meta-analysis study including 29 articles revealed that the presence of Skip N2 is a good prognostic indicator .
Studies to elucidate the molecular mechanism of Skip N2 are very limited in the literature. In two studies, epidermal growth factor receptor mutation was found to be higher in patients with Skip N2, but both could not suggest a mechanism for the possible contribution of tyrosine kinase receptors to the pathogenesis [3,22]. In a study investigating the relationship between tumor long diameter and Ki67 expression levels, which is a proliferation marker, with Suvmax in lung cancer, it was shown that Ki67 was higher in the presence of Skip N2, but this was not statistically significant due to the insufficient number of samples . In this study, Skip N2 encodes a protein that plays an important role in EMT, such as ZEB1, Slug, in which different mRNA expression such as SLUG is detected in the examinations performed on metastatic tissues. In the studies conducted, while the factors that contribute to both ZEB1 and SLUG EMT and cause the suppression of E-Cadherin expression, the intracellular targets of both differ from each other. For example, while ZEB1 plays a more effective role in the change of epithelial cell polarity and promotion of proliferation, Slug stands out with its suppression of Claudin-1 expression and its role in the development of resistance to apoptosis (24). In the literature, where the differences and additional functions of proteins involved in EMT were compiled, it was reported that Slug expression increased in hypoxic lung adenocarcinoma tissue, but ZEB1 expression did not change [24,25]. It has been reported that Fragile Histidine Triad (FHIT) pathway disorder, which is known to play a role in Cisplatin resistance and is frequently reported to be inactivated in lung cancer, especially in smokers, increases cell invasion through Slug, but a similar interest has not yet been observed with ZEB1 . On the other hand, there are studies showing that the expression of these two proteins, which work in the same pathway, is regulated by different microRNAs in the translational process. For example, while miR-218 suppresses protein levels by targeting SLUG and ZEB2 in lung cancer, it does not have such an effect on ZEB1 . In addition, in a study conducted in head and neck squamous cell carcinoma, it was shown that the 𝚫Np63 isoform of the p63 protein encoded by the TP63 gene is necessary and sufficient for Slug-mediated EMT activation . Bioinformatic analyzes of our study revealed a significant correlation between TP63 expression and SLUG expression in non-small cell lung cancer samples, suggesting that the two proteins act together in the EMT process. Recent studies also indicate that 𝚫Np63 isoform of p63, also regulates collective invasion package (CIP) during lung cancer metastasis , and Vimentin is required for CIP, which is also poor prognostic indicator in lung adenocarcinoma cells , compatible with our findings in this research.
This research also indicated genes other than TP63 that were co-downregulated with SNAI2 in several online Oncomine Lung Cancer datasets. These were SIAH2 (Siah E3 Ubiquitin Protein Ligase 2), FXR1 (Fragile X Mental Retardation Syndrome - Related Protein 1), TP53AIP1 (Tumor Protein P53 Regulated Apoptosis Inducing Protein 1) and SEMA6A (Transmembrane semaphorin), respectively. There is no research in the literature regarding the interactions of these proteins with Slug, and it seems to be open for further research.