SYK(L) Interacts With YY1 And Coordinately Suppresses Slug-Mediated EMT In Non-Small Cell Lung Cancer Cells

Objective∶ Spleen tyrosine kinase(SYK) is most highly expressed by haematopoietic cells and plays a crucial role in mediating many immune receptor signaling. However, recent reports have indicated that it is also expressed by various cancer cell types outside of the hematopoietic system. SYK acts both as an oncoprotein and as an oncosuppressor during tumorigenesis. In solid tumor cells, SYK provides a pro-survival signal, while suppresses tumor progression by restraining epithelial to mesenchymal transition and strengthening cell-cell adhesion. Until now, SYK inhibitors have not been used in the clinical trials because of its functional diversity. Lung cancer is the most life-threatening neoplasia in our country even around the world with high incidence and mortality rates, and non-small cell lung cancer accounts for 80-85% of cases. However, the cellular activity and the biological significance of SYK are still not well defined in human non-small cell lung cancer cells. It is not clearly that whether SYK(L) is involved in gene transcriptional regulation. In this study, differential expression of SYK was detected and the association of SYK expression with clinicopathological features was investigated in lung cancers. In addition, the mechanism of transcriptional regulation that SYK(L) participated during tumor metastasis was explored to provide the experimental basis for clinical lung cancer patients’ diagnosis and treatment.Methods: 1. Expression level of SYK was detected by RT-PCR and Western blot assays in a panel of human lung cancer cell lines. Immunohistochemistry(IHC) method was used to detect SYK expression in lung squamous carcinoma tissues. Cell adhesion assay, wound healing assay and transwell invasion assays were explored to test the effect of SYK on biological behaviour of NSCLC cells. Lentiviral sh RNA was conducted to knockdown of SYK to detect the expression of epithelial-mesenchymal transition(EMT) markers using q RT-PCR, Western blot and immunofluorescence assays. 2. Immunofluorescence staining and Western blot assays were used to examine the distribution of SYK in cells. Mass spectrometry and co-immunoprecipitation assays were used to identify specific SYK(L) binding partners in cells. Expression level of YY1 was detected by RT-PCR and Western blot in a panel of human lung cancer cell lines. And co-immunoprecipitation assay was used to validate the interaction of full length or serial truncation of SYK(L) and YY1. The conjugation between YY1 and SYK(L) was also confirmed by fluorescence cross-correlation spectroscopy and Duolink assays. Phos-taq SDS-PAGE and co-immunoprecipitation assays were used to detect whether SYK could change YY1 phosphorylation status. EMT markers were tested by Western blot in SYK or/and YY1 depleted cells. Chromatin immunoprecipitation(Ch IP), EMSA and luciferase reporter assays were performed to analyze the function of YY1-SYK(L) complex in transcriptional regulation of SNAI2 promoter region. 3. The Bisulfite-sequencing method and DNA methyltransferase inhibitor 5-aza-Cd R were used to detect the methylation status of SYK promoter region in lung cancer cell lines. Ch IP, EMSA and luciferase reporter assays were further conducted to investigate the function of YY1 in transcriptional regulation of SYK promoter region.Results: 1. SYK is differentially expressed in lung cancer cell lines, and is positively correlated with the prognosis of lung squamous cancer patients(P = 0.032). Loss of SYK in NSCLC cells increased cell invasion and migration abilities but decreased cell adhesive ability. Knockdown SYK decreased E-cadherin and β-catenin level and increased Slug and Vimentin in H1155 cells. However, SYK depletion had no effect on ZEB1 and Snail expression. Immunofluresence staining of β-catenin showed that knockdown of SYK resulted in translocation of β-catenin from cytoplasm to nucleus. 2. SYK(L) and SYK(S) has different subcellular localization, SYK(L) was present both in nuclear and cytoplasmic extraction while SYK(S) was predominantly cytoplasmic. The peptide sequences by mass spectrometry indicate that SYK(L) might associate with transcription factors YY1, and the binding between YY1 and SYK(L) is predominantly in the cell nucleus. Co-IP and Duolink assays showed that SYK(L) interacted directly with YY1, and two N-terminal SH2 domains of SYK but not other domains were necessary for YY1 association. SYK(L) interacts with YY1 without changing YY1 phosphorylation status as Phos-taq SDS-PAGE assay indicated. YY1 depletion promoted EMT in A549 cells lacking of SYK expression. In addition, YY1 depletion increased SYK(L) but not SYK(S) expression in A549 cells. However, neither enforced SYK(L) nor SYK(S) expression could change A549 cells phenotype and the EMT biomarkers. Knockdown of YY1 suppressed EMT in H1155 cells expressing endogenous SYK. Knockdown of SYK or double knockdown of SYK and YY1 upregulated Slug protein level but suppressed E-cadherin protein level. Both SYK(L) and YY1 occupied SNAI2 promoter by Ch IP assays in H1155 cells. Depletion of YY1 decreased SNAI2 promoter activity and SYK knockdown showed the opposite effect by luciferase assays in H1155 cells. Further EMSA assay also indicated that YY1 directly combined with SNAI2 promoter region in vitro. 3. Less methylated SYK promoter was found in H209 cells, which express SYK, than Beas-2B and H209 cell lines that lack SYK expression. However, SYK expression was not upregulated in Beas-2B cells by the DNA methyltransferase inhibitor 5-AZA-Cd R treatment. YY1 occupied cis-element of the endogenous SYK promoter region. Luciferase assay also showed that depletion of YY1 reduced SYK promoter activity in H1155 cells. Further EMSA assay also detected the direct association of YY1 with SYK promoter in vitro.Conclusions: SYK is differentially expressed in NSCLC cells and its expression positively correlated with patients’ survival time. Loss of SYK in NSCLC cells increased their malignance with increased cell invasion and migration, indicated that SYK acts as a tumor suppressor in NSCLC. The two isoforms of SYK have different subcellular localization. SYK(L) is expressed in both nuclear and cytoplasmic whereas SYK(S) is predominantly cytoplasmic. SYK(L) directly interacts with transcription factor YY1 without changing YY1 phosphorylation status in the nucleus. Endogenous SYK(L)-YY1 complex inhibits Slug expression and EMT progression in NSCLC cells. These data provide an insight into the mechanisms of the SYK(L) in lung cancer metastasis. DNA methylation is not the only mechanism that silences SYK expression. And YY1 can suppress SYK expression level through transcriptional regulation, which provides a new approach for SYK expression research.