HIF-1α induced NID1 expression promotes pulmonary metastases via the PI3K-AKT pathway in salivary gland adenoid cystic carcinoma.

This study aimed to investigate the potential role of nidogen 1 (NID1), a basement membrane component, in the growth and metastasis of salivary gland adenoid cystic carcinoma (SACC) and the underlying molecular mechanism. High-throughput next-generation sequencing was used to compare the gene expression profiles of SACC with and without lung metastasis. Luciferase gene reporter assays were used to measure the NID1 promoter activity. BALB/c nude mice were used to establish a lung metastasis model of SACC to evaluate the prometastatic activity of NID1. ChIP and dual-luciferase reporter assays were performed to confirm the HIF-1α-binding site in the NID1 promoter. NID1 expression in SACC was significantly increased and associated with lung metastasis (P = 0.011). The elevated NID1 expression was a predictor of poor outcomes in patients with SACC (P < 0.05). Overexpression of NID1 promoted cancer cell migration and invasion through PI3K/AKT pathway activation and subsequent epithelial-mesenchymal transition (EMT), as indicated by the upregulation of N-cadherin and vimentin. Furthermore, in vivo live monitoring of a mouse model of lung cancer demonstrated the pro-metastatic role of NID1 in SACC cell lung metastasis. Hypoxia-inducible factor 1α (HIF-1α) upregulation via transfection of an HIF-1α-overexpressing plasmid enhanced HIF-1α binding to the NID1 promoter and the subsequent transcriptional activity and expression of NID1. HIF-1α-activated NID1 overexpression promotes SACC cell metastasis via PI3K/AKT pathway activation and EMT. Thus, NID1 could be a novel biomarker and therapeutic target for preventing metastasis and treating patients with SACC in future.

Han N ，Li X ，Wang Y ，Li H ，Zhang C ，Zhao X ，Zhang Z ，Ruan M ，Zhang C ... -  《-》

Hypoxia promotes vasculogenic mimicry formation by vascular endothelial growth factor A mediating epithelial-mesenchymal transition in salivary adenoid cystic carcinoma.

To investigate the role of hypoxia in vasculogenic mimicry (VM) of salivary adenoid cystic carcinoma (SACC) and the underlying mechanism involved. Firstly, wound healing, transwell invasion, immunofluorescence and tube formation assays were performed to measure the effect of hypoxia on migration, invasion, EMT and VM of SACC cells, respectively. Then, immunofluorescence and RT-PCR were used to detect the effect of hypoxia on VE-cadherin and VEGFA expression. And pro-vasculogenic mimicry effect of VEGFA was investigated by confocal laser scanning microscopy and Western blot. Moreover, the levels of E-cadherin, N-cadherin, Vimentin, CD44 and ALDH1 were determined by Western blot and immunofluorescence in SACC cells treated by exogenous VEGFA or bevacizumab. Finally, CD31/ PAS staining was performed to observe VM and immunohistochemistry was used to determine the levels of VEGFA and HIF-1α in 95 SACC patients. The relationships between VM and clinicopathological variables, VEGFA or HIF-1α level were analysed. Hypoxia promoted cell migration, invasion, EMT and VM formation, and enhanced VE-cadherin and VEGFA expression in SACC cells. Further, exogenous VEGFA markedly increased the levels of N-cadherin, Vimentin, CD44 and ALDH1, and inhibited the expression of E-cadherin, while the VEGFA inhibitor reversed these changes. In addition, VM channels existed in 25 of 95 SACC samples, and there was a strong positive correlation between VM and clinic stage, distant metastases, VEGFA and HIF-1α expression. VEGFA played an important role in hypoxia-induced VM through regulating EMT and stemness, which may eventually fuel the migration and invasion of SACC.

Wang HF ，Wang SS ，Zheng M ，Dai LL ，Wang K ，Gao XL ，Cao MX ，Yu XH ，Pang X ，Zhang M ，Wu JB ，Wu JS ，Yang X ，Tang YJ ，Chen Y ，Tang YL ，Liang XH ... -  《-》

EGF/EGFR Promotes Salivary Adenoid Cystic Carcinoma Cell Malignant Neural Invasion via Activation of PI3K/AKT and MEK/ERK Signaling.

Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant cancers of the salivary gland, and 32.4-72.0% of SACC cases exhibit neural invasion (NI); however, the molecular mechanism underlying the high invasion potential of SACC remains unclear. The present study investigated the role of epidermal growth factor receptor (EGFR) in the AKT inhibition- or mitogen-activated protein kinase kinase (MEK)-induced NI and epithelialmesenchymal transition (EMT) in SACC cells using EGFR, PI3K, and MEK inhibitors. SACC-83 cell viability was assessed using an MTT assay, and a wound healing assay was performed to evaluate cell migration. Immunohistochemical staining with streptavidin peroxidase was used to detect the positive expression rate of EMT, AKT, phosphorylated (p)-AKT, ERK, and p-ERK proteins. The impact of EGFR, PI3K, and MEK inhibitors on tumor growth and NI was examined in a xenograft model in nude mice. EGF and EGFR are effective in increasing cell viability, migration, and invasion. SACC metastasis is affected by the PI3K/AKT and MEK/ERK pathways, both of which are initiated by EGF/EGFR. The EMT and NI are regulated by the EGF/EGFR, PI3K/AKT, and MEK/ERK pathways. The present findings demonstrate the importance of suppressed EGFR/AKT/MEK signaling in NI in SACC by neural-tumor co-culture in vitro. Furthermore, our preclinical experiment provides solid evidence that injection of EGFR, PI3K, and MEK inhibitors suppressed the tumor growth and NI of SACC cells in nude mice. It was identified that inhibitors of EGFR, PI3K/AKT or MEK/ERK suppressed the proliferation, migration, and NI of SACC-83 cells via downregulation of the PI3K/AKT or MEK/ERK pathways. It was also demonstrated that inhibition of EGFR abolishes EMT in SACC by inhibiting the signaling of PI3K/AKT and MEK/ERK. The present results suggest the potential effectiveness of targeting multiple oncogenes associated with downstream pathways of EGF/EGFR, as well as potential therapeutic targets to limit NI in SACC by PI3K/AKT or MEK/ERK inhibition.

Ren Y ，Hong Y ，He W ，Liu Y ，Chen W ，Wen S ，Sun M ... -  《-》

MiR-200b-5p inhibits tumor progression in salivary adenoid cystic carcinoma via targeting BTBD1.

Salivary adenoid cystic carcinoma (SACC) is a rare malignant tumor of the salivary gland. Studies have suggested that miRNA may play a crucial role in the invasion and metastasis of SACC. This study aimed to investigate the role of miR-200b-5p in SACC progression. Reverse transcription-quantitative PCR and western blot assay were used to detect the expression levels of miR-200b-5p and BTBD1. The biological functions of miR-200b-5p were evaluated via wound-healing assays, transwell assays, and xenograft nude mice model. The interaction between miR-200b-5p and BTBD1 was assessed using luciferase assay. Results showed that miR-200b-5p was downregulated in the SACC tissues while BTBD1 was upregulated. miR-200b-5p overexpression suppressed SACC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Bioinformatics prediction and luciferase reporter assay revealed that miR-200b-5p could directly bind to BTBD1. Besides, miR-200b-5p overexpression could rescue the tumor-promoting effect of BTBD1. miR-200b-5p inhibited tumor progression by modulating EMT-related proteins, targeting BTBD1 and inhibiting PI3K/AKT signaling pathway. Overall, our findings indicate that miR-200b-5p can suppress SACC proliferation, migration, invasion, and EMT by regulating BTBD1 and PI3K/AKT axis, providing a promising therapeutic target for SACC treatment.

Tang Y ，Zhu Q ，Yang L ，Meng Y ，Zhang G ，Zhou T ，Wang C ，Song X ，Su YX ，Ye J ... -  《-》

Upregulation of lncRNA ADAMTS9-AS2 Promotes Salivary Adenoid Cystic Carcinoma Metastasis via PI3K/Akt and MEK/Erk Signaling.

Neurotropic infiltrative growth and distant metastasis are the main causes of death in salivary adenoid cystic carcinoma (SACC) patients. Long noncoding RNAs (lncRNAs) are involved in many human neoplasms, however, their potential roles in SACC are unclear. In our study, we found that ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9) antisense RNA 2 (ADAMTS9-AS2) was significantly upregulated in SACC patients with metastasis and SACC-lung metastasis (LM) cells. Moreover, ADAMTS9-AS2 expression was closely associated with the prognosis and distant metastasis in SACC patients. Next, we found that c-myc could specifically bind to the promoter of ADAMTS9-AS2 and activated its transcription. Knockdown of ADAMTS9-AS2 significantly inhibited migration and invasion of SACC cells in vitro and distant lung metastasis in vivo. Furthermore, ADAMTS9-AS2, which mainly expressed in the cytoplasm, shared microRNA (miRNA) response elements with Integrin α6 (ITGA6). Overexpression of ADAMTS9-AS2 competitively bound to miR-143-3p that inhibited ITGA6 from miRNA-mediated degradation, and thus it activated the activity of PI3K/Akt and MEK/Erk signaling and facilitated SACC metastasis. In summary, ADAMTS9-AS2 promotes migration and invasion in SACC by competing with miR-143-3p. This sheds a new insight into the regulation mechanism of ADAMTS9-AS2, and it provides a possible application for the SACC treatment.

Xie S ，Yu X ，Li Y ，Ma H ，Fan S ，Chen W ，Pan G ，Wang W ，Zhang H ，Li J ，Lin Z ... -  《-》