Salivary Adenoid Cystic Carcinoma Cells Research Articles

Long non-coding RNA MRPL23-AS1 suppresses anoikis in salivary adenoid cystic carcinoma in vitro.

Distant lung metastasis is the main factor that affects the survival rate of patients with salivary adenoid cystic carcinoma (SACC). Anoikis resistance is a feature of tumor cells that easily metastasize. The long non-coding RNA (lncRNA) MRPL23 antisense RNA 1 (MPRL23-AS1) is related to lung metastasis in SACC, but its role in anoikis resistance is unknown. After altering MPRL23-AS1 expression in SACC cells, anoikis resistance was detected by calcein AM/PI staining and annexin V/PI flow cytometry. The apoptosis marker activated caspase-3 and the bcl-2/bax ratio were detected by Western blotting. The relationship between MPRL23-AS1 and the promoter of the potential downstream target gene p19INK4D was identified by chromatin immunoprecipitation (ChIP)-PCR assay. p19INK4D expression in patient tissues was determined using qRT-PCR and immunohistochemistry. The functional experiments showed that MPRL23-AS1 could promote anoikis resistance in vitro. MRPL23-AS1 recruited the EZH2 to the promoter region of p19INK4D, inhibited p19INK4D expression, and promoted tumor cell anoikis resistance. p19INK4D overexpression did not affect anoikis in attached cells; however, it attenuated the anoikis resistance effect of MPRL23-AS1 in suspension cells. p19INK4D expression was significantly lower in SACC tissues than in normal tissues. The novel MRPL23-AS1/p19INK4D axis may be a potential SACC biomarker or therapeutic target.

Circular RNA hsa_circ_0011946 promotes the malignant process of salivary adenoid cystic carcinoma by downregulating miR-1205 expression.

Circular RNA (circRNA/circ) hsa_circ_0011946 has been reported to serve an important role in a number of cancer types; however, to the best of our knowledge, its role in salivary adenoid cystic carcinoma (SACC) has not been reported. In the present study, the primary focus was the effects of hsa_circ_0011946 on the invasion, migration and epithelial-mesenchymal transformation (EMT) of SACC cells, and the specific mechanisms involved. The expression levels of hsa_circ_0011946 and microRNA (miR)-1205 in cancer tissues and paracancerous tissues of patients with SACC were analyzed using reverse transcription-quantitative (RT-q)PCR. The cell proliferation rate was determined using a Cell Counting Kit-8 assay. Wound healing assays were performed to analyze the cell migratory ability, while a transwell assay was used to measure the cell invasion ability. Western blotting was used to analyze the expression levels of EMT-related proteins. Cell transfection was used to knockdown hsa_circ_0011946 and knockdown or overexpress miR-1205. Subcellular localization assays for hsa_circ_0011946 were performed using RT-qPCR. A dual-luciferase reporter gene assay was used to verify the binding between hsa_circ_0011946 and miR-1205. The results of the present study revealed that the expression levels of hsa_circ_0011946 were significantly upregulated in cancer tissues from patients with SACC. The knockdown of hsa_circ_0011946 expression inhibited the proliferation, invasion and migration of SACC cells, thereby inhibiting the EMT process, which was achieved by downregulating miR-1205 expression. In conclusion, circRNA hsa_circ_0011946 was discovered to promote the malignant process of SACC by downregulating miR-1205 expression.

Sphk1 promotes salivary adenoid cystic carcinoma progression via PI3K/Akt signaling

The progression of salivary adenoid cystic carcinoma (SACC) is closely related to abnormal gene expression. Herein, the role of Sphk1 in SACC was explored. Sphk1 was overexpressed in SACC tissues. In SACC cell lines, Sphk1 induced cell proliferation, inhibited apoptosis, and promoted cell migration. Moreover, Sphk1 overexpression induced up-regulation of the PI3K protein level and AKT phosphorylation level. Rescue assays further showed that activation of the Sphk1 /PI3K/Akt pathway affected various biological functions of SACC cells. Together, these findings suggested that Sphk1 promotes salivary tumorigenesis by activating the PI3K/ Akt pathway, which may provide novel intervention targets for SACC treatment.

Protein kinase D1 induced epithelial–mesenchymal transition and invasion in salivary adenoid cystic carcinoma via E‐cadherin/Snail regulation

Salivary adenoid cystic carcinoma (SACC) is a malignant tumor, which is characterized by a higher incidence of distant metastasis. The aim of this study was to investigate the role and mechanism of protein kinase D1 (PKD1) in regulating the epithelial-mesenchymal transition (EMT) and promotes the metastasis in SACC. We analyzed the expression of PKD1 in 40 SACC patients and different metastatic potential cell lines. Then, we investigated whether the migration and growth of SACC were regulated by PKD1 using shRNA interference or inhibition of kinase active in vitro cell. Moreover, the mechanism by which PKD1 regulates the stability of Snail protein was determined. Finally, nude mice were used to testify the function of PKD1 via tail vein injection. PKD1 was correlated with metastasis and poor prognosis of SACC patients. PKD1 inhibition attenuated proliferation, migration, invasion, and EMT of SACC cells. Conversely, kinase active PKD1 could induce EMT and promoted cell migration in human HSG cell. Furthermore, downregulation of PKD1 regulated Snail via phosphorylation at Ser-11 on Snail protein and promotion of proteasome-mediated degradation, and reduced lung metastasis in vivo. Our results suggest that PKD1 induces the EMT and promotes the metastasis, which illustrate that PKD1 may be a potential prognostic biomarker and serve as a potential therapeutic target for SACC patients.

CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187.

CXCR5 played critical roles in tumorigenesis and metastasis. Nevertheless, little was known about the involvement of CXCR5 in perineural invasion (PNI) of salivary adenoid cystic carcinoma (SACC). Here, we confirmed upregulation of CXCR5 in SACC specimens and cells and identified that CXCR5 exhibited a significant positive correlation with PNI. Functionally, knockdown of CXCR5 suppressed SACC cells migration, invasion and PNI ability, whereas CXCR5 overexpression displayed the opposite effects. Moreover, CXCR5 downregulated microRNA (miR)-187, which could competitively sponge S100A4. The PNI-inhibitory effect of CXCR5 knockdown or miR-187 overexpression could be reversed by elevated expression of S100A4. Conjointly, our data revealed that CXCR5 facilitated PNI through downregulating miR-187 to disinhibit S100A4 expression in SACC.

The expression of the GATA6 gene in oral carcinoma cell lines

BackgroundThis study aimed to investigate the expression level of the GATA6 gene in different oral cancer cells.MethodsIn this study, we sub-cultured normal oral epithelial cell lines HOK, human tongue squamous cell carcinoma cell lines CAL-27 and SCC-4, and human salivary gland adenoid cystic carcinoma cell lines SACC-LM and SACC-83. Subsequently, we used reverse transcription-polymerase chain reaction RT-PCR and Western blot methods to detect the mRNA and the protein expressions of GATA6 in normal oral epithelial cells, human tongue squamous cell carcinoma cells, and human salivary gland adenoid cystic carcinoma cells.ResultsThe results of this study showed that the mRNA expression levels of GATA6 in CAL-27, SCC-4, and SACC-LM cells were significantly increased when compared with the HOK cells. However, the mRNA expression level of GATA6 in the SACC-83 cells had no significant difference compared with the HOK cells. The protein expression levels of GATA6 in the SCC-4 and SACC-LM cells were, however, significantly increased whereas the protein expression levels of GATA6 in the CAL-27 and SACC-83 cells had no significant difference when compared with the HOK cells.ConclusionThe GATA6 gene may be related to the occurrence and progression of certain oral cancers.

Inhibition of DEC2 is necessary for exiting cell dormancy in salivary adenoid cystic carcinoma

BackgroundPatients were prone to have poor prognosis once dormant tumor cells being reactivated. However, the molecular mechanism of tumor cell dormancy remains poorly understood. This study aimed to investigate the function of DEC2 in the dormancy of salivary adenoid cystic carcinoma (SACC) in vitro and vivo.MethodsThe function of DEC2 in tumor dormancy of SACC was investigated in nude mice by establishing primary and lung metastasis model. Meanwhile, the interaction between hypoxia and SACC dormancy and the role of DEC2 were demonstrated through CoCl2 induced hypoxia–mimicking microenvironments. Furthermore, the expression of DEC2 was detected by immunohistochemical staining in primary SACC samples with and without recurrence.ResultsIn the primary SACC, DEC2 overexpression inhibited cell proliferation, increased cell population arrested in G0/G1 phase, and participated in dormancy regulation, which limited tumor growth. Intriguingly, in the model of lung metastasis, the level of DEC2 was reduced significantly and resulted in dormancy exit and growth resumption of SACC cells. Then, we found that DEC2 may associate with hypoxia in contributing to tumor dormancy, which might provide a possible cue to explain the different roles of DEC2 in primary and metastasis lesions. And overexpression of DEC2 induced dormancy and promoted migration and invasion through activating EMT program. Finally, DEC2 positive expression was shown to be significantly correlated with recurrence and dormancy of SACC patients.ConclusionsThese findings provide a novel insight into the role of DEC2 gene in tumor dormancy and metastasis.

The Role of MRPL23 Antisense RNA 1 (MRPL23-AS1) in the Pre-Metastatic Microenvironment of Malignancy During the Process of Epithelial-Mesenchymal Transition

We aimed to explore MRPL23-AS1’s role in the pre-metastatic microenvironment of malignancy during epithelial-mesenchymal transition (EMT). Identification and verification of lncRNA-interacting proteins in salivary adenoid cystic carcinoma (SACC) cells were conducted via RNA-pulldown, silver staining, and Western blotting. RIP and RIP-seq were sequentially administered to verify the binding partners of lncRNA. CHIRP was performed to detect the promoter DNA in the downstream of lncRNA-protein complex. Ultimately CHIP-qPCR detected the effects of lncRNA on the binding degree of its interacting protein to the promoter DNA in the downstream genes and the methyla-tion level of histones in the promoter region. The exosomes secreted by different SACC cells were extracted from culture supernatant to measure lncRNA expression via qPCR. MRPL23-AS1 interacted with EZH2 protein and promoted EZH2 binding to E-cadherin gene promoter region along with the H3K27 methylation. MRPL23-AS1 could promote EMT of SACC cells and increase pulmonary vascular endothelial cells permeability via exosomes secretion. MRPL23-AS1 up-regulated VEGFA, while down-regulated E-cadherin and VE-cadherin in endothelial cells. Exosomes rich in MRPL23-AS1 could boost lung metastasis in vivo. MRPL23-AS1 inhibits E-cadherin level and promotes EMT of SACC cells, suggesting that it might be a biomarker and therapeutic target for lung cancer.

MiR-5191 acts as a tumor suppressor in salivary adenoid cystic carcinoma by targeting Notch-2.

This study sought to investigate the effect of miR-5191 on proliferation, invasion and metastasis in salivary adenoid cystic carcinoma (SACC). The differential expression level of miR-5191 between 5 primary tumor and adjacent non-neoplastic samples, and in two SACC cell lines was detected by quantitative real-time PCR. Cell proliferation, invasion, and migration were performed, followed by luciferase reporter assay and western analysis. The effect of miR-5191 on cell proliferation and apoptosis was evaluated by cell growth and apoptosis assay. The function of miR-5191 in SACC tumorigenesis and metastasis in vivo was investigated by nude mice experiment. The associations between miR-5191/Notch-2 expression and clinicopathological features were analyzed. miR-5191 was downregulated in primary tumor tissues and SACC-LM cells. By targeting Notch-2, miR-5191 expression level affected the migration, invasion, and proliferation of SACC cells. Overexpression of miR-5191 inhibited the expression levels of Notch-2, followed by the decreased expression of c-Myc, Bcl-2, Hes-1, Hey-1, and Cyclin D1. In vivo, miR-5191 overexpression suppressed the SACC tumorigenesis and pulmonary metastasis in mice. In SACC patients, higher expression of miR-5191 was related to better prognoses and lower possibility of metastasis. miR-5191 acts as a tumor suppressor in SACC by targeting Notch-2.

Dll4/Notch1 signalling pathway is required in collective invasion of salivary adenoid cystic carcinoma.

High expression of δ-like ligand 4 (Dll4) is reportedly related to the invasion, metastasis, and clinical prognosis of various malignant tumours. Our previous study revealed that collective cell invasion was a common pattern in salivary adenoid cystic carcinoma (SACC). However, the roles of the Dll4/Notch1 signalling pathway in the collective invasion of SACC remain unclear. The present study revealed that Dll4 expression was higher at the invasive front of SACC, and that this upregulation was associated with solid tumour type, high TNM grade, and high rates of metastasis and recurrence. Furthermore, the expression levels of Notch1 and Dll4 were positively correlated at the invasive front, and a three-dimensional (3D) culture model revealed that leader cells showed high expression of Dll4, while follower cells showed high expression of Notch1. Moreover, silencing of Dll4 expression using small interfering RNA reduced the migration, invasion, and collective invasion of SACC cells, and these abilities were rescued by Notch1 overexpression. Finally, SACC collective invasion was increased via the Dll4/Notch1 signalling pathway in experiments that involved a stiff 3D gel, hypoxia and co-culture with human endothelial cells. These findings indicated that the Dll4/Notch1 signalling pathway may be involved in the collective invasion of SACC, which may help to provide possible targets for the treatment of SACC.

CASC9 plays a role in salivary adenoid cystic carcinoma in vitro by upregulation of ACLY.

The study was designed to explore the role of cancer susceptibility candidate 9 (CASC9) in salivary adenoid cystic carcinoma (SACC) (SACC-83 and SACC-LM) cell malignant phenotypes. Colony formation assay was used to measure cell proliferation. Transwell assay was used to detect cell migration and invasion. Flow cytometry analysis was applied to determine cell cycle distribution and apoptosis. FISH assay revealed the subcellular location of CASC9. Downregulation of CASC9 inhibited SACC cell proliferation, migration, and invasion, led to cell arrest at G0/G1 phase, and facilitated cell apoptosis. In mechanism, CASC9 bound with microRNA 146b-5p (miR-146b-5p) and negatively modulated miR-146b-5p expression. MiR-146b-5p directly targeted 3' untranslated region of ATP-Citrate Lyase (ACLY) to degrade ACLY in SACC cells. CASC9 upregulated ACLY expression through competitively binding with miR-146b-5p. Furthermore, rescue assays indicated that ACLY overexpression counteracted the effects triggered by CASC9 knockdown on cell proliferation, migration, invasion, and apoptosis in SACC cells. CASC9 facilitated the malignant phenotypes of SACC cells by the regulation of the miR-146b-5p/ACLY axis. These findings might lay foundation for SACC research.

The circular RNAs differential expression profiles in the metastasis of salivary adenoid cystic carcinoma cells.

In order to reveal circular RNAs (circRNAs) differential expression profiles and investigate the function and mechanism of circRNAs in the metastasis of salivary adenoid cystic carcinoma (SACC), microarray was used to detect differentially expressed circRNAs in SACC-83 and SACC-lung metastasis (LM) cell lines. Up-regulated circRNAs were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to further predict their function. Expression of candidate circRNA and microRNA (miRNA) was determined using quantitative real-time polymerase chain reaction (qRT-PCR). Constructed circRNA-miRNA-mRNA co-expression network was based on TargetScan, miRanda databases. Wound healing and transwell assays were completed to examine the effects of hsa_circRNA_001982 and miR-181a-5p on cell migration and invasion. qRT-PCR confirmed hsa_circRNA_092556, hsa_circRNA_101379, and hsa_circRNA_001982 up-regulation in SACC-LM. miR-181a-5p was down-regulated in SACC-LM and correlated with up-regulated hsa_circRNA_001982. Wound healing and transwell assays indicated that silencing hsa_circRNA_001982 inhibited the migration and invasion of the SACC-LM cells. Furthermore, over-expression of hsa_circRNA_001982 promoted the migration and invasion of SACC-83 cells. Interestingly, up-regulation or down-regulation of miR-181a-5p led to the opposite result in wound healing and transwell assays. Overall, differential expression circRNA profiles in SACC-83 and SACC-LM cells may reveal potential targets and a novel mechanism of circRNAs in the metastasis of SACC. Moreover, the interaction of hsa_circRNA_001982/miR-181a-5p is closely related to the metastasis of SACC cells.

Establishment of PDX-derived salivary adenoid cystic carcinoma cell lines using organoid culture method.

To generate a reliable preclinical model system exhibiting the molecular features of salivary adenoid cystic carcinoma (ACC) whose biology is still unclear due to the paucity of stable cell cultures. To develop new in vitro and in vivo models of ACC, the techniques of organoid culture and patient-derived tumor xenograft (PDX), which have attracted attention in other malignancies in recent years, were applied. Tumor specimens from surgically resected salivary ACC were proceeded for the preparation of PDX and organoid culture. The orthotopic transplantation of patient-derived or PDX-derived organoids was demonstrated into submandibular glands of NSG mice and those histology was evaluated. PDX-derived organoid cells were evaluated for the presence of MYB-mediated fusion genes and proceeded for in vitro drug sensitivity assay. Human ACC-derived organoids were successfully generated in three-dimensional culture and confirmed the ability of these cells to form tumors by orthotopic injection. Short-term organoid cell cultures from two individual ACC PDX tumors were also established that maintain the characteristic MYBL1 translocation and histological features of the original parent and PDX tumors. Finally, the establishment of drug sensitivity tests on these short-term cultured cells was confirmed using three different agents. This is the first to report an approach for the generation of human ACC-derived organoids as in vitro and in vivo cancer models, providing insights into understanding of the ACC biology and creating personalized therapy design for patients with ACC.

Fatty acid synthase contributes to epithelial-mesenchymal transition and invasion of salivary adenoid cystic carcinoma through PRRX1/Wnt/β-catenin pathway.

Fatty acid synthase (FASN) has been shown to be selectively up‐regulated in cancer cells to drive the development of cancer. However, the role and associated mechanism of FASN in regulating the malignant progression of salivary adenoid cystic carcinoma (SACC) still remains unclear. In this study, we demonstrated that FASN inhibition attenuated invasion, metastasis and EMT of SACC cells as well as the expression ofPRRX1, ZEB1, Twist, Slug and Snail, among which the level of PRRX1 changed the most obviously. Overexpression of PRRX1 restored migration and invasion in FASN knockdown cells, indicating that PRRX1 is an important downstream target of FASN signalling. Levels of cyclin D1 and c‐Myc, targets of Wnt/β‐catenin pathway, were significantly decreased by FASN silencing and restored by PRRX1 overexpression. In addition, FASN expression was positively associated with metastasis and poor prognosis of SACC patients as well as with the expression of PRRX1, cyclin D1 and c‐Myc in SACC tissues. Our findings revealed that FASN in SACC progression may induce EMT in a PRRX1/Wnt/β‐catenin dependent manner.

Abstract LB-310: Whole-exome sequencing reveals genetic underpinnings of salivary adenoid cystic carcinoma and tongue carcinoma in Chinese population

Abstract Background: Salivary adenoid cystic carcinoma (ACC) and oral tongue squamous cell carcinoma (OTSCC) are rare malignancies with low incidence and dismal prognosis. The molecular underpinnings of these remain poorly understood, particularly in Asian population. Methods: Through a whole-exome sequencing analysis of 13 OTSCC and 30 ACC patients, a systematic overview of the mutational features in single-nucleotide variations, copy number variations and chromosomal variations were illustrated. Results: In addition to the well-characterized alterations previously reported in Caucasian patients, such as TP53 mutations (11/13, 84.6%) in OTSCC and MYB-NFIB fusion in ACC (11/30, 36.7%), we also observed several novel genetic changes, including FOXP1-TEX261 fusion (1/13, 7.7%) and MYB-FMO5 fusion (1/30, 3.3%) in OTSCC and ACC respectively, which might play a role in facilitating tumor progression. The data exhibited promising translational value as numerous actionable genes were identified with mutations. 76.9% OTSCC and 20% salivary ACC harbored at least one alteration in actionable genes, mutations in CDKN2A (4/13, 30.8%), NTRK3 (3/13, 23.1%) in OTSCC and FGFR2 (2/30, 6.7%), BRAF (2/30, 6.7%) in salivary gland ACC, suggesting targeted immunotherapy might be an effective intervention. Instead of MYB-independent NFIB fusions previously reported in ACC, only NFIB-independent MYB fusions were found in our cohort, indicating that alterations in NFIB rather than MYB might be essential to ACC tumorigenesis. Conclusions: This study reveals the genetic underpinnings of two rare malignancies in Chinese population, illustrating consistent mutational features with published literature as well as demonstrating several distinct genetic alterations, warranting further investigations. Citation Format: Ning Li, Shuhang Wang, Yue Yu, Yuan Fang, Huiyao Huang, Dawei Wu, Hong Fang, Chao Sun, Anqi Yu, Qi Fan, Shi-Yuan Cheng. Whole-exome sequencing reveals genetic underpinnings of salivary adenoid cystic carcinoma and tongue carcinoma in Chinese population [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-310.

MicroRNA‑103a‑3p promotes metastasis by targeting TPD52 in salivary adenoid cystic carcinoma.

Salivary adenoid cystic carcinoma (SACC) exhibits slow continuous growth, frequent local recurrences and a high incidence of blood metastasis, with advanced lung metastasis frequently occurring and being among the primary causes of mortality. MicroRNAs(miR) serve a significant role in the initiation and development of cancer and may be tumour‑specific molecular targets. However, the role of miR‑103a‑3p in SACC remains largely unknown. In the present study, the expression levels of miR‑103a‑3p and tumour protein D52 (TPD52) were detected by reverse transcription‑quantitative PCR. In addition, wound‑healing assays, Transwell assays and mouse models of lung metastasis were used to investigate the biological functions exerted by miR‑103a‑3p. The present results suggested that miR‑103a‑3p expression was significantly upregulated in SACC samples. Gain‑of‑function and loss‑of‑function studies in SACC cells demonstrated that miR‑103a‑3p acted as an oncogene by promoting tumour cell migration invitro and lung metastasis invivo. Dual‑luciferase reporter gene assays indicated that miR‑103a‑3p exerted its regulatory functions by binding to the 3'untranslated region of TPD52 mRNA. TPD52 overexpression rescued the effect of miR‑103a‑3p on promoting SACC cell migration, suggesting that miR‑103a‑3p acted as an oncogene to promote cancer metastasis by directly targeting TPD52. Thus, the newly identified miR‑103a‑3p/TPD52 axis contributes to the understanding of SACC pathogenesis, providing insights into the identification of novel biomarkers or potential therapeutic targets in SACC.

C6-ceramide induces salivary adenoid cystic carcinoma cell apoptosis via IP3R-activated UPR and UPR-independent pathways

C6-ceramide is an exogenous short-chain ceramide which can induce apoptosis of multiple cancer cells. Salivary adenoid cystic carcinoma (SACC) is a common salivary gland cancer, which possesses of high rate of local recurrence and distant metastasis. The mechanism of ceramide-induced SACC-83 and SACC-LM cell apoptosis has not been revealed. In our study, gene expression microarray was used to discover that the unfolded protein response (UPR) pathway, especially PRKR-like endoplasmic reticulum kinase (PERK) pathway, was the major activated pathway after treatment of c6-ceramide. D1ER, an endoplasmic-reticulum-targeted Ca2+ indicator, was used to measure Ca2+ release from endoplasmic reticulum (ER) dynamically. We found that inositol 1,4,5-trisphosphate receptor 3 (IP3R3) was activated, leading to Ca2+ release from ER, soon after c6-ceramide treatment. IP3R3 silencing could block UPR, although it could not prevent SACC-83 and SACC-LM cells from apoptosis. Moreover, we found that C/EBP-homologous protein could upregulate in a UPR-independent way. Mitochondria outer membrane permeabilization might play an important role in inducing SACC cell apoptosis.

The NOTCH1-HEY1 pathway regulates self-renewal and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells.

Our previous study demonstrated a close relationship between the NOTCH signaling pathway and salivary adenoid cystic carcinoma (SACC). Its receptor gene, NOTCH1, and its downstream gene, HES1, contribute to the proliferation, invasion and metastasis of SACC. Accumulating evidence supports HEY1 as another effector of the signaling pathway. The purpose of this study was to explore the effects of the NOTCH1-HEY1 pathway on the proliferation, invasion and metastasis of SACC cells. Our results verified that HEY1 is a specific molecular target of the NOTCH signaling pathway in SACC cells and that its expression in carcinoma is much higher than that in paracarcinoma tissues. The expression of NOTCH1 and HEY1 are positively correlated in the salivary adenoid cystic carcinoma tissues. NOTCH1 is significantly related to the activation of HEY1 in SACC, and that HEY1 reciprocally regulates NOTCH1 expression in SACC. HEY1 promotes cell proliferation and spheroid formation and inhibits cell apoptosis in vitro. In addition, HEY1 enhances the tumorigenicity of SACC in vivo. Furthermore, HEY1 increases cell invasion and metastasis by driving the expression of epithelial-mesenchymal transition (EMT)-related genes and MMPs. The results of this study indicate that the NOTCH1-HEY1 pathway is specifically upregulated in SACC and promotes cell proliferation, self-renewal, invasion, metastasis and the expression of EMT-related genes and MMPs. Our findings suggest that a NOTCH1-HEY1 pathway inhibitor might therefore have potential therapeutic applications in treating SACC patients by inhibiting cancer cell growth and metastasis.

Claudin-7 Inhibits Proliferation and Metastasis in Salivary Adenoid Cystic Carcinoma Through Wnt/β-Catenin Signaling

The aim of this study was to investigate claudin-7 (CLDN7) expression in salivary adenoid cystic carcinoma (SACC) and its function in SACC cells. We determined CLDN7 expression in SACC tumors via immunohistochemistry and western blotting and evaluated the association between CLDN7 expression and clinicopathologic variables. Besides this, we constructed a stably transfected CLDN7 knockdown SACC-LM cell line via RNAi and assessed its biological behavior changes (cell viability, migration, and invasion). The correlation between CLDN7 and epithelial-mesenchymal transition (EMT) was analyzed. Additionally, a subcutaneous tumor formation model was used to assess SACC-LM cells tumorigenicity after the CLDN7 knockdown. In the present study, we found the CLDN7 expression of tumor group was lower than that in normal salivary glands and was significantly correlated with lymph node metastasis, recurrence, and gender. CLDN7 knockdown could add the proliferation and metastasis ability of SACC by regulating EMT through Wnt/β-catenin signaling pathway. In addition, CLDN7 knockdown in SACC promoted tumor growth in nude mice. CLDN7 inhibits cell proliferation and metastasis by inactivating the Wnt/β-catenin signaling in SACC. Thus, CLDN7 expression might be a useful marker to identify the potential for progression in SACC.

RhoG/Rac1 signaling pathway involved in migration and invasion of salivary adenoid cystic carcinomacells.

This study aimed to explore whether RhoG/Rac1 was involved in migration and invasion of salivary adenoid cystic carcinoma (SACC). RhoG and Rac1 were evaluated in two SACC cell lines, namely SACC-83 and SACC-LM, with low and high rates of lung metastasis, respectively. Functional changes were evaluated using cell proliferation, transwell, and wound-healing assays, and molecular events were investigated using real-time PCR and Western blot assays. RhoG and Rac1 were highly expressed and more activated in SACC-LM cells than in SACC-83 cells. RhoG overexpression promoted SACC-83 cell migration and invasion through activating Rac1. The knockdown of RhoG or Rac1 partially blocked epiregulin-induced migration and invasion in SACC-83 cells. Epiregulin-induced activation of RhoG/Rac1 in SACC-83 cells was blocked by a Src inhibitor, or an AKT inhibitor or AKT siRNA, or an ERK1/2 inhibitor. Moreover, the epiregulin-induced phosphorylation of AKT and ERK1/2 in SACC-83 cells was blocked by a Src inhibitor, and the epiregulin-induced phosphorylation of ERK1/2 was blocked by an AKT inhibitor or AKT siRNA. Overexpression of activated AKT induced activation of ERK1/2 and RhoG. RhoG/Rac1 signaling pathway was involved in SACC cell migration and invasion. RhoG/Rac1 at least partially mediated epiregulin/Src/AKT/ERK1/2 signaling to promote SACC cell migration and invasion.