Transcription Factor MYB as Therapeutic Target: Current Developments.

The MYB protein is a pivotal player in the cellular transcriptional network, influencing major important processes such as cell proliferation, differentiation, and apoptosis. Because of its role in oncogenesis, MYB is now a compelling target for therapeutic interventions in cancer research. This review summarizes its molecular functions and current therapeutic approaches aiming to inhibit its oncogenic activity.

TGFß signaling pathway in salivary gland tumors.

OBJECTIVE: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß cascade. DESIGN: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. RESULTS: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFß signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. CONCLUSIONS: Our study demonstrated the differential expression of TGFß cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

Novel Epigenetic Modifiers of Histones Presenting Potent Inhibitory Effects on Adenoid Cystic Carcinoma Stemness and Invasive Properties.

Adenoid cystic carcinoma (ACC) is a rare neoplasm known for its indolent clinical course, risk of perineural invasion, and late onset of distant metastasis. Due to the scarcity of samples and the tumor's rarity, progress in developing effective treatments has been historically limited. To tackle this issue, a high-throughput screening of epigenetic drugs was conducted to identify compounds capable of disrupting the invasive properties of the tumor and its cancer stem cells (CSCs). ACC cells were screened for changes in tumor viability, chromatin decondensation, Snail inhibition along tumor migration, and disruption of cancer stem cells. Seven compounds showed potential clinical interest, and further validation showed that Scriptaid emerged as a promising candidate for treating ACC invasion. Scriptaid demonstrated a favorable cellular toxicity index, effectively inhibited Snail expression, induced hyperacetylation of histone, reduced cell migration, and effectively disrupted tumorspheres. Additionally, LMK235 displayed encouraging results in four out of five validation assays, further highlighting its potential in combating tumor invasion in ACC. By targeting the invasive properties of the tumor and CSCs, Scriptaid and LMK235 hold promise as potential treatments for ACC, with the potential to improve patient outcomes and pave the way for further research in this critical area.

Anti-NOTCH1 therapy with OMP-52 M51 inhibits salivary adenoid cystic carcinoma by depressing epithelial-mesenchymal transition (EMT) process and inducing ferroptosis.

Salivary adenoid cystic carcinoma (ACC) is a common type of salivary gland cancer, and the mechanisms underlying its progression still remain poorly understood without efficient therapies. NOTCH1, an evolutionally conserved cell-cell signaling pathway, is involved in the progression of ACC. In our study, we attempted to explore whether NOTCH1 suppression using the monoclonal anti-NOTCH1 antibody OMP-52 M51 could be of potential for ACC treatment. Here, we identified NOTCH1 elevation in human ACC tissues compared with the matched normal samples. Patients with metastasis expressed much higher NOTCH1. We then found that OMP-52 M51 markedly reduced the expression of NOTCH1 and its intracellular active form NICD1 (NOTCH1 intracellular domain). Importantly, OMP-52 M51 markedly reduced the proliferation, migration and invasion of ACC cells. RNA-Seq and in vitro studies further showed that OMP-52 M51 significantly induced ferroptosis in ACC cells, indicated by the increased cellular malondialdehyde (MDA), iron contents and lipid ROS production, and decreased glutathione (GSH) levels. Further, remarkable glutathione peroxidase 4 (GPX4) reduction was detected in ACC cells with OMP-52 M51 treatment. However, promoting NOTCH1 expression markedly abolished the function of OMP-52 M51 to induce ferroptosis. Intriguingly, low-dose OMP-52 M51 strongly facilitated the capacity of ferroptosis inducer erastin to trigger ferroptotic cell death, revealing that OMP-52 M51 could improve the sensitivity of ACC cells to ferroptosis. In vivo, OMP-52 M51 administration suppressed tumor growth and induced ferroptosis in the constructed ACC xenograft mouse model. Collectively, our findings demonstrated that NOTCH1 inhibition by OMP-52 M51 represses the proliferation and epithelial-mesenchymal transition (EMT) in ACCs, and promotes ferroptosis, revealing the potential therapeutical application of OMP-52 M51 in ACC.

Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation.

Geranylgeranyltransferase type I (GGTase-I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase-I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase-I in mediating the malignant progression of SACC. The level of GGTase-I gene in cells was stably knocked down by short hairpin RNA-EGFP-lentivirus. The effects of GGTase-I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase-I and RhoA genes of SACC cells after GGTase-I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase-I on the above-mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p-MLC, E-cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1 . Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse-transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase-I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase-I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p-MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E-cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase-I is expected to serve as a novel exploration site of SACC.

The mitotic checkpoint kinase BUB1 is a direct and actionable target of MYB in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.

Locally Advanced Adenoid Cystic Carcinoma of the Breast-A Case Report with a Review of the Literature.

Breast cancer (BC) is a heterogeneous disease distinguished by four main subtypes based on the expression of estrogen, progesterone receptors, and human epidermal growth factor-2 on the cancer cells. Triple-negative breast cancer (TNBC) consists of approximately 10-20% of all BCs and is characterized by a poor prognosis. Adenoid cystic carcinoma (ACC) of the breast is a rare, special type of TNBC with low metastatic potential and usually favorable prognosis. There are no established recommendations concerning systemic therapy in advanced ACC. We present a case of a 70-year-old woman with locally advanced ACC with progression after radical mastectomy, and review the literature concerning the treatment of metastatic disease focused on systemic therapy.

Induction of perineural invasion in salivary adenoid cystic carcinoma by circular RNA RNF111

Objective Local recurrence, distant metastasis, and perineural invasion (PNI) viciously occur in salivary adenoid cystic carcinoma (SACC), resulting in a poor prognosis. This study aimed to explore the mechanism by which circular RNA RNF111 (circ-RNF111) regulates PNI in SACC by targeting the miR-361-5p/high mobility group box 2 (HMGB2) axis. Method Circ-RNF111 and HMGB2 were highly expressed in SACC specimens, while miR-361-5p was underexpressed. Functional experiments showed that ablating circ-RNF111 or promoting miR-361-5p hindered the biological functions and PNI of SACC-LM cells. Results HMGB2 overexpression induced the reversal of SACC-LM cell biological functions and PNI caused by circ-RNF111 knockout. Furthermore, reduction of circ-RNF111 suppressed PNI in a SACC xenograft model. Circ-RNF111 regulated HMGB2 expression through targeted modulation of miR-361-5p. Conclusion Taken together, circ-RNF111 stimulates PNI in SACC by miR-361-5p/HMGB2 axis and may serve as a potential therapeutic target for SACC (AU)

Extracting Potential New Targets for Treatment of Adenoid Cystic Carcinoma using Bioinformatic Methods.

Background: Adenoid cystic carcinoma is a slow-growing malignancy that most often occurs in the salivary glands. Currently, no FDA-approved therapeutic target or diagnostic biomarker has been identified for this cancer. The aim of this study was to find new therapeutic and diagnostic targets using bioinformatics methods. Methods: We extracted the gene expression information from two GEO datasets (including GSE59701 and GSE88804). Different expression genes between adenoid cystic carcinoma (ACC) and normal samples were extracted using R software. The biochemical pathways involved in ACC were obtained by using the Enrichr database. PPI network was drawn by STRING, and important genes were extracted by Cytoscape. Real-time PCR and immunohistochemistry were used for biomarker verification. Results: After analyzing the PPI network, 20 hub genes were introduced to have potential as diagnostic and therapeutic targets. Among these genes, PLCG1 was presented as new biomarker in ACC. Furthermore, by studying the function of the hub genes in the enriched biochemical pathways, we found that insulin-like growth factor type 1 receptor and PPARG pathways most likely play a critical role in tumorigenesis and drug resistance in ACC and have a high potential for selection as therapeutic targets in future studies. Conclusion: In this study, we achieved the recognition of the pathways involving in ACC pathogenesis and also found potential targets for treatment and diagnosis of ACC. Further experimental studies are required to confirm the results of this study.

A Review of the Molecular Landscape of Adenoid Cystic Carcinoma of the Lacrimal Gland.

Adenoid cystic carcinoma (ACC) has a worldwide incidence of three to four cases per million population. Although more cases occur in the minor and major salivary glands, it is the most common lacrimal gland malignancy. ACC has a low-grade, indolent histological appearance, but is relentlessly progressive over time and has a strong proclivity to recur and/or metastasise. Current treatment options are limited to complete surgical excision and adjuvant radiotherapy. Intra-arterial systemic therapy is a recent innovation. Recurrent/metastatic disease is common due to perineural invasion, and it is largely untreatable as it is refractory to conventional chemotherapeutic agents. Given the rarity of this tumour, the molecular mechanisms that govern disease pathogenesis are poorly understood. There is an unmet, critical need to develop effective, personalised targeted therapies for the treatment of ACC in order to reduce morbidity and mortality associated with the disease. This review details the evidence relating to the molecular underpinnings of ACC of the lacrimal gland, including the MYB-NFIB chromosomal translocations, Notch-signalling pathway aberrations, DNA damage repair gene mutations and epigenetic modifications.

Immunohistochemical Expression Of Human Epidermal Growth Factor Receptor-2 (Her-2) In Common Salivary Gland Carcinomas.

BACKGROUND: Carcinomas of the salivary gland are known to be aggressive in nature, making them difficult to manage. The therapeutic options offered include excision of the gland (maxillectomy in cases of palatal tumours), with or without lymph node dissection, proceeded with radiotherapy. Chemotherapy has not produced promising outcomes and has a minimal impact as a therapeutic alternative. Targeted therapy against human epidermal growth factor receptor 2 (HER-2), which is a commonly used treatment modality for their mammary analogues, is not being offered to these patients since scant literature is available showing its usefulness and no promising evidence is present regarding their efficacy and efficiency in such cases. The study aimed to evaluate and quantify the immunohistochemical expression of HER-2 in cases of adenoid cystic carcinoma (AdCC), mucoepidermoid carcinoma (MEC) and salivary duct carcinoma (SDC), which are analogues of similar tumours arising in breast tissue. METHODS: A retrospective, cross-sectional study was carried out in the department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, duration of which was six months. A total of 45 cases (15 of each tumour) were taken, and sampled using non-probability convenience technique. The immunohistochemical marker, monoclonal HER-2 antibody (Leica microsystem Germany) was applied on appropriate blocks of all included cases. The staining pattern and intensity were recorded after visualizing the slides under a light microscope. RESULTS: Seven cases of salivary duct carcinoma and a single case of mucoepidermoid carcinoma expressed positivity for HER-2, while no expression could be seen in the case of adenoid cystic carcinoma. A statistically significant difference was seen when HER-2 expression was compared among the aforementioned tumours. CONCLUSIONS: The use of targeted therapy against HER-2 is limited to patients of salivary duct carcinoma and a fraction of patients suffering from mucoepidermoid carcinoma.

CELA3B immunostaining is a highly specific marker for acinar cell carcinoma of the pancreas.

Chymotrypsin-like elastase family member 3B (CELA3B, elastase-3B) is a pancreatic enzyme with digestive function in the intestine. Since RNA analyses of normal tissues suggest that CELA3B expression is limited to the pancreas, the potential diagnostic utility of CELA3B immunohistochemistry for the distinction of pancreatic from extrapancreatic cancers and in the distinction of acinar cell carcinoma from ductal adenocarcinoma was assessed. CELA3B expression was successfully analyzed in 13,223 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types by immunohistochemistry in a tissue microarray format (TMA). In normal tissues, CELA3B immunostaining was only seen in acinar cells and in a fraction of ductal cells of the pancreas as well as on some apical membranes of surface epithelial cells of the intestine. Among tumors, CELA3B immunostaining was seen in 12 of 16 (75%) acinar cell carcinoma of the pancreas including 6 cases with strong staining (37.5%) as well as in 5 of 13,207 other tumors (0.04%). These included 1.2% of 91 adenoid cystic carcinomas, 1.2% of 246 mucoepidermoid carcinomas and 0.8% of 127 acinic cell carcinomas of salivary glands. Our data show a good sensitivity (75%) and a high specificity (99.9%) of CELA3B immunohistochemistry for diagnosing acinar cell carcinoma of the pancreas.

Identification of MicroRNA Expression Profiles Related to the Aggressiveness of Salivary Gland Adenoid Cystic Carcinomas.

Adenoid cystic carcinoma (ACC) has been reported as the second most common carcinoma of the salivary glands. Few studies have associated miRNA expression with ACC aggressiveness. In this study, we evaluated the miRNA profile of formalin-fixed, paraffin-embedded (FFPE) samples of salivary gland ACC patients using the NanoString platform. We studied the miRNA expression levels associated with the solid growth pattern, the more aggressive histologic feature of ACCs, compared with the tubular and cribriform growth patterns. Moreover, the perineural invasion status, a common clinicopathological feature of the disease that is frequently associated with the clinical progression of ACC, was investigated. The miRNAs showing significant differences between the study groups were selected for target prediction and functional enrichment, which included associations with the disease according to dedicated databases. We observed decreased expression of miR-181d, miR-23b, miR-455, miR-154-5p, and miR-409 in the solid growth pattern compared with tubular and cribriform growth patterns. In contrast, miR-29c, miR-140, miR-195, miR-24, miR-143, and miR-21 were overexpressed in patients with perineural invasion. Several target genes of the miRNAs identified have been associated with molecular processes involved in cell proliferation, apoptosis, and tumor progression. Together, these findings allowed the characterization of miRNAs potentially associated with aggressiveness in salivary gland adenoid cystic carcinoma. Our results highlight important new miRNA expression profiles involved in ACC carcinogenesis that could be associated with the aggressive behavior of this tumor type.

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.

[Study on enhanced sensitivity to DNA damage in POLQ knocking-down salivary of adenoid cystic carcinoma-83 cells].

PURPOSE: To investigate the effects of POLQ inhibition on proliferation, colony formation, cell cycle, DNA damage and repair in salivary adenoid cystic carcinoma-83 (SACC-83) cell line. METHODS: POLQ knocking-down SACC-83 cells were constructed using short hairpin RNA (shRNA) transient transfection, and the inhibition efficiency was detected by qRT-PCR and Western blot. DNA damage in SACC-83 cells was induced by different concentration of DNA damage agent etoposide (VP-16-213), and the levels of γH2AX expression were detected by Western blot to evaluate DNA double-strain breaks. Under different concentration of etoposide-induced DNA damage condition, CCK-8 assay was used to evaluate the effect of POLQ inhibition on cell proliferation in SACC-83 cell line. Under etoposide-induced DNA damage condition, plate colony assay was performed to detect the effect of POLQ inhibition on cell clone formation ability in SACC-83 cell line, and flow cytometry was used to detect the effect of POLQ inhibition on cell cycle in SACC-83 cell line. Furthermore, under etoposide-induced DNA damage condition, Western blot was used to analyze POLQ, γH2AX, RAD51 and PARP1 protein expression. SPSS 20.0 software package was used for statistical analysis. RESULTS: The mRNA and protein expression of POLQ was inhibited by shRNA transient transfection. Increased γH2AX in SACC-83 was closely coupled with increased concentrations of etoposide. The results of CCK-8 assay showed that POLQ knocking-down suppressed cell proliferation ability in SACC-83 cell line, and the inhibitory effect was mitigated with increased concentration of etoposide(P＜0.001). The result of plate colony assay demonstrated that under etoposide-induced DNA damage condition, compared with the control group, POLQ knocking-down suppressed cell colony ability in SACC-83 cell line(P＜0.001). Moreover, the results of flow cytometry demonstrated that under etoposide-induced DNA damage conditions, compared with the control group, POLQ knocking-down induced S phase arrest(P＜0.01). Mechanistically, the results of Western blot showed that POLQ regulated DNA damage and repair by promoting expression of γH2AX(P＜0.05) and homologous recombination (HR) pathway-related protein RAD51 (P＜0.05), respectively, and down-regulating the alternative non-homologous end joining (alt-NHEJ) pathway-related protein PARP1(P＜0.01). CONCLUSIONS: POLQ knocking-down promotes the sensitivity of SACC-83 cell line to DNA damage.

Induction of perineural invasion in salivary adenoid cystic carcinoma by circular RNA RNF111.

OBJECTIVE: Local recurrence, distant metastasis, and perineural invasion (PNI) viciously occur in salivary adenoid cystic carcinoma (SACC), resulting in a poor prognosis. This study aimed to explore the mechanism by which circular RNA RNF111 (circ-RNF111) regulates PNI in SACC by targeting the miR-361-5p/high mobility group box 2 (HMGB2) axis. METHOD: Circ-RNF111 and HMGB2 were highly expressed in SACC specimens, while miR-361-5p was underexpressed. Functional experiments showed that ablating circ-RNF111 or promoting miR-361-5p hindered the biological functions and PNI of SACC-LM cells. RESULTS: HMGB2 overexpression induced the reversal of SACC-LM cell biological functions and PNI caused by circ-RNF111 knockout. Furthermore, reduction of circ-RNF111 suppressed PNI in a SACC xenograft model. Circ-RNF111 regulated HMGB2 expression through targeted modulation of miR-361-5p. CONCLUSION: Taken together, circ-RNF111 stimulates PNI in SACC by miR-361-5p/HMGB2 axis and may serve as a potential therapeutic target for SACC.

MYB RNA detection by in situ hybridisation has high sensitivity and specificity for the diagnosis of adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common primary salivary gland cancers. ACC has several benign and malignant mimics amongst salivary gland neoplasms. An accurate diagnosis of ACC is essential for optimal management of the patients and their follow-up. Upregulation of MYB has been described in 85-90% of ACC, but not in other salivary gland neoplasms. In ACC, MYB upregulation can occur as a result of a genetic rearrangement t(6;9) (q22-23;p23-24), MYB copy number variation (CNV), or enhancer hijacking of MYB. All mechanisms of MYB upregulation result in increased RNA transcription that can be detected using RNA in situ hybridisation (ISH) methods. In this study, utilising 138 primary salivary gland neoplasms including 78 ACC, we evaluate the diagnostic utility of MYB RNA ISH for distinguishing ACC from other primary salivary gland neoplasms with a prominent cribriform architecture including pleomorphic adenoma, basal cell adenoma, basal cell adenocarcinoma, epithelial myoepithelial carcinoma, and polymorphous adenocarcinoma. Fluorescent in situ hybridisation and next generation sequencing were also performed to evaluate the sensitivity and specificity of RNA ISH for detecting increased MYB RNA when MYB gene alterations were present. Detection of MYB RNA has 92.3% sensitivity and 98.2% specificity for a diagnosis of ACC amongst salivary gland neoplasms. The sensitivity of MYB RNA detection by ISH (92.3%) is significantly higher than that of the FISH MYB break-apart probe (42%) for ACC. Next generation sequencing did not demonstrate MYB alterations in cases that lacked MYB RNA overexpression indicating high sensitivity of MYB RNA ISH for detecting MYB gene alterations. The possibility that the sensitivity may be higher in clinical practice with contemporary samples as compared with older retrospective tissue samples with RNA degradation is not entirely excluded. In addition to the high sensitivity and specificity, MYB RNA testing can be performed using standard IHC platforms and protocols and evaluated using brightfield microscopy making it a time and cost-efficient diagnostic tool in routine clinical practice.

miR-183-5p overexpression orchestrates collective invasion in salivary adenoid cystic carcinoma through the FAT1/YAP1 signaling pathway.

The invasion of cancer cells into interstitial tissues in a cohesive unit is termed collective invasion, and it is important for the invasion and metastasis of salivary adenoid cystic carcinoma (SACC). However, the underlying mechanisms regulating SACC collective invasion are still poorly understood. Here, we found that SACC tissues exhibited remarkable FAT1 downregulation and YAP1 upregulation at the invasive front, which was closely associated with collective invasion and distant metastasis. Decreasing FAT1 expression significantly activated the YAP1 signaling pathway and promoted collective invasion. Moreover, miR-183-5p was identified as the candidate regulator of FAT1 by bioinformatic analysis and an online database algorithm. A dual luciferase reporter experiment further confirmed that miR-183-5p directly targeted the FAT1 3'-UTR to reduce FAT1 expression. Increasing or decreasing miR-183-5p expression promoted or attenuated collective invasion, which was reversed by YAP1 siRNA or FAT1 siRNA, respectively. In addition, knocking down miR-183-5p reduced tumor burden and attenuated collective invasion in vivo. Together, these results suggest that the miR-183-5p/FAT1/YAP1 signaling pathway is a critical driver of SACC collective invasion, revealing a novel therapeutic target.

Increased retinoic acid signaling decreases lung metastasis in salivary adenoid cystic carcinoma by inhibiting the noncanonical Notch1 pathway.

MYB-NFIB fusion and NOTCH1 mutation are common hallmark genetic events in salivary gland adenoid cystic carcinoma (SACC). However, abnormal expression of MYB and NOTCH1 is also observed in patients without MYB-NFIB fusion and NOTCH1 mutation. Here, we explore in-depth the molecular mechanisms of lung metastasis through single-cell RNA sequencing (scRNA-seq) and exome target capture sequencing in two SACC patients without MYB-NFIB fusion and NOTCH1 mutation. Twenty-five types of cells in primary and metastatic tissues were identified via Seurat clustering and categorized into four main stages ranging from near-normal to cancer-based on the abundance of each cell cluster in normal tissue. In this context, we identified the Notch signaling pathway enrichment in almost all cancer cells; RNA velocity, trajectory, and sub-clustering analyses were performed to deeply investigate cancer progenitor-like cell clusters in primary tumor-associated lung metastases, and signature genes of progenitor-like cells were enriched in the "MYC_TARGETS_V2" gene set. In vitro, we detected the NICD1-MYB-MYC complex by co-immunoprecipitation (Co-IP) and incidentally identified retinoic acid (RA) as an endogenous antagonist of genes in the "MYC_TARGETS_V2" gene set. Following this, we confirmed that all-trans retinoic acid (ATRA) suppresses the lung metastasis of SACC by correcting erroneous cell differentiation mainly caused by aberrant NOTCH1 or MYB expression. Bioinformatic, RNA-seq, and immunohistochemical (IHC) analyses of primary tissues and metastatic lung tissues from patients with SACC suggested that RA system insufficiency partially promotes lung metastasis. These findings imply the value of the RA system in diagnosis and treatment.

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.