Adenoid Cystic Carcinoma Cell Line Research Articles

Abstract LB022: Preclinical anti-tumorigenic evaluation of AXL targeting antibody-drug-conjugate in an adenoid cystic carcinoma cell line xenograft model

Abstract Adenoid cystic carcinoma (ACC) is a rare type of salivary gland cancer. Despite the many advances made in helping understand the disease behavior and characteristics, the prevailing challenge in managing patients with ACC is the lack of clinically approved systemic therapy drugs to successfully treat those with advanced recurrent or metastatic disease. There are multiple promising candidate agents being evaluated preclinically and in early phase clinical trials, yet an effective drug with consistent overall response remains to be identified. AXL is a member of the TAM family of tyrosine kinase receptors, and its overexpression and activation is generally associated with poor prognosis and chemoresistance in many cancer types. Previous reports show AXL is significantly upregulated in a subset of ACC tumors. Here, we evaluate the tumor growth inhibition effects of a newly developed antibody-drug-conjugate targeting AXL (ADCT-601) in a xenograft ACC cell line model. MDA-ACC-01 cells express high levels of cell surface AXL as confirmed by biochemical analysis. The tumor growth inhibition of MDA-ACC-01 cell xenograft was tested in a single-dose drug administration study. Immunodeficient mice bearing ACC tumors were either injected with vehicle (n=7) or with ADCT-601 at 0.5 (n=9) or 1.0 mg/kg (n=7). ADCT-601 treatment with either 0.5 mg/kg or 1.0 mg/kg resulted in complete response (CR) for all tumor implants. To reaffirm the drug’s efficacy, we challenged the vehicle-treated tumor, prior to exceeding the tumor volume endpoint, with a single-dose of ADCT-601 at 1.0 mg/kg. Strikingly, by day 6 post-treatment, tumor volume began to shrink and eventually resulted in CR except in all but one case. These results demonstrate ADCT-601 is an effective drug with significant anti-tumor activity in our preclinical models and is worthy of additional investigation to determine its translational potential. Citation Format: Joseph O. Humtsoe, Anita Pothukuchi, Hyunseok Kang, Patrick Ha. Preclinical anti-tumorigenic evaluation of AXL targeting antibody-drug-conjugate in an adenoid cystic carcinoma cell line xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB022.

TGFβ-induced EN1 promotes tumor budding of adenoid cystic carcinoma in patient-derived organoid model.

Adenoid cystic carcinoma (ACC) and basal cell adenoma (BCA) share many histological characteristics and often need a differential diagnosis in clinical pathology. Recently, we found homeobox protein engrailed-1 (EN1) was a potential diagnostic marker for ACC in an organoids library of salivary gland tumors (SGTs). Here we aim to confirm EN1 as a differential diagnostic marker for ACC, and further investigate the regulatory mechanism and biological function of EN1 in tumor progression. The transcriptional analysis, quantitative polymerase chain reaction, Western blot and immunohistochemistry staining were performed and revealed that EN1 was specifically and highly expressed in ACC, and accurately differentiated ACC from BCA. Furthermore, TGFβ signaling pathway was found associated with ACC, and the regulation of EN1 through TGFβ was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFβ-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFβ1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFβ1 in ACC was significantly associated with the clinical stage. In summary, our study verified EN1 is a good diagnostic marker to differentiate ACC from BCA. TGFβ-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

Abstract LB084: AXL as a therapeutic target in adenoid cystic carcinoma: preclinical evaluation of AXL targeting antibody-drug conjugate (ADCT-601)

Abstract Background: Adenoid cystic carcinoma (ACC) is a rare cancer of salivary gland cancer with a significant unmet clinical need. Conventional systemic therapy has limited efficacy and there are currently no FDA approved drugs for these patients with metastatic disease. Advances in molecular profiling led identification of potential therapeutic targets, and previous studies with proteomic analysis identified AXL as a potential therapeutic target in ACC. AXL, a member of the TAM tyrosine kinase receptor family has been generally associated with poor prognosis, and its overexpression and activation is implicated in conferring resistance to conventional systemic therapies in solid tumors. ADCT-601 is an antibody drug conjugate targeting human AXL carrying novel pyrrolobenzodiazepine dimer cytotoxin as the payload. Methods: The goal of this study is to determine the in vitro and in vivo anti-tumor activity of ADCT-601 in ACC cell lines and patient-derived xenograft (ACCX) models with varying AXL expression determined by Western blot. ADCT-601 treated cell lines in vitro were analyzed by MTT-based cytotoxic assay and immunoblotting, and anti-tumorigenicity was assessed in two ACCX models. Results: ADCT-601 induced a potent dose-dependent cytotoxic effect in ACC cell lines, but its cytotoxic activity was attenuated after AXL knockdown. Biochemical data indicated that ADCT-601 treatment led to DNA damage and induction of apoptotic factors accompanied by phosphorylation of gamma-H2AX. In high AXL expressing ACCX6 model, a single dose of either 0.5 or 1.0 mg/kg ADCT-601 significantly suppressed tumor growth resulting 3/5 partial responders (PRs) at 1mg/kg and 2/5 PRs at 0.5 mg/kg, compared to the vehicle or isotype-ADC control treated mice (0/5 PR and 1/5 PR, respectively). ADCT-601 appears to eliminate tumor, as no evidence of recovery in tumor growth was observed during a 60-day period. In low AXL expressing ACCX9 model, ADCT-601 treatment (at either the 0.5 or 1.0 mg/kg dose) delayed tumor growth compared to isotype-ADC control, but did not lead to durable partial responses. Conclusion: This study demonstrated that ADCT-601 induced a potent and specific in vitro and in-vivo anti-tumor activities in AXL expressing ACC models and suggests further development of ADCT-601 in biomarker driven clinical trials. Citation Format: Joseph O. Humtsoe, Leilani Jones, Shorook Naara, Francesca Zammarchi, Nicole Spardy Burr, Patrick H. van Berkel, Patrick K. Ha, Hyunseok Kang. AXL as a therapeutic target in adenoid cystic carcinoma: preclinical evaluation of AXL targeting antibody-drug conjugate (ADCT-601) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB084.

Cytotoxic effects of targeted agent alone or with chemotherapy in the treatment of adenoid cystic carcinoma: a preclinical study

Adenoid cystic carcinoma (ACC) is a rare malignancy characterized by high incidence of relapse. When relapsing, ACC has an indolent but relentless behaviour, thus leading to a poor long-term prognosis. The treatment of choice of relapsing ACC remains surgery followed by radiotherapy, whenever feasible. Therapeutic weapons are limited to systemic drugs. The most widely used chemotherapy regimen is the combination of cisplatin and doxorubicin, however with low response rate and not long lasting; there is also a lack of alternatives for second line therapies in case of disease progression. Therefore, a more comprehensive strategy aimed at identifying at preclinical level the most promising drugs or combination is clearly needed. In this study, the cytotoxic effects of two standard chemotherapy drugs, cisplatin and doxorubicin, and of five targeted therapy-drugs was tested in vitro, on an h-TERT immortalized ACC cell line, and in vivo, on zebrafish embryos with ACC tumoral cell xenograft. Then, combinations of one standard chemotherapy drug plus one targeted therapy drug were also evaluated, in order to find the best treatment strategy for ACC. Data obtained demonstrated that both vorinostat and olaparib significantly increased the standard chemotherapy cytotoxic effects, suggesting new interesting therapeutic options for ACC.

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.

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.

GOLT1A-KISS1 fusion is associated with metastasis in adenoid cystic carcinomas

Genetic alterations can drive carcinogenesis. Numerous studies have shown that gene fusion is associated with cancer progression and could provide valuable biomarkers for clinical diagnosis or targets for cancer therapy. Adenoid cystic carcinoma (ACC) is a rare form of adenocarcinoma, characterized by frequent local recurrence and high rates of distant metastasis, ultimately resulting in low survival rates. Owing to the lack of effective therapeutic targets and limited biomarkers for diagnosis, a deeper understanding of the molecular basis of ACC is urgently needed. Here, we show that gene fusion is associated with ACC metastasis. We identified a metastasis suppressor KISS1 fused with a close-by gene, GOLT1A, in highly metastatic ACC cell lines and human specimens. Such fusion blocks KISS1 translation, but not transcription, by introducing 5’ upstream open reading frames (uORFs) in the GOLT1A-KISS1 fusion transcript. Deletion of these uORFs rescued KISS1 expression and reduced invasion and migration of metastatic ACC cells. We also detected GOLT1A-KISS1 fusion transcripts in other types of highly metastatic cancer cell lines. Taken together, our results highlight the significance of this novel GOLT1A-KISS1 gene fusion in tumor metastasis and provide a valuable biomarker for clinical diagnosis and future therapeutic targeting of ACC.

The endocytic pathways of carbon dots in human adenoid cystic carcinoma cells.

ObjectivesThis study aimed at investigating cellular uptake pathways of carbon dots (CDs) in human adenoid cystic carcinoma cell line ACC‐2.Materials and methodsWe synthesized CDs using a hydrothermal method with citric acid and polyethylenimine (PEI, Mw = 25 000). The CDs incubated with the ACC‐2 cells showed their bioimaging capabilities using a confocal microscopy test. Flow cytometry was used to analyse cellular uptake pathways of CDs in ACC‐2 cells.ResultsOur findings indicated that CDs possessed good biocompatibility in ACC‐2 cells. CDs were endocytosed mainly via micropinocytosis and energy‐dependent pathways.ConclusionsIn general, these findings suggested that CDs had excellent biomedical imaging properties for ACC‐2 cells and there was a potential opportunity to develop biomedical applications.

Transfection of T-Box Transcription Factor BRACHYURY and SOX2 Synergistically Promote Self-Renewal and Invasive Phenotype in Oral Cancer Cells.

Recent studies suggest that epithelial–mesenchymal transition (EMT) correlates with cancer metastasis. In addition, there is growing evidence of the association of EMT with cancer stem cells (CSCs). Recently, we showed that the T-box transcription factor BRACHYURY could be a strong regulator of EMT and the CSC phenotype, which were effectively suppressed by a BRACHYURY knockdown in an adenoid cystic carcinoma cell line. In this study, we further tested whether BRACHYURY is a regulator of cancer stemness by means of forced expression of BRACHYURY in oral cancer cell lines. BRACHYURY, SOX2, or both were stably transfected into oral carcinoma cell lines. We analysed these transfectants with respect to self-renewal phenotypes using a sphere-formation assay, and we assessed the expression levels of EMT markers and stem cell markers using real-time reverse transcription-polymerase chain reaction (RT-PCR). Cell migration and invasiveness in vitro were evaluated using a wound healing assay and a tumour cell dissemination assay, respectively. Forced expression of BRACHYURY or SOX2 slightly increased expression of EMT and stem cell markers and the self-renewal phenotype. The expression levels, however, were much lower compared to those of cancer stem cell-like cells. Forced co-expression of BRACHYURY and SOX2 strongly upregulated EMT and stem cell markers and the self-renewal phenotype. Cell migration and invasiveness in vitro were also remarkably enhanced. These synergistic effects increased expression levels of FIBRONECTIN, SNAIL, SLUG, ZEB1, and TGF-β2. In particular, the effects on FIBRONECTIN and TGF-β2 were significant. We found that BRACHYURY and SOX2 synergistically promote cancer stemness in oral cancer cells. This finding points to the importance of gene or protein networks associated with BRACHYURY and SOX2 in the development and maintenance of the CSC phenotype.

UM-HACC-2A: MYB-NFIB fusion-positive human adenoid cystic carcinoma cell line

ObjectivesLimited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. Material and MethodsImmediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. ResultsThe University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. ConclusionUM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

Adenoid cystic carcinoma cells produce exosomes that promote tumor cell proliferation

To observe the effect of exosomes released by adenoid cystic carcinoma (ACC) cell line SACC-83 on the proliferation of ACC cells. Exosomes were isolated from SACC-83 cell culture supernatants using total exosome isolation reagents. The whole-mount exosomes were characterized using transmission electron microscope and Western blotting. The exosomes were labeled with green fluorescent dye PKH67 and co-cultured with SACC-83 cells for 48 h, followed by staining with Alexa Fluor 594 phalloidin and DAPI to observe exosome uptake by the cells using laser scanning confocal microscopy (LSCM). The cell proliferation was assessed using MTT assay and wound healing assay, and the expressions of ERK and P-ERK in the co-cultured SACC-83 cells were detected using Western blotting. The exosomes isolated from SACC-83 cells showed a size range of 30-100 nm and expressed the exosomal markers CD9, CD63 and TSG101. LSCM showed exosome uptake by SACC-83 cells, which exhibited accelerated proliferation and significantly enhanced P-ERK expression (P < 0.05) without significant changes in ERK expression. SACC-83 cells produce exosomes that promote the tumor cell proliferation and enhances the cellular expression of P-ERK, suggesting a potential role of MAPK/ERK pathway activation in exosome-mediated acceleration of ACC cell proliferation.

Effect of survivin downregulation by simvastatin on the growth and invasion of salivary adenoid cystic carcinoma.

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is been used in the clinic due to its pleiotropic effects, such as breast cancer, prostate cancer, pancreatic cancer. Simvastatin has recently been demonstrated to serve a potential role in the prophylaxis and therapeutics of a number of human cancers. The majority of reports concerning simvastatin treatment in the majority of human cancers have demonstrated that survivin is significantly decreased as a result and has been implicated in tumorigenesis. However, only a limited number of studies have investigated the use of simvastatin for the treatment of salivary gland adenoid cystic carcinoma (SACC). Therefore, this agent is a candidate for further investigation. The aim of the present study was to investigate the effects of simvastatin on the proliferation, invasion and apoptosis of the human salivary adenoid cystic carcinoma cell line, SACC-83, as well as survivin expression in the cells. The Cell Counting kit-8 assay results revealed that simvastatin inhibited the proliferation of SACC-83 cells in a dose-dependent (10 to 50 µM) and time-dependent (24 to 48 h) manner when compared with the untreated cells. Flow cytometry analysis indicated that simvastatin increased the percentage of cells in early and late apoptosis. Invasion assays revealed that simvastatin treatment inhibited the invasiveness of SACC-83 cells in a dose-dependent manner. In addition, simvastatin downregulated survivin expression in SACC-83 cells. In conclusion, simvastatin significantly inhibited the proliferation and invasion of SACC-83 cells, induced apoptosis, and reduced the expression of survivin, which suggests that simvastatin may be a novel target for SACC therapy.

Substrate stiffness regulated migration and invasion ability of adenoid cystic carcinoma cells via RhoA/ROCK pathway.

Human salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumours of the salivary gland and has strong migratory and invasive ability, which often lead to poor prognosis and lower survival rate. Tumour tissue tends to stiffen during solid tumour progression. This study aimed to investigate the influence of various substrate stiffness on the migration and invasion of SACC. Salivary adenoid cystic carcinoma cell line ACC2 cells were cultured on polydimethylsiloxane substrates (PDMS) with varying stiffness for investigating the effects of substrate stiffness on the activities of MMPs and TIMPs. The underlying mechanism was also explored. When ACC2 cells were cultured on various stiffness of PDMS, the expressions of matrix metalloproteinases 2 (MMP2), MMP9, MMP14, RhoA, Rac1, Rho-associated protein kinase 1 (ROCK1) and ROCK2 were up-regulated with increasing substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 and TIMP4 were down-regulated with increasing substrate stiffness. Our results showed that substrate stiffness regulated the activities of MMPs and TIMPs and then modulate migratory and invasive ability of ACC2 cells via RhoA/ROCK pathway. This work indicate that matrix stiffness played an important role in progression of SACC, which not only can help understand the strong invasive ability of SACC, but also suggested that therapeutically targeting matrix stiffness may help reduce migration and invasion of SACC and improve effective therapies.

Deregulation of Nicotinamide N-Methyltransferase and Gap Junction Protein Alpha-1 Causes Metastasis in Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (AdCC) is a malignant tumor that occurs in the salivary glands and frequently metastasizes. The aim of this study was to identify factors mediating AdCC metastasis. We established three AdCC cell lines by orthotropic transplantation and in vivo selection: parental, highly metastatic (ACCS-M-GFP), and lymph node metastatic (ACCS-LN-GFP) cells. We examined the three cell lines. DNA microarray indicated significantly altered processes in ACCS-LN-GFP cells: particularly, the expression of nicotinamide N-methyltransferase (NNMT) was enhanced the most. NNMT is associated with tumorigenesis and is a potential tumor biomarker. Concomitantly, we found-significant down-regulation of gap junction protein alpha-1. We suggest that ACCS-LN-GFP cells acquire cancer stem cell features involving the up-regulation of NNMT and the loss of gap junction protein alpha-1, leading to epithelial-mesenchymal transition and consequent AdCC metastasis. NNMT is a potential biomarker of AdCC.

Regulation of β-Catenin Phosphorylation by PR55β in Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (AdCC) is a rare cancer of the salivary gland with high risk of recurrence and metastasis. Wnt signalling is critical for determining tumor grade in AdCC, as it regulates invasion and migration. β-catenin dephosphorylation plays an important role in the Wnt pathway, but its underlying molecular mechanism remains unclear. Because the regulatory subunits of protein phosphatase 2A (PP2A) drive Wnt signalling via target molecules, including β-catenin, we used qRT-PCR and immunoblot analysis to investigate the expression of these subunits in an AdCC cell line (ACCS) and a more aggressive subline (ACCS-M). PR55β was highly expressed in ACCS-M, suggesting its functional importance. In addition, PR55β expression was associated with tumor grade, with ACCS-M exhibiting higher PR55β levels. More importantly, knockdown of PR55β in ACCS-M cells significantly reduced invasiveness and metastatic ability. Furthermore, dephosphorylation and total levels of β-catenin were dependent on PR55β in ACCS-M. Finally, we confirmed a correlation between PR55β staining intensity and histopathological type in human AdCC tissues. Our study provides new insight into the interaction between PR55β and β-catenin and suggests that PR55β may be a target for the clinical treatment of AdCC.

Abstract 64: Abnormal splicing events in adenoid cystic carcinoma

Abstract Salivary gland adenoid cystic carcinoma (ACC) is a relatively rare and understudied malignancy. While this disease can often be controlled locally by surgery and radiation therapy, distant metastases occur in nearly 50% of cases. Interestingly, these metastases can remain asymptomatic for several years, and there are currently no factors that are known to contribute to their dormancy or aggressiveness. Furthermore, there are no targeted chemotherapeutic agents available to treat any stage of this disease. To identify aberrations that contribute to ACC tumorigenesis, we performed whole genome methylation profiling, RNA sequencing, and exome sequencing on 18 primary ACC tumors and matched normal salivary gland tissue. A novel analysis methodology of Splice Expression Variability Analysis (SEVA) coupled with outlier statistical analyses of mRNA splice junctions revealed the presence of several abnormal splicing events (ASEs) in the studied tumors. Interestingly, the ASEs were significantly overrepresented in tumors as compared to matched normal salivary gland tissue. In vitro analyses found that many of the ASEs are also expressed in the only validated ACC cell line, MDA-ACC01, and multiple patient-derived xenograft (PDX) models of ACC. Overall, these studies have identified an unanticipated aberration in ACC and developed a model in which to study ASEs. Future work will be aimed at determining the functional impact that ASEs have on tumor initiation and growth. Citation Format: Brandon Leonard, Joseph O. Humtsoe, Hyun-Su Kim, Shizhang Ling, Luigi Marchionni, Elana J. Fertig, Michael Considine, Patrick Ha. Abnormal splicing events in adenoid cystic carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 64.

Tumor-derived exosomes enhance invasion and metastasis of salivary adenoid cystic carcinoma cells.

Tumor-derived exosomes (TDE) have been shown to participate in different steps of the dissemination of cancer cells. However, the role of salivary adenoid cystic carcinoma-derived (SACC-derived) exosomes had not been documented in SACC. The study aims to explore the functions of SACC-derived TDE in SACC progression and investigate potential mechanisms. Salivary adenoid cystic carcinoma cell line SACC-83 was used to generate TDE. Afterward, SACC-83 or HUVECs were cocultured with or without TDE. Tumor migration, tumor invasion, and endothelial permeability were examined by wound healing assay, tumor invasion assay, endothelial permeability assay, and tumor cell transendothelial migration assay, respectively. Moreover, the expression levels of cell junction-related proteins were examined by qRT-PCR and Western blot. Salivary adenoid cystic carcinoma -83-derived exosomes were taken up by their host cells. Meanwhile, TDE increased migration and invasion capacity of SACC-83 cells and enhanced endothelial cell permeability. Furthermore, we demonstrated that the expression of cell junction-related proteins (Claudins and ZO-1) was downregulated, whichis presumablyinvolved in the TDE-mediated promotion of migration, invasion, and metastasis. The results suggested that SACC cell-derived exosomes were loaded with individual components that could enhance invasiveness and induce microenvironment changes, thus promoting SACC aggression.

Effect of HBXIP on biological function and PI3K/Akt signaling pathway of adenoid cystic carcinoma cell line ACC-M

To study the effect of hepatitis B virus X protein binding protein (HBXIP) on proliferation, migration and invasion of adenoid cystic carcinoma cell line ACC-M, and the possible mechanism of PI3K/Akt signaling pathway. HBXIP plasmid was transfected into ACC-M. The cells were divided into experimental group (transfected with plasmid pEGFP-N1-HBXIP) control group (non-transfected group) and blank control group (vector group, pEGFP-N1). RT-PCR was used to detect the expression HBXIP in ACC-M; MTT assay, transwell chamber experiments and scratches over the proliferation of HBXIP were utilized individually to evaluate the influence of HBXIP on ACC-M expression, migration and invasion; Western blotting was used to detect the protein expression of Akt, p-Akt, PI3K, p-PI3K and S100A4 after overexpression of HBXIP. Statistical analysis was performed using SPSS 18.0 software package. MTT results showed that the number of surviving cells of experimental group was significantly higher than the control group (P<0.05); Scratch test results showed that the cell mobility of the experimental group was significantly higher than the control group (P<0.01); Transwell chamber experiments showed that the number of cell invasion of the experimental group was significantly higher than the control group (P<0.01); Western blotting results showed that compared with the control group, the expression of p-Akt, p-PI3K and S100A4 in the experimental group with overexpressed HBXIP was relatively increased. Overexpression of HBXIP gene promotes ACC-M proliferation, invasion and migration. Further, ACC-M proliferation, invasion and migration may be promoted by increased Akt, PI3K phosphorylation and S100A4 protein expression.

Doxorubicin-loaded environmentally friendly carbon dots as a novel drug delivery system for nucleus targeted cancer therapy

Chemotherapy is widely applied against various kinds of carcinoma. Generally, chemotherapeutic agents, such as Doxorubicin (DOX), Paclitaxel (PTX), 5-Fluorouracil (5-FU), Methotrexate (MTX), and Vinblastine (VLB) are combined with a view to maximizing their efficacy. Unfortunately, chemotherapeutics are indiscriminate and also kill normal healthy cells, resulting in serious side effects. This non-productive and destructive distribution of chemotherapeutics is regarded as one of the largest problems associated with chemotherapy. Recently, the application of carbon dots (CDs) in cancer therapy has attracted considerable attention due to their attractive properties, such as biocompatibility and low toxicity. We report herein on the fabrication of CD-DOX antitumor drug complexes, from the combination of CDs and DOX, with a view to providing a novel and efficient strategy for cancer treatment. CDs were synthesized by hydrothermal treatment of milk, a simple and environmentally friendly synthetic process. DOX was conjugated to the CDs through electrostatic interactions via the multiple surface CD functional groups. The CD-DOX complexes exhibited pH-dependent DOX release behavior. A cytotoxicity study demonstrated that the CDs were non-cytotoxic in the range of concentrations used. Compared to free DOX, the CD-DOX complexes were significantly more destructive to the adenoid cystic carcinoma cell line (ACC-2), but exhibited lower toxicity to a mouse fibroblast cell line (L929). Confocal microscopy and flow cytometry confirmed that CD-DOX complexes increased cancer therapy efficiency through the localization of a much higher quantity of drugs in the nuclei of tumor cells and induced a higher rate of apoptosis in ACC-2 cells, compared to DOX alone.