Establishment of a patient‑derived mucoepidermoid carcinoma cell line with the CRTC1‑MAML2 fusion gene
- Authors:
- Published online on: February 2, 2022 https://doi.org/10.3892/mco.2022.2508
- Article Number: 75
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Copyright: © Noguchi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Mucoepidermoid carcinoma (MEC), representing 5% of all salivary gland tumors and 26% of malignant salivary gland tumors registered for the last 39 years in Hiroshima, Japan, is the most common malignant tumor of the major and minor salivary glands (1,2). MEC is characterized by its cellular heterogeneity and consists of mucin-producing, epidermoid and intermediate cells. Clinical and pathological parameters (age, tumor size, presence of cervical lymphadenopathy, distant spread, perineural invasion and histological grade) of MEC have been associated with tumor biological behavior and patient management (3). Pathological classification of MEC is graded as low-, intermediate- or high-grade based on adverse features, such as perineural invasion, angiolymphatic invasion, coagulative necrosis, infiltrative growth, high mitotic rate, anaplasia and cystic components of <20% (4).
An important genetic abnormality in MEC is the translocation between chromosomes 11q and 19p, which has been hypothesized to be an early event in the pathogenesis of MEC (5,6), and has been reported in >50% of MEC tumors (7). Low-grade tumors have a higher incidence rate of this fusion compared with that in high-grade tumors (8) and patients with fusion-positive cancer tend to have improved survival time, with significantly lower risks of recurrence, metastases or cancer-related mortality (9). The majority of fusion genes in MEC are associated with a specific chromosomal t(11;19) (q14-21;p12-13) translocation that joins exon 1 of the cAMP response element-binding (CREB) protein-binding domain of CREB-regulated transcription coactivator 1 (CRTC1) gene to exons 2-5 of the Notch coactivator mastermind-like gene 2 (MAML2) gene, resulting in the expression of a new CRTC1-MAML2 fusion gene (10). This translocation generates a fusion protein comprised of CRTC1 (also called MECT1, TORC1 or WAMP1) at 19q21 and the C-terminal transcriptional activation domain of MAML2 at 11q21 (11-14). Previous analysis suggested that another member of the CRTC family, at 15q26, CRTC3, also fused with MAML2 (15). Okabe et al (16) and Nakayama et al (17) showed that CRTC1-MAML2 or CRTC3-MAML2 fusions occurred in 40-80% of primary salivary gland MECs, and was associated with a distinct tumor subset that had favorable clinicopathological features and an indolent clinical course.
Previously, amphiregulin (AREG), a member of the epidermal growth factor (EGF) family, was identified as a target of the CRTC1-MAML2 fusion gene and secreted AREG was shown to activate EGF receptor (EGFR) signaling in an autocrine manner (18). Furthermore, mutations in EGFR itself are rare in salivary gland carcinomas (19), while copy number alternations in EGFR are frequently found in high-grade MEC, regardless of fusion gene positivity (20). The molecular pathology and oncology of MEC are still poorly understood. Established authentic cell lines are essential to determine the biological characteristics of MEC, and a number of cell cultures and models have emerged; however, the cell line usability is limited (21). The present study reports the establishment of a MEC cell line (HCM-MEC010) carrying the CRTC1-MAML2 fusion gene and activated EGFR. The potential uses for this cell line will also be discussed to understand the biological characteristics of MEC.
Materials and methods
Cell line generation and cell culture
A patient with MEC provided consent in accordance with Hyogo College of Medicine (Hyogo, Japan) institutional policies. Tumor samples were obtained according to an approved Institutional Review Board protocol of Hyogo College of Medicine (approval no. 276; Hyogo, Japan). The present study was also conducted in accordance with the Declaration of Helsinki. Clinical and pathological data were collected from the medical records of the patient. Tumor tissues were minced into 1-2-mm pieces with a disposable scalpel and placed in primary culture. To separate the stromal cells from the mass culture, a magnetic-activated cell sorting (MACS) system was used. Briefly, MACS buffer, containing 1X PBS, 0.5% BSA, 2 mM EDTA (pH 7.2) (cat. no. 130-042-901; Miltenyi Biotec Inc.), was pre-cooled to 4˚C. To remove the fibroblasts, the single cell suspension was centrifuged at 300 x g for 10 min at room temperature. and positive selection was performed using CD326 (EpCAM) MicroBeads and a MidiMACSTM Separator (Miltenyi Biotic GmbH), according to the manufacturer's instructions. The obtained primary human MEC cells were seeded in F-medium (22) with 10 µM Y-27632 (FUJIFILM Wako Pure Chemical Corporation). After 1 week, the culture medium was replaced with fresh medium, which was changed every 4 days thereafter. At the same time, the fibroblasts derived from the tumor tissue of the same patient, were obtained and grown in F-medium. Once cells reached confluence (80%), they were washed with PBS (Mg2+ and Ca2+ free) (23) and detached with 0.05% EDTA/trypsin for 5 min at 38˚C (24). After centrifugation at 167 x g for 5 min at 4˚C, the MEC cells were resuspended in F-medium, containing Y-27632 and seeded (0.3x106 cells) in 60 mm dishes. An epithelial cell line was successfully established from the sample of the patient and was termed HCM-MEC010. The morphology of the exponentially proliferating cells in a monolayer was reviewed and documented using inverted phase contrast microscopy. The cells were also tested for mycoplasma infection using the MycoAlert® Assay (Lonza Group, Ltd.) and the cell culture growth medium and with fluorescent microscopy using the Mycoplasma Hoechst Stain Assay (MP Biomedicals, LLC).
Short tandem repeat (STR) authentication of the MEC cell line
To verify the identity of the cell line, genomic DNA was extracted from the blood of the patient, whose tumor sample was used to generate the HCM-MEC010 cell line, as well as from the cell line using the QIAamp DNA Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. DNA genotyping using STR profiling was performed using the GenePrint 10 System (Promega Corporation) and the Applied Biosystems 3130xl Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.) and analyzed by BEX Co., Ltd. The evaluation value (EV) was determined using the following equation: EV=(number of coincidental peaks) x 2/total number of peaks in cell A and total number of peaks in cell B.
Reverse transcription (RT)-PCR of the CRTC1-MAML2 fusion oncogene
The HCM-MEC010 cell line was plated in 100-mm dishes and cultured to 90% confluence. RNA was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) and RT-PCR was performed using the PrimeScript RT-PCR kit (Takara Bio, Inc.) according to the manufacturer's instructions. The following primers were used: CRTC1 forward 1, 5'-TTCGAGGAGGTCATGAAGGA-3' and 2, 5'-ATGGCGACTTCGAACAATCCGCGGAA-3'; MAML2 reverse 1, 5'-TTGCTGTTGGCAGGAGATAG-3' and 2, 5'-GGGTCGCTTGCTGTTGGCAGGAG-3' (18), which amplified 101 and 194 bp fragments, respectively. Amplification of the GAPDH gene (forward, 5'-CAATGACCCCTTCATTGACC-3' and reverse, 5'-GACAAGCTTCCCGTTCTCAG-3') was performed as a control. Successfully amplified RT-PCR products of the CRTC1-MAML2 fusion gene were purified and sequenced (24) using BigDye™ Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.) and 2% agarose gel electrophoresis.
Western blot analysis
The culture medium was removed and the cells were washed with PBS (Mg2+ and Ca2+ free). RIPA buffer was added (cat. no. sc-24948; Santa Cruz, Inc.) and the cells were incubated at 4˚C for 60 min, then centrifuged at 12,000 x g for 20 min 4˚C. The supernatant was the total cell lysate. Proteins were extracted from the HCM-MEC010 and human tongue squamous cell carcinoma (SAS; purchased from the Japanese Collection of Research Bioresources Cell Bank) cell lines as previously described (25). Protein concentration was measured using a Bradford assay (26) Western blot analysis was performed as previously described (25). The primary and secondary antibodies are listed in Table I. The protein expression ratio, compared with that in SAS cells, was measured using ImageJ v1.53e software (National Institutes of Health). The data are presented as the mean ± SD. The experiment was repeated three times.
Immunofluorescence staining
The cultured HCM-MEC010 and SAS cell lines were fixed in 3.7% formaldehyde for 20 min at room temperature. After permeabilization with 0.2% Triton-X/PBS for 5 min at room temperature, the cells were blocked with 2% (w/v) BSA (Nacalai Tesque, Inc.)/PBS, then washed with PBS (Mg2+ and Ca2+ free) and incubated with the primary antibodies overnight at 4˚C. The cells were washed with PBS (Mg2+ and Ca2+ free), then incubated with the secondary antibody and Rhodamine phalloidin (Cytoskeleton, Inc.) for 2 h at room temperature. The samples were mounted in Vecta shield containing DAPI (Vector Laboratories). Fluorescent images were captured using a confocal laser-scanning microscope (LSM780; Zeiss AG). The primary and secondary antibodies are listed in Table I.
RNA analysis
RNA-Sequencing (RNA-Seq) libraries were generated using RNA extracted from the HCM-MEC010 cell line, as previously described (27), with the TruSeq Stranded mRNA Library Prep kit for Illumina, Inc., following the manufacturer's instructions, then sequenced on a NovaSeq 6000 System (Illumina, Inc.). The analysis was performed by Takara Bio, Inc.
Hematoxylin and eosin-staining
A section of the hard palate was fixed in 10% formalin solution at room temperature for 24 h and embedded in paraffin. Sections (5-µm thick) were cut from the paraffin blocks and stained with hematoxylin (0.09%) for 5 min and eosin (0.13%) for 9 min at room temperature according to standard methods (28). The images were captured using a light microscope (BX51; Olympus Corporation).
Patient
A 45-year-old Japanese female noticed spontaneous dull pain and swelling in her hard palate for 1 month and was referred to Hyogo College of Medicine, Nishinomiya, Hyogo, Japan on January, 2019. On examination, diffuse swelling was observed in the right hard palate. There was no trismus. The surface of the mass was smooth and was soft on palpation (Fig. 1A). Bilateral cervical lymph nodes were palpable, but painless and mobile. Magnetic resonance imaging showed an irregular mass measuring 30x20x18 mm in the right hard palate, and resorption in the nasal septum and posterior wall of the maxillary sinus (Fig. 1B). The clinical diagnosis was a malignant tumor of the hard palate. A biopsy was performed intraorally and the lesion was pathologically diagnosed as low-grade MEC using Armed Forces Institute of Pathology (29).
Results
The patient was admitted to Hyogo College of Medicine, Nishinomiya, Hyogo, Japan and treated by partial resection of the hard palate, supraomohyoid neck dissection and reconstruction using an anterolateral thigh flap under general anesthesia. Hematoxylin and eosin-stained tumor tissue microscopically showed an overlying stratified squamous epithelium, mucous cells and squamous cells that were polygonal-to-ovoid in shape with eosinophilic cytoplasms (Fig. 1C). The mucous cells were cuboidal or goblet-like and tended to line the cysts. The squamous cells formed solid sheets. The tumor was diagnosed as mucoepidermoid carcinoma, low-grade type, pT4aN0M0 MEC of the hard palate. All dissected cervical lymph nodes showed no metastatic cells. At the 30-month follow up, the patient's prognosis was excellent and she had maintained a disease-free status.
Establishment of a MEC cell line from a patient tumor
A new MEC cell line, termed HCM-MEC010 was established, which maintained a cobblestone epithelial-like morphology for at least 30 passages (Fig. 2A and B). To confirm that the HCM-MEC010 cell line was derived from the tumor sample of the patient, STR profiling was performed using the DNA extracted from the high-passage HCM-MEC010 cell line and the blood from the patient. Genotypic analysis confirmed that the cell line was derived from the tumor and no contamination with other cell types was detected (EV, 1.0). (Table SI; Figs. S1 and S2).
RT-PCR analysis reveals that HCM-MEC010 cells express the CRTC1-MAML2 fusion gene
As the CRTC1-MAML2 gene fusion is common in MEC (9), the fusion event was analyzed in the HCM-MEC010 cell line using RT-PCR. Fig. 3A shows the translocation event between chromosomes 11 and 19, while Fig. 3B shows the RT-PCR amplified fragments (lane 1, 101 bp and lane 2, 196 bp) using primer sets 1 or 2, respectively. The fusion transcript of CRTC1 and MAML2 genes was confirmed using Sanger sequencing (Fig. 3C). This revealed the fusion products of CRTC1 exon 1 and MAML2 exon 2 with the predicted splicing event, indicating that a translocation event had occurred between the first introns of CRTC1 and MAML2.
Protein expression in the HCM-MEC010 cell line
Next, the protein expression of the epithelial and mesenchymal markers in the HCM-MEC010 cell line was confirmed using immunofluorescent staining. EGFR and E-cadherin were expressed on the cell membrane in the HCM-MEC010 cells, while N-cadherin expression was only faintly detected. Vimentin expression was also detected in HCM-MEC010 cells (Fig. 4).
HCM-MEC010 cells express AREG and show EGFR activation
As the AREG-EGFR signaling cascade has been identified as a CRTC1-MAML2 fusion gene target (18), AREG expression and the status of the EGFR cascade was analyzed in the HCM-MEC010 cell line. The human tongue SAS cell line was used as a comparison as the SAS cell line contains a mutation in the HER4 gene, which encodes one of the other types of human EGFR, and the authentic EGFR pathway is not involved in cell proliferation (30). EGFR was expressed in both cell types, but the AREG expression level was much higher in the HCM-MEC010 cell line compared with that in the SAS cell line (Fig. 5). Furthermore, EGFR was phosphorylated (p) in the HCM-MEC010 cell line compared with that in the SAS cell line, indicating the activation of the EGFR pathway. In addition, the expression level of AKT and p-AKT was lower in the HCM-MEC010 cell line compared with that in the SAS cell line. In the SAS cell line, AKT can be phosphorylated by both the AREG-EGFR and HER4 pathways (31,32), and high levels of AKT phosphorylation in the SAS cell line must represent an additive effect of HER4 pathway activation (33). E-cadherin was expressed at higher levels in the HCM-MEC010 cell line compared with that in the SAS cell line. Vimentin expression was detected in small amounts in both the HCM-MEC010 and SAS cell lines (Fig. 5).
RNA-seq analysis of the HCM-MEC010 cell line revealed epidermoid characteristics
To further characterize the HCM-MEC010 cell line, RNA-Seq analysis was performed. MEC is known to be composed of a mixture of mucous, epidermoid, and intermediate cells (34). RNA-Seq analysis revealed the high expression level of genes in the keratin family, including KRT5, KRT14, KRT6A, KRT17, and KRT7. Table II lists the top 200 expressed genes. However, expression of the mucous cell marker MUC was not detected. These results, together with the cell morphology results, suggest that the HCM-MEC010 cell line is considered to be of epidermoid, but not mucinous, origin.
Discussion
The isolation of primary tumor cells from patient samples is the first step for several genetic, biochemical and pharmacological experiments relevant to personalized cancer treatment (35). However, such studies are limited due to cell availability. The establishment of a cancer cell line is a traditional, but still powerful and informative method of studying human cancer. The present study reports the establishment of a MEC cell line with a CRTC1-MAML2 fusion gene.
Several studies have shown that the presence of the CRTC1/3-MAML2 fusion gene confers an improved prognosis, with improved disease-free survival and fewer distant metastasis in MEC (36,37). There are rare exceptions to this rule, including fusion-positive high-grade MEC with multiple additional genetic variations, such as mutations in CDKN2A, that have been associated with a poor prognosis (38).
The function of the CRTC1-MAML2 fusion gene has been intensively studied. Its transformation ability was identified using the RK3E cell line (39) and its importance for tumor state maintenance has also been demonstrated. Initially, it was hypothesized to cause tumor growth by the constitutive activation of Notch signaling via the MAML2 gene portion. Furthermore, the N terminus CRTC1 domain-mediated aberrant activation of cAMP/CREB signaling has also been identified as a cause of tumor formation (14,40). The interaction between AP-1 and MYC oncoprotein with CRTC1–MAML2 fusion proteins has been reported (41), suggesting that the CRTC1-MAML2 fusion gene regulates several different signaling pathways. AREG is a known cAMP/CREB-regulated gene, whose expression positively correlates with that of CRTC1-MAML2 in MEC (42). As AREG-EGFR signaling was identified as one of the CRTC1-MAML2 fusion gene targets, EGFR signaling could represent the mechanism of action by which the fusion gene promotes carcinogenesis.
These observations suggest an overall role for EGFR in the pathogenesis of MEC and the EGFR pathway could be a possible therapeutic target. As several drugs target this pathway, AREG–EGFR signaling was analyzed in the HCM-MEC010 cell line in the present study. The HCM-MEC010 cell line was found to express AREG and phosphorylate EGFR. Immunofluorescence analysis localized EGFR expression to the HCM-MEC010 cell membrane. These data suggest that the EGFR ligand, AREG, activated EGFR in an autocrine manner; therefore, antibodies that block AREG-EGFR binding or drugs that interfere with EGFR activation could be used for CRTC1-MAML2 fusion-positive MEC treatment. However, further analysis is required to identify suitable therapies.
MECs are composed of mucin-producing, epidermoid, and intermediate cells; however, RNA-Seq analysis of the HCM-MEC010 cell line detected little expression of MUC genes in the mucous cell marker family, indicating that mucin-producing cells and intermediate cells may have been removed during culture. MECs develop in excretory duct cells (43) and the mixture of three different cell types in MECs predicts their common origin. Duct and acinar cell differentiation are typically lineage-restricted; however, after irradiation, both duct and acinar cells can differentiate into different cell types (44). It is conceivable that established epidermoid-like cells are competent to differentiate into acinar cells, which is a predicted characteristic of injured duct stem cells. Further analysis will assist in the clarification into the origin of MECs. Cancer stem cells have been hypothesized to be involved in tumor formation (43). The results of the present study potentially indicate these cells may be of the same origin.
In conclusion, a MEC cell line, HCM-MEC010, with a CRTC1-MAML2 gene fusion was established. This cell line showed typical MEC characteristics, including AREG expression and EGFR activation; therefore, it could be used to assist in the identification of EGFR-targeted drugs for the treatment of CRTC1-MAML2 fusion gene-harboring MEC.
Supplementary Material
Results of STR test in cells. STR, short tandem repeat.
Result of STR test in blood. STR, short tandem repeat.
Resultsfrom short tandem repeat analysis.
Acknowledgements
The authors would like to thank Ms. Shinobu Osawa (Department of Oral and Maxillofacial Surgery, Hyogo College of Medicine, Nishinomiya, Japan) for preparation of the experiments and Ms. Takako Nanba (Department of Oral and Maxillofacial Surgery, Hyogo College of Medicine, Nishinomiya, Japan) for the management of the grants. The authors would also like to thank Nikki March and Sarah Williams for editing a draft version of the manuscript.
Funding
Funding: This study was supported by JSPS Grants-in-Aid for Scientific Research (grant nos. 16H11737 and 19H 10277), a Grant-in-Aid for Graduate Students, and a Hyogo College of Medicine and Hyogo Health Foundation Cancer Research Award.
Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due to a pending patent application, but are available from the corresponding author on reasonable request.
Authors' contributions
KN, SK, KaY, KT, HK and YN conceived and designed the present study. KN, SK, KaY, YF, KyY and YN performed the experiments. KN, SK, KoY and YN analyzed the data. KN, SK and YN wrote, reviewed, and revised the manuscript. All authors read and approved the final manuscript. KN and SK confirm the authenticity of all the raw data.
Ethics approval and consent to participate
The current study was approved by the Institutional Review Board of Hyogo College of Medicine (Hyogo, Japan) and was conducted in accordance with the Declaration of Helsinki. The patient provided written informed consent to participate.
Patient consent for publication
The patient provided written informed consent for the publication of their case study.
Competing interests
The authors declare that they have no competing interests.
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