Research ArticleExperimental Studies
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Epigenetic Modification of Muscarinic Acetylcholine Receptors in Squamous Cell Carcinoma of the Head and Neck

NATSUKI SUGIYAMA, SATOSHI YAMADA, YUKI NAKAMURA, KOTARO MORITA, KOTARO KANO, KOTARO ISHIDA, KAZUTAKA TAKEUCHI, JUN-YA KITA, SOSUKE SAHARA, KOSUKE SUGAWARA, KEN-ICHIRO NAGAI, SHIGERU MATSUDA, AKITOSHI HAYASHI, YUSEI MAKOSHI, DAIKI MOCHIZUKI, HIROSHI NAKANISHI, ATSUSHI IMAI, YOSHINORI TAKIZAWA, YUKI MISAWA and KIYOSHI MISAWA
Anticancer Research January 2025, 45 (1) 81-95; DOI: https://doi.org/10.21873/anticanres.17395

Abstract

Background/Aim: The five members of the mammalian muscarinic acetylcholine receptor family are encoded by the cholinergic receptor, muscarinic, 1-5 (CHRM1-5) genes. CHRM genes are incriminated in formation of various cancer types, but their roles in head and neck squamous cell carcinoma (HNSCC) are improperly understood. Aberrant epigenetic modifications of specific tumor-suppressor genes and oncogenes are known to promote cancer development. We aimed to analyze the connection between methylation of CHRM genes and HNSCC recurrence, with specific reference to human papillomavirus (HPV)-positive oropharyngeal carcinoma. Materials and Methods: We investigated the methylation state of CHRM1-CHRM5 genes expressed in 305 samples of primary HNSCCs by quantitative methylation-specific polymerase chain reaction. We explored associations between methylation of these genes and the clinicopathological characteristics of HNSCC (location of the tumor as well as recurrence) Results: We found 1.08±0.94 methylated genes per sample (range=0-5), with promoters of CHRM1-5 genes methylated in 85.9%, 47.5%, 10.2%, 17.7%, and 19.3%, respectively, of the evaluated samples. Methylation levels of CHRM2 were significantly raised in HNSCC samples compared to corresponding normal tissues (p<0.001). Although there was no significance of tumor location, analysis by Kaplan-Meier estimate and multivariate Cox proportional hazard methods showed that methylated CHRM2 was significantly associated with increased recurrence of HNSCC with HPV-positive oropharyngeal cancer. Conclusion: CHRM methylation status is associated with HNSCC pathogenesis.

Key Words:

In most cases, cancers of the head and neck originate from squamous cells lining local mucosal surfaces (for example, within the oral cavity, oropharynx, hypopharynx, or larynx). Together, such cancers are called head and neck squamous cell carcinoma (HNSCC) (1). Approximately 680,000 new cases of HNSCC are diagnosed annually, making it the eighth most commonly-diagnosed malignancy worldwide (2). In spite of the heterogeneous nature of HNSCC, with smoking and drinking being well established as major risk factors, a significant percentage of cases of HNSCC of the oropharynx are associated as well with human papillomavirus (HPV) infection. Although molecular studies can distinguish HPV-positive HNSCC from HPV-negative cases, molecular characterization methods remain to be validated (3). Therefore, screening for sensitive molecular markers that predict patient prognosis and identifying effective molecular targets to improve prognosis is the focus of research worldwide (4).

Muscarine binds to the muscarinic acetylcholine receptors (mAChRs) in the parasympathetic nervous system, without affecting nicotinic-type receptors. Muscarine is a quaternary amine that cannot pass across the blood–brain barrier; therefore, it has no toxic effects on the central nervous system (5). The mAChRs are members of the Aα family of G-protein coupled receptors (GPCRs) and are broadly expressed in a range of physiological systems, including the nervous and cardiovascular systems, plus the gastrointestinal tract. They play a vital role in the parasympathetic peripheral nervous system, where they stimulate contraction of smooth muscle and glandular secretion, while lowering the heart rate (6).

The mAChR family comprises five clearly-defined subtypes, M1-M5 (encoded by genes CHRM1-5): mAChRs M1, M3, and M5 mainly bind to the Gq/11 subfamily of G proteins, while M2 and M4 predominantly bind Gi/o proteins (7, 8).

Based on similarities of sequence and function, GPCRs have been classified into six groups, designated classes A-F. The four chief subfamilies are class A (rhodopsin-like receptors), class B (secretin family), class C (metabotropic glutamate receptors), and class F (frizzled and smoothened receptors) (9). The class A receptors are further divided into four sub-groups: α, β, γ, and δ. All five mAChRs are members of the class Aα subgroup and encode neuropeptide receptors (10). The class Aα GPCRs are a family of rhodopsin-like proteins that includes prostanoid, adenosine, histamine, and adrenergic receptors (11). We previously demonstrated that aberrant methylation of the prostanoid receptor family genes prostaglandin (PTG) D2 receptor 1 (PTGDR1), PTG E2 receptor 4 (PTGER4), PTG I2 receptor (PTGIR), and thromboxane A2 receptor (TBXA2R) contributes to recurrence of HNSCC (12). The prostanoid receptor genes encode neuropeptide receptors of the GPCR class Aα subgroup. All five mAChRs are implicated in the initiation and progression of many forms of cancer. However, there have been few studies concerning methylation of the genes encoding mAChRs or their effect on HNSCC prognosis.

We explored association between methylation of CHRM1-5 genes and the clinicopathological characteristics of HNSCC (location of the tumor as well as recurrence) in affected patients. To the best of our knowledge, we are the first to demonstrate an association of CHRM methylation status with HNSCC pathogenesis.

Materials and Methods

Clinical tumor samples. For this study, tissue samples were gathered from patients with HNSCC who had undergone major surgical resection (n=305) at the Department of Otolaryngology of Hamamatsu University School of Medicine (Hamamatsu, Shizuoka, Japan). The Ethics Committee of Hamamatsu University School of Medicine approved the study (approval date: October 2, 2015; ethics code: 25-149). All participating patients provided their written informed consent. All the procedures described were accomplished in accordance with the principles outlined in the Declaration of Helsinki. The male:female ratio was 261:44, and the patients had a mean age of 66.7 (range=32-92) years. The primary tumors were all carcinomas and included 98 of the oropharynx, 78 of the hypopharynx, 57 of the larynx, and 72 affecting the oral cavity. The patients’ medical records were used as a source of detailed clinical information.

Bisulfite modification and quantitative methylation-specific polymerase chain reaction (Q-MSP). A QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract DNA from fresh tissues. Next, sodium bisulfite conversion was carried out using a MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (TaKaRa, Tokyo, Japan). Abnormal DNA methylation, which is often found around the transcription start site within CpG islands, was evaluated using Q-MSP. Specific primer sequences for the CHRM genes are listed in Table I. A standard curve was constructed for Q-MSP using five serially-diluted standard solutions of EpiScop-methylated HeLa DNA (TaKaRa Bio). Analysis of normalized methylation values (NMVs) for PCR conditions was performed as described previously (13). The NMV was defined as: NMV=(CHRM-S/CHRM-FM)/(ACTB-S/ACTB-FM), where CHRM-S and CHRM-FM represent the CHRM methylation level in the sample and in the fully-methylated control DNA, respectively, and ACTB-S and ACTB-FM represent the corresponding levels for β-actin.

View this table:
Table I.

List of primers used for quantitative methylation-specific polymerase chain reaction for muscarinic cholinergic receptor 1-5 (CHRM) genes.

Detection of high-risk HPV DNA by PCR. An HPV Typing Set (Takara Bio, Tokyo, Japan), a PCR primer set which is specifically designed to identify HPV genotypes 16, 18, 31, 33, 35, 52 and 58 in genomic DNA, was used to evaluate HPV status in the clinical samples. HPV typing was carried out using PCR in accordance with the manufacturer’s instructions. The products of PCR were separated by electrophoresis on a 9% polyacrylamide gel, then subjected to ethidium bromide staining.

Analysis of publicly-available data from The Cancer Genome Atlas. Data on abnormal DNA methylation was obtained from The Cancer Genome Atlas (TCGA-HNSC dataset) and collected from MEXPRESS, a repository of data on DNA methylation and gene expression in Head and Neck cancer (http://mexpress.ugent.be) (14). The data on DNA methylation were collated using the Infinium HumanMethylation450 platform (Illumina, Inc., San Diego, CA, USA), and presented here as β values.

Data analyses. We analyzed the receiver operating characteristic (ROC) curves and measured the area under the ROC curve to obtain diagnostic values. Calculation of the Youden index was used to identify the optimal cut-off value, sensitivity, and specificity, and was performed using the Stata/SE 13.0 system (Stata Corporation, College Station, TX, USA). Cut-off values which corresponded to the highest accuracy were identified based on the sensitivity and specificity. The methylation index (MI) was defined as the number of genes with methylated promoters. For frequency analysis in contingency tables, associations between variables were analyzed by Fisher’s exact test. The association between clinical variables and the MI was evaluated using the Mann-Whitney U-test, while the Kaplan-Meier method and the log-rank test were used to assess disease-free survival (DFS) from 2000 to 2015. DFS was measured from the date of the initial surgical treatment to the date of diagnosis of locoregional recurrence or distant metastasis. Kaplan-Meier curves were created to calculate the probability of survival. Identification of multivariate predictive values of prognostic factors was achieved through Cox proportional hazards regression analysis that included age, sex, alcohol intake, smoking status, tumor stage, and methylation status. A value of p<0.05 was considered statistically significant.

Results

Characterization of 50 matched pairs of head and neck tumor and adjacent noncancerous mucosal tissue samples. Distinctive methylation patterns were observed: (a) Methylation of the CHRM2 gene was increased in HNSCC samples compared to normal mucosal samples (p<0.001); (b) methylation of CHRM1, CHRM3, CHRM4 and CHRM5 genes was similar in HNSCC and normal mucosal samples (p≥0.05) (Figure 1).

Figure 1.
Figure 1.

Methylation patterns of promoters of genes encoding muscarinic-type acetylcholine receptors (CHRMs) in 50 matched pairs of head and neck tumor (T) tissues and adjacent normal mucosal tissues (N). Normalized methylation values (NMVs) for the CHRM1 (A), CHRM2 (B), CHRM3 (C), CHRM4 (D) and CHRM5 (E) promoters are shown, the mean values are indicated by a horizontal bar. NMVs were significantly higher in head and neck tumor tissues than in paired adjacent normal mucosal tissues. Significance of differences in means was determined using Student’s t-test.

Hypermethylation of the five CHRM gene promoters resulted in marked differences in ROC curves (Figure 2). Using the ROC curves, CHRM1, CHRM2, CHRM3, CHRM4 and CHRM5 were considered to be methylated in samples when their NMVs exceeded 0.01581, 0.2064, 0.2622, 0.0011 and 0.1305, respectively.

Figure 2.
Figure 2.

Receiver operating characteristic curves for methylation of promoters of genes encoding muscarinic-type acetylcholine receptors (CHRMs) in cancer tissue versus adjacent normal mucosal tissue. Based on the receiver operating characteristics curve analysis, the area under the curve (AUC) was determined for CHRM1 (A), CHRM2 (B), CHRM3 (C), CHRM4 (D), and CHRM5 (E). Hypermethylation of the five CHRM gene promoters resulted in marked differences in ROC curves. CHRM1, CHRM2, CHRM3, CHRM4 and CHRM5 were considered to be methylated in samples when their NMVs exceeded 0.01581, 0.2064, 0.2622, 0.0011 and 0.1305, respectively.

Analysis of methylation status in HNSCC tissue samples. The methylation status of the five CHRM genes was investigated by Q-MSP analysis using 305 samples of primary HNSCC (Figure 3A). The average number of methylated genes per sample was 1.08±0.94 (range=0-5) (Figure 3B). MI according to age of onset, tumor size, lymph node status, clinical stage, HPV status, recurrence, patient sex, smoking habits, and alcohol consumption are illustrated in Figure 3C. The MI was significantly higher in females (p=0.031), non-smokers (p=0.007), and non-drinkers (p=0.039). No significant differences in MI were observed with respect to age at onset, tumor stage, lymph node status, or clinical stage (Figure 3C). As shown in Table II, a trend of increasing recurrence for patients with methylation of the CHRM2 gene was observed (p=0.06).

Figure 3.
Figure 3.

Promoter methylation of five genes encoding muscarinic-type acetylcholine receptors (CHRMs) in samples from 305 patients with head and neck squamous cell carcinoma. (A) Bar graph depicting the methylation frequency of each of the five CHRM genes. (B) Bar graph depicting the frequency of methylated target genes in the patient cohort. (C) Bar graph depicting the methylation index according to selected clinical parameters. Differences in mean methylation index for each parameter were determined using Student’s t-tests. *Significantly different at p<0.05. HPV: Human papilloma virus.

View this table:
Table II.

Selected epidemiologic and clinical characteristics according to methylation status of muscarinic cholinergic receptor (CHRM) genes.

Site-specific analysis of methylation status. Analysis of methylation frequencies for the five CHRM genes and with clinicopathological classifications in the oral cavity (Figure 4A), oropharynx (Figure 4B), hypopharynx (Figure 4C), and larynx (Figure 4D) was performed. The results of the site-specific analysis of MI are given in Figure 4E. We observed no significant association between CHRM promoter hypermethylation and the primary tumor site.

Figure 4.
Figure 4.
Figure 4.

Site-specific methylation index and clinicopathological factors. Comparison of methylation statuses of the promoters of five genes encoding muscarinic-type acetylcholine receptors (CHRMs) in patients with oral (A), oropharyngeal (B), hypopharyngeal (C) or laryngeal cancer (D). Corresponding clinicopathological factors for each patient are shown. Data are the mean±standard deviation. The mean methylation index for patients according to tumor site were compared using Student’s t-tests (E).

Prognostic value of CHRM gene promoter methylation status. Figure 5 and Figure 6 show the results of Kaplan-Meier analysis of DFS according to methylation status of CHRM genes and tumor location, respectively. Among the 305 patients with HNSCC, we found no difference in DFS between patients with methylated genes versus those with unmethylated genes (Figure 5A and C-F), with the notable exception that DFS was significantly shorter when the CHRM2 promoter (log-rank test, p=0.026) was methylated (Figure 5B). Among the 98 patients with oropharyngeal cancer, DFS in patients with methylated CHRM2 was significantly shorter than that of those with the unmethylated gene (log-rank test, p=0.012) (Figure 6B). In 56 patients with HPV-associated oropharyngeal cancer, DFS was lower in those with methylation of the CHRM2 gene than in patients without (log-rank test, p=0.014; Figure 6E, left panel). Among the 42 patients with oropharyngeal cancer which was HPV-negative, DFS was lower cases in which CHRM2 was methylated (log-rank test, p=0.046) (Figure 6E, right panel).

Figure 5.
Figure 5.

Kaplan-Meier survival curves based on the promoter methylation status of five genes encoding muscarinic-type acetylcholine receptors (CHRMs) in 305 patients with head and neck squamous cell carcinoma. Disease-free survival is shown with respect to promoter methylation of CHRM1 (A), CHRM2 (B), CHRM3 (C), CHRM4 (D) and CHRM5 (E), and according to the number of CHRM genes with promoter methylation (F).

Figure 6.
Figure 6.

Kaplan-Meier disease-free survival curves for patients with head and neck squamous cell carcinoma based on muscarinic-type acetylcholine receptor 2 (CHRM2) promoter methylation status. Disease-free survival according to promoter methylation by site: (A) oral cancer, (B) oropharyngeal cancer, (C) hypopharyngeal cancer, (D) laryngeal cancer, and (E) oropharyngeal cancer positive (left panel), and negative (right panel) for human papillomavirus (HPV).

Analysis of the relationship between methylation status and recurrence risk was performed by multivariate analysis with a Cox proportional hazards regression model which was adjusted for clinical stage, age, sex, alcohol consumption and smoking status. Considering the whole cohort of 305 patients, there was no significant correlation between CHRM gene methylation and recurrence. We also determined the adjusted odds ratio (ORs) for recurrence based on tumor origin for the four sites analyzed (oral cavity, oropharynx, hypopharynx, and larynx). In patients with laryngeal cancer with methylation of CHRM5, the OR was 5.09 (95% confidence interval=1.52- 17.1), and in patients diagnosed with oropharyngeal cancer who also exhibited methylation of the CHRM2 promoter, the OR was 2.47 (95% confidence interval=1.18-5.14, p=0.016) (Figure 7A). In patients with oropharyngeal cancer and methylation of the CHRM2 promoter, the adjusted OR for recurrence for those with HPV-positive disease was 5.50 (95% confidence interval=1.43-21.11, p=0.013) (Figure 7B).

Figure 7.
Figure 7.

Risk of recurrence based on promoter methylation status of muscarinic-type acetylcholine receptor (CHRM) genes in tumors of different origins. (A) Odds ratios for recurrence were determined using a Cox proportional hazards model adjusted for age, sex, alcohol intake, smoking status, tumor stage and methylation status. (B) Cox’s proportional hazards regression analysis of patients with oropharyngeal squamous cell carcinoma according to human papillomavirus (HPV) status. CI: Confidence interval. *Significant at p<0.05.

Analysis of publicly-available data from TCGA. Aberrant methylation of the CHRM1, CHRM2, CHRM3, CHRM4 and CHRM5 promoters was detected in 516 samples of HNSCC but only in 50 samples of normal tissue. The mean β values for CHRM2, CHRM3, CHRM4 and CHRM5 methylation were significantly increased in the samples from HNSCC compared to normal tissue samples (tumor values: 0.473, 0.551, 0.793 and 0.455, respectively; normal values: 0.404, 0.515, 0.896 and 0.436, respectively; p<0.001, p=0.001, p<0.001 and p=0.011, respectively) (Figure 8A). The mRNA expression levels of CHRM1, CHRM2 and CHRM5 in HNSCC were significantly lower than levels in normal samples (p<0.001, p<0.001 and p=0.001, respectively) (Figure 8B). Only the CHRM2 gene was found to be both highly methylated and consequently expressed at lower levels in cancer tissues than in normal tissues.

Figure 8.
Figure 8.

DNA methylation and mRNA expression data in the Head and Neck Squamous Cell Carcinoma dataset from The Cancer Genome Atlas for genes encoding muscarinic-type acetylcholine receptors (CHRMs). (A) DNA methylation levels in tumor and normal samples. (B) mRNA expression levels in tumor and normal samples. Significant differences were determined using Student’s t-test.

Discussion

HNSCC is the most common head and neck malignancy and originates from the mucosal epithelium of the oral cavity, pharynx, or larynx (15). It is classified into several subtypes based on anatomical location, etiology, and biological features (16). Head and neck tumorigenesis is a process of multiple steps involving the accumulation of alterations, both genetic and epigenetic (17). DNA methylation is one of the most significant epigenetic modifications that plays a major role in regulating gene expression (18).

Epigenetic changes to mAChR-encoding genes may play roles in the development and recurrence of tumors. We studied the methylation state of the CHRM genes in 305 cases of HNSCC which originated from the oral cavity, oropharynx, larynx or hypopharynx. Additionally, we compared data on the methylation status of genes between HNSCC samples and matched normal control samples using TCGA database. Our analysis showed that abnormal methylation of the CHRM2 gene promoter exhibited a positive correlation with HNSCC recurrence. Further, site-specific analysis showed that hypermethylation of CpG islands of CHRM2 was independently associated with aggressive clinical behavior in HPV-positive cases of oropharyngeal cancer. To the best of our knowledge, our report is the first to investigate whether primary HNSCC tumors with their genesis at different anatomical sites might show similar changes in DNA methylation or if such changes differ between different anatomical sites.

mAChRs are metabotropic receptors that, when activated, can induce diverse signaling pathways which are known as canonical or non-canonical pathways in various tissues (19). Humans possess only two endogenous, naturally-occurring muscarinic receptor agonists – acetylcholine and conjugated secondary bile acids. Muscarinic receptors are expressed in a variety of organ systems, and these signaling molecules are thought to be ubiquitously expressed (20). Muscarinic agonists stimulate tumor growth while muscarinic receptor antagonists impede tumor growth, as has been revealed in various cancer types including brain, breast, ovarian, prostate, lung, pancreatic, gastric, and colon, as well as melanoma [reviewed in (21)]. Recent studies have suggested the muscarinic pathway as a potential target for future antineoplastic therapies (22, 23).

Accumulating evidence suggests an association between mAChRs and various cancer types. M1 has been shown to be involved in regulating prostate cancer migration and invasion, mediated via the hedgehog signaling pathway (24). Signaling via muscarinic receptor M1 directly suppresses the development of pancreatic tumors by downregulating mitogen-activated protein kinase (MAPK) signaling and phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT) signaling pathways in cancer cells (25). Antagonization of the local cholinergic loop, achieved by blocking M2 signaling, was shown to impede the growth of lung cancer cells by reducing phosphorylation of MAPK and AKT, and reverse the epithelial–mesenchymal transition in vitro as well as in vivo (26). In contrast, M2 agonist treatment reduced cell counts in a dose- and time-dependent manner by reducing cell proliferation and enhancing cell death in ovarian and glioblastoma cancer cells (27, 28). Larabee et al. (29) have reported that M3 signaling through the PKC-p38 MAPK pathway selectively instigated expression of the oncogenic microRNAs miR-21, miR-221, and miR-222 in colon cancer cells, while Song et al. (30) stated that treating small-cell lung carcinoma cells with M3 antagonists led to inhibition of cell growth both in vitro and in vivo, and reduced MAPK phosphorylation in vivo in tumors in nude mice. Activated nerve growth factor upregulated M4, which connects AKT signaling with MYCN stimulation, enhancing neuroendocrine prostate cancer reprogramming (31).

Rhodopsin-like class A GPCRs form the most extensive and best-understood GPCR family. This family includes several members with important roles in tumor biology, such as acting as protease-activated receptors (32). Our recent findings implicate hypermethylation of the GPCR class Aα members, PTGDR1, PTGER4, PTGIR and TBXA2R, in reducing DFS, and suggest that this modification may be a critical step in hypopharyngeal, laryngeal, oropharyngeal, and oral cancer, respectively (12). Through crosstalk with modifiers of histones, DNA methylation can either block binding of the transcriptional apparatus or create conditions favorable to transcription (33). Previous studies have reported that miR-490-3p, together with its host gene CHRM2, are both downregulated in patients with glioblastoma, and that epigenetic modifications, including EZH2-based histone methylation, may play a critical role in its regulation (34). Ma et al. (35) showed that there was a strong association between six CpG sites, namely in CHRM2, laminin subunit alpha 4 (LAMA4), collagen type XI alpha 1 chain (COL11A1), fibroblast growth factor 10 (FGF10), insulin-like growth factor 1 (IGF1), and TEK receptor tyrosine kinase (TEK) genes and HPV-16-positive cervical cancer, suggesting their possible diagnostic role in cervical carcinogenesis.

Conclusion

We ascertained the association between the methylation status of the genes encoding mAChRs and HNSCC recurrence, thus shining new light on this issue which will help with the development of molecular-targeted therapies. This is, we believe, the first study to provide evidence of an association between CHRM2 methylation and poor survival in patients with oropharyngeal cancer. However further research will be needed involving prospective studies in larger HNSCC cohorts to confirm the use of methylation markers in clinical practice.

Acknowledgements

The Authors would like to thank Ms. Yuko Mohri for her excellent technical support and Editage (www.editage.jp) for English language editing.

Footnotes

  • Authors’ Contributions

    K. Misawa and Y. Misawa conceived the study. N. Sugiyama, K. Misawa, A. Imai and S. Yamada designed the experiments. N. Sugiyama, Y. Nakamura, K. Morita, K. Kano, K. Ishida, K. Takeuchi, J. Kita, S. Sahara, A. K. Sugawara, K. Nagai, S. Matsuda, A. Hayashi, Y. Makoshi, D. Mochizuki, H. Nakanishi and Y. Takizawa analyzed the data and prepared figures and tables. All Authors contributed to writing the manuscript, reviewing its drafts, approving its final version and agreed with its submission.

  • Funding

    This study was funded by a Grant-in-Aid for Scientific Research (No. 20K09689, 20K18250, 20K18277, 20K18249, 21K09559, 22K16927, 23H03054, 23K08911, 23K08981 and 23K08960) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received November 7, 2024.
  • Revision received November 27, 2024.
  • Accepted December 3, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Epigenetic Modification of Muscarinic Acetylcholine Receptors in Squamous Cell Carcinoma of the Head and Neck
NATSUKI SUGIYAMA, SATOSHI YAMADA, YUKI NAKAMURA, KOTARO MORITA, KOTARO KANO, KOTARO ISHIDA, KAZUTAKA TAKEUCHI, JUN-YA KITA, SOSUKE SAHARA, KOSUKE SUGAWARA, KEN-ICHIRO NAGAI, SHIGERU MATSUDA, AKITOSHI HAYASHI, YUSEI MAKOSHI, DAIKI MOCHIZUKI, HIROSHI NAKANISHI, ATSUSHI IMAI, YOSHINORI TAKIZAWA, YUKI MISAWA, KIYOSHI MISAWA
Anticancer Research Jan 2025, 45 (1) 81-95; DOI: 10.21873/anticanres.17395
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