水谷 泰嘉

OBJECTIVES: SMARCA4, a core component of the SWI/SNF chromatin remodeling complex, is frequently mutated in non-small cell lung cancer (NSCLC). SMARCA4-deficient cancer cells are associated with increased replication stress, one of the major causes of genomic instability, which may lead to cancer. SMARCAD1, a chromatin remodeler, is known as replication fork progressor, and SMARCAD1 dysregulation is also closely related to cancer development. This study aimed to investigate the role of the SMARCA4-SMARCAD1 axis in the toleration of replication stress in NSCLC, focusing on the regulatory relationship between SMARCA4 and SMARCAD1 during replication stress conditions. METHODS: Human NSCLC cell lines (Calu-6, NCI-H1975, Calu-1, and NCI-H460) were used for experiments. SMARCA4 and SMARCAD1 expression levels were analyzed by quantitative RT-PCR and immunoblotting. Transcriptional regulation of SMARCAD1 was analyzed by chromatin immunoprecipitation assay. Immunofluorescent analysis was performed to assess SMARCAD1 accumulation at stalled replication forks. Clonogenic assays were conducted to evaluate the roles of SMARCA4 and SMARCAD1 in cell survival. RESULTS: SMARCAD1 was highly expressed in SMARCA4-depleted cells under replication stress. Immunofluorescent analysis revealed significant accumulation of SMARCAD1 at stalled replication forks in SMARCA4-depleted cells. Chromatin immunoprecipitation assays demonstrated that SMARCA4 bound to the transcriptional regulatory region of SMARCAD1, and that this efficacy was decreased under replication stress, suggesting that SMARCA4 is a transcriptional suppressor of SMARCAD1. In a clonogenic analysis either SMARCA4 or SMARCAD1 is required for cell survival. CONCLUSIONS: The SMARCA4-SMARCAD1 axis is a novel mechanism that provides tolerance for replication stress.

BACKGROUND: Precision oncology leverages the molecular and genetic characteristics of tumors to enable accurate diagnosis and effective treatment selection. However, recent clinical trials have highlighted the limitations of current approaches and underscored the need to integrate static molecular profiling with functional analyses using patient-derived xenograft (PDX) models-particularly for cancers such as head and neck cancer (HNC), where driver mutations are rare and prognosis remains poor. METHODS: Here, we aimed to establish a large-scale PDX library for HNC, termed the Fujita Xenograft Library (FXeL), annotated with detailed clinical information. Since 2022, tumor specimens from over 100 surgical cases at Fujita Health University Hospital have been transplanted into immunodeficient mice, resulting in the successful establishment of 62 PDX models. RESULTS: Advanced clinical stage was significantly associated with successful engraftment, and serial passaging led to progressively accelerated tumor growth. Comparative analyses of genomic profiles between patient tumors and PDXs demonstrated that major cancer-related mutations were largely preserved in PDXs, while clonal selection and evolution occurred during engraftment. Histopathological features, including keratinization and nuclear atypia, were retained, whereas stromal components such as cancer-associated fibroblasts exhibited compositional shifts. Furthermore, drug sensitivity assays revealed that PDX responses to cisplatin (CDDP) closely mirrored the clinical outcomes of the corresponding patients. CONCLUSIONS: The FXeL represents a robust and scalable platform for investigating HNC biology and therapeutic response. Despite limitations such as stromal remodeling and the absence of an immune microenvironment, these models provide valuable translational insights and support the advancement of functional precision oncology.

Objectives: Genetic risk models have substantially advanced our understanding of germline pathogenic variants (GPVs) in some malignancies, whereas their clinical significance in lung cancer remains unclear. The present study aimed to better understand potential contribution of GPVs to lung cancer etiology. Methods: A targeted sequencing panel of 143 cancer-related genes was applied to analyze 26 distinct lung adenocarcinoma (LUAD) tumors from 11 patients histopathologically diagnosed with multiple primary lung cancers (MPLC). Tumor classification was performed through integrated evaluation of mutation profiles, and variants shared among tumor lesions were further validated as likely germline or somatic mutations using Sanger sequencing. Results: Mutation profiles were compared to reveal clonal relationships among lesions in each patient. Nine of the 11 cases (81.8%) were classified as MPLC, 1/11 (9.1%) as intrapulmonary metastasis (IM), and 1/11 (9.1%) exhibited features of both MPLC and IM. Among the nine MPLC cases, eight (88.9%) harbored matching variants across independent tumor lesions that were also detected in tumor-adjacent regions, suggesting classification as likely germline variants. Importantly, among the eight cases with shared variants, one possessed a novel truncating BRCA2 DNA repair associated (BRCA2) variant (p.N900IfsTer4), while the others harbored variants of uncertain significance (VUS) in the tumor protein p53 (TP53), caspase recruitment domain family member 11 (CARD11), platelet derived growth factor receptor beta (PDGFRB), lysine methyltransferase 2D (KMT2D), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), neuregulin 1 (NRG1), androgen receptor (AR), and KIT proto-oncogene, receptor tyrosine kinase (KIT) genes. To determine whether a similar BRCA2 variant was present in other lung cancer patients, 123 LUAD cases were analyzed, and one (0.81%) possessing a truncating BRCA2 variant (p.Q1429FfsTer20) without any typical driver mutations was identified. Conclusions: BRCA2 GPVs may represent putative pathogenic mutations, and thus be potential molecular targets for future treatment of LUAD.

CERS6 is associated with metastasis and poor prognosis in non-small cell lung cancer (NSCLC) patients through d18:1/C16:0 ceramide (C16 ceramide)-mediated cell migration, though the detailed mechanism has not been elucidated. In the present study, examinations including co-immunoprecipitation, liquid chromatography, and tandem mass spectrometry analysis were performed to identify a novel binding partner of CERS6. Among the examined candidates, LASP1 was a top-ranked binding partner, with the LIM domain possibly required for direct interaction. In accord with those findings, CERS6 and LASP1 were found to co-localize on lamellipodia in several lung cancer cell lines. Furthermore, silencing of CERS6 and/or LASP1 significantly suppressed cell migration and lamellipodia formation, whereas ectopic addition of C16 ceramide partially rescued those phenotypes. Both LASP1 and CERS6 showed co-immunoprecipitation with actin, with those interactions markedly reduced when the LASP1–CERS6 complex was abolished. Based on these findings, it is proposed that LASP1–CERS6 interaction promotes cancer cell migration.

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y-box as a cis-acting element. As a parallel analysis of database records for 149 non-small-cell lung cancer (NSCLC) cancer patients, we screened for trans-acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer-binding protein γ (CEBPγ) or Y-box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide-dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y-box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Diagnosis of gastric adenocarcinoma using small biopsy samples is occasionally difficult. Various markers have been employed for improving the diagnostic accuracy, but there remains room for improvement. A total of 129 endoscopically biopsied samples were studied, consisting of 104 intramucosal tubular adenocarcinomas, 24 non-cancerous lesions and one cancer sample originally suspected of non-cancer but revised as cancer after immunostaining. We evaluated the association between histopathology and immunohistochemical expression of MUC1, HER2, p53, CEA, E-cadherin, β-catenin and claudin-18. Regarding β-catenin and claudin-18, not only membranous expression (β-catenin(M) and claudin-18(M)) but also nuclear expression (β-catenin(N) and claudin-18(N)) were analyzed. When subtyped with mucin core protein expression, the gastric-type cancers dominantly expressed claudin-18(M), while claudin-18(N) was significantly encountered in intestinal- and mixed-types. Expression of MUC1 (P = 0.0010), HER2 (P = 0.0173), p53 (P = 0.0002), CEA (P = 0.0019) and claudin-18(N) (P < 0.0001) revealed significant correlation with gastric cancers. Negative correlation of claudin-18(M) (P = 0.0125) was also noted. MUC1 and p53 were negative in non-cancer lesions. The non-cancer group exceptionally expressed HER2 and β-catenin(N). Membranous expression of E-cadherin was consistent in both groups. Logistic regression analysis showed that MUC1 (P = 0.0086), p53 (P = 0.0031), claudin-18(M) (P = 0.0158) and claudin-18(N) (P = 0.0190) were independently associated with gastric cancers. Nuclear expression of claudin-18 should be the novel diagnostic marker for gastric cancer.

<italic>Background</italic>.<italic>In situ</italic>hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver.<italic>Methods</italic>. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients.<italic>Results</italic>. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma.<italic>Conclusion</italic>. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.