Atsuko Niimi

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.

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.

Alanine-serine-cysteine transporter 2 (ASCT2) has been associated with increased levels of metabolism in various malignant tumors. However, its biological significance in the proliferation of prostate cancer (PCa) cells remains under investigation. We used the cBioPortal database to assess the effect of ASCT2 expression on the oncological outcomes of 108 PCa patients. To evaluate the function of ASCT2 in castration-sensitive PCa (CSPC) and castration-resistant PCa (CRPC), LNCaP cells and the ARV7-positive PCa cell line, 22Rv1, were assessed using cell proliferation assays and Western blot analyses. The ASCT2 expression level was associated with biochemical recurrence-free survival after prostatectomy in patients with a Gleason score ≥ 7. In vitro experiments indicated that the growth of LNCaP cells after combination therapy of ASCT2 siRNA and enzalutamide treatment was significantly reduced, compared to that following treatment with enzalutamide alone or ASCT2 siRNA transfection alone (p < 0.01, 0.01, respectively). After ASCT2 inhibition by siRNA transfection, the growth of 22Rv1 cells was significantly suppressed as compared with negative control siRNA via downregulation of ARV7 both in fetal bovine serum and androgen-deprivation conditions (p < 0.01, 0.01, respectively). We demonstrated that ASCT2 inhibition significantly reduced the proliferation rates of both CSPC and CRPC cells in vitro.