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REST Inactivation and Coexpression of ASCL1 and POU3F4 Are Necessary for the Complete Transformation of RB1/TP53-Inactivated Lung Adenocarcinoma into Neuroendocrine Carcinoma

  • Meitetsu Masawa
    Affiliations
    Department of Respiratory Medicine, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Hanako Sato-Yazawa
    Correspondence
    Address correspondence to Hanako Sato-Yazawa, Ph.D., or Takuya Yazawa, M.D., Ph.D., Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, 880 Kita-kobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan.
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Korehito Kashiwagi
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Jun Ishii
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Chie Miyata-Hiramatsu
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Masami Iwamoto
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan

    Department of Pathology, The Jikei University School of Medicine, Minato-ku, Japan
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  • Kakeru Kohno
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan

    Institute of Life Innovation Studies, Toyo University, Itakura-machi, Japan
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  • Tadasuke Miyazawa
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Masato Onozaki
    Affiliations
    Department of Diagnostic Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Shuhei Noda
    Affiliations
    Department of Diagnostic Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Yasuo Shimizu
    Affiliations
    Department of Respiratory Medicine, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Seiji Niho
    Affiliations
    Department of Respiratory Medicine, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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  • Takuya Yazawa
    Correspondence
    Address correspondence to Hanako Sato-Yazawa, Ph.D., or Takuya Yazawa, M.D., Ph.D., Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, 880 Kita-kobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan.
    Affiliations
    Department of Pathology, Dokkyo Medical University School of Medicine and Graduate School of Medicine, Mibu-machi, Japan
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Open AccessPublished:March 29, 2022DOI:https://doi.org/10.1016/j.ajpath.2022.03.007
      Although recent reports have revealed the importance of the inactivation of both RB1 and TP53 in the transformation from lung adenocarcinoma into neuroendocrine carcinoma (NEC), the requirements for complete transformation into NEC have not been elucidated. To investigate alterations in the characteristics associated with the inactivation of RB1/TP53 and define the requirements for transformation into NEC cells, RB1/TP53 double-knockout A549 lung adenocarcinoma cells were established, and additional knockout of REST and transfection of ASCL1 and POU class 3 homeobox transcription factors (TFs) was conducted. More than 60 genes that are abundantly expressed in neural cells and several genes associated with epithelial-to-mesenchymal transition were up-regulated in RB1/TP53 double-knockout A549 cells. Although the expression of chromogranin A and synaptophysin was induced by additional knockout of REST (which mimics the status of most NECs), the expression of another neuroendocrine marker, CD56, and proneural TFs was not induced. However, coexpression of ASCL1 and POU3F4 in RB1/TP53/REST triple-knockout A549 cells induced the expression of not only CD56 but also other proneural TFs (NEUROD1 and insulinoma-associated 1) and induced NEC-like morphology. These findings suggest that the inactivation of RB1 and TP53 induces a state necessary for the transformation of lung adenocarcinoma into NEC and that further inactivation of REST and coexpression of ASCL1 and POU3F4 are the triggers for complete transformation into NEC.
      A variety of carcinomas can originate from the lung; the major histologic types of lung carcinomas consist of adenocarcinoma, squamous cell carcinoma, and neuroendocrine carcinoma (NEC); NEC is characterized as small-cell lung carcinoma (SCLC) or large-cell neuroendocrine carcinoma (LCNEC). Novel therapeutic strategies targeting genes with driver mutations have been especially developed to treat lung adenocarcinomas.
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      However, lung cancer remains the leading cause of cancer-related death worldwide. One reason is that NECs, which have the worst prognosis of carcinomas and frequently express proneural transcription factors (TFs), such as ASCL1, NEUROD1, insulinoma-associated 1 (INSM1), and POU class 3 homeobox (POU-III) TFs, commonly arise in the lung.
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      The WHO Classification of Tumours Editorial Board (Ed): Lung neuroendocrine neoplasms. Thoracic Tumours. WHO Classification of Tumours. ed 5. Lyon.
      These reports suggest that some lung NECs are generated from preexisting lung adenocarcinomas and that the functional loss of both RB1 and TP53 is essential for lung NEC tumorigenesis.
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      Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.
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      However, A549, an adenocarcinoma cell line expressing wild-type RB1 and TP53 molecules, could not be differentiated into neuroendocrine cells in spite of the insertion of POU-III TF transgenes, suggesting the existence of a cellular phenotype-maintaining system or mechanism of escape from transformation toward another cellular phenotype in differentiating lung carcinoma cells.
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      In this study, RB1 and/or TP53 in A549 adenocarcinoma cells was first knocked out to define phenotypic alterations associated with the abrogation of RB1 and/or TP53 signaling. Second, the transformation of A549 adenocarcinoma cells into NEC cells was attempted via REST gene editing and forced expression of the proneural TFs ASCL1 and POU-III. Knockout of RB1 and/or TP53 did not accelerate cell growth, RB1 knockout increased the expression levels of genes that neural cells abundantly express, TP53 knockout up-regulated the gene expression of epithelial-to-mesenchymal transition (EMT)–associated molecules, and coexpression of ASCL1 and POU3F4 under REST-knockout conditions in RB1/TP53 double-knockout A549 cells strongly promoted transformation into NEC.

      Materials and Methods

      Cells and Cell Culture

      The adenocarcinoma cell line A549, which expresses wild-type RB1 and TP53 molecules and has a homozygous mutation in KRAS [p.Gly12Ser (c.34 G>A)], was used in this study.
      • Ishii J.
      • Sato H.
      • Yazawa T.
      • Shishido-Hara Y.
      • Hiramatsu C.
      • Nakatani Y.
      • Kamma H.
      Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.
      SCLC cell lines (Lu134A and Lu139) and an LCNEC cell line (H810) were used as references.
      • Ishii J.
      • Sato H.
      • Yazawa T.
      • Shishido-Hara Y.
      • Hiramatsu C.
      • Nakatani Y.
      • Kamma H.
      Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.
      ,
      • Carbone D.P.
      • Koros A.M.
      • Kinnoila R.I.
      • Jewett P.
      • Gazdar A.F.
      Neural cell adhesion molecule expression and messenger RNA splicing patterns in lung cancer cell lines are correlated with neuroendocrine phenotype and growth morphology.
      The human embryonic kidney cell line Lenti-X 293T (Takara Bio, Shiga, Japan) was used for the transgene experiment. Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) was used to cultivate A549 and Lenti-X 293T cells, and RPMI 1640 medium (Sigma-Aldrich) was used to cultivate NECs; both media were supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and cultured at 37°C in 5% CO2.

      Gene Editing

      To knock out the RB1, TP53, or REST gene, guide RNA (gRNA) for CRISPR-Cas9 was designed by CRISPRdirect (http://crispr.dbcls.jp, last accessed April 15, 2020). The gRNA sequences targeting exon 2 of RB1, exon 2 of TP53, and exon 3 of REST were 5′-GAGAGAGAGCTTGGTTAACT-3′, 5′-CTCAGAGGGGGCTCGACGCT-3′, and 5′-AGACATATGCGTACTCATTC-3′, respectively. The schematic diagram of gene and protein, gRNA and PAM sequences, and localization of the epitopes recognized by the antibodies of RB1, TP53, and REST are shown in Supplemental Figures S1 through S3, respectively. The gRNA was assembled into the pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid, which was a gift from Feng Zhang (Addgene, Watertown, MA). PX459 carrying the gRNA was transiently transfected with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA). Forty-eight hours after transfection, the cells were treated with 1 μg/mL puromycin for positive selection. After 48 hours of selection, bulk RB1-knockout A549 cells (A549-RB1-KO), TP53-knockout A549 cells (A549-TP53-KO), RB1/TP53 double-knockout A549 cells (A549-RB1/TP53-DKO), and RB1/TP53/REST triple-knockout A549 cells (A549-RB1/TP53/REST-TKO) were trypsinized (Sigma-Aldrich) and diluted to 2 × 104 cells/mL. The cell suspension was sent to On-chip SPiS (On-chip Biotechnologies, Tokyo, Japan) for cloning. The cell concentration was automatically optimized for single-cell sorting, and the optimized cell suspension was dispensed into 96-well microplates at one cell per well. Single-cell clones were obtained after 3 weeks of culture and subjected to sequencing analysis of the genome-editing regions and Western blot analysis to examine whether the RB1, TP53, and/or REST genes were knocked out. A549-RB1/TP53-DKO cells were established by TP53 gene editing of A549-RB1-KO cells, and A549-RB1/TP53-REST-TKO cells were established by REST gene editing of A549-RB1/TP53-DKO cells.

      DNA Sequencing

      Genomic DNA was extracted by the conventional phenol/chloroform method. The genetically modified regions of RB1, TP53, or REST were amplified using PCR with appropriate primer sets (Table 1). Amplification reactions were conducted using GoTaq Green (number M7122; Promega, Madison, WI) and TaKaRa PCR Thermal Cycler Dice Touch (number TP-350; Takara Bio). PCR products were purified and inserted into the cloning vector pMD20 (number 3280; Takara Bio). All sequence analyses were performed using a 3730xl DNA analyzer (Applied Biosystems, Foster, CA) and M13M4 (5′-GTTTTCCCAGTCACGAC-3′) and M13RV (5′-CAGGAAACAGCTATGAC-3′) primers.
      Table 1Primers for PCR, RT-PCR, and Quantitative RT-PCR
      GeneForward/reverseSequenceProduct size, bp
      RB1Forward5′-TCACAGAAGTGTTTTGCTGCTTTGA-3′908
      Size of wild type.
      Reverse5′-TGGTGGGAGGCATTTATGGAGGAA-3′
      TP53Forward5′-GCTTGGGTTGTGGTGAAACATTGG-3′522
      Size of wild type.
      Reverse5′-TGAAAAGAGCAGTCAGAGGACCAG-3′
      RESTForward5′-CTGTGAGAATTCGGAAAGTCAA-3′503
      Size of wild type.
      Reverse5′-CCTTTGGATTCCATGTTAGGAA-3′
      CHGAForward5′-TCCCTGTGAACAGCCCTATGAATAA-3′78
      Reverse5′-AAAGTGTGTCGGAGATGACCTCAA-3′
      SYPForward5′-GGCTCTGGCCACCTACATCTTC-3′116
      Reverse5′-CCGATGAGCTAACTAGCCACATGA-3′
      CD56Forward5′-GATGCGACCATCCACCTCAA-3′113
      Reverse5′-TCTCCGGAGGCTTCACAGGTA-3′
      ASCL1Forward5′-TGGTGCGAATGGACTTTGGA-3′50
      Reverse5′-TAAAGATGCAGGTTGTGCGATCA-3′
      NEUROD1Forward5′-TTACCTTGGCATATGCTCTTGTC-3′115
      Reverse5′-TGCCGTCCAGTCCCATATTC-3′
      INSM1Forward5′-TGGTCTAGAAATGCGGTCTGGTC-3′174
      Reverse5′-GACTCCAGCAGTTCACAAGCCATA-3′
      POU3F1Forward5′-GGTGAGGTGGGAACCATGTAAATA-3′181
      Reverse5′-ATGGGTCAGAAATTCGGAAATC-3′
      POU3F2Forward5′-ACACTGACGATCTCCACGCAGTA-3′85
      Reverse5′-GAGGGTGTGGGACCCTAAATATGAC-3′
      POU3F3Forward5′-CACACTCTACGGCAACGTGTTC-3′112
      Reverse5′-CTCCAGCCACTTGTTCAGCAG-3′
      POU3F4Forward5′-GCCAACCTCTGATGAGTTGGAA-3′143
      Reverse5′-CGAACCTGCAGATGGTGGTC-3′
      RPS18Forward5′-TTTGCGAGTACTCAACACCAACATC-3′89
      Reverse5′-GAGCATATCTTCGGCCCACAC-3′
      Forward, forward primer; reverse, reverse primer.
      Size of wild type.

      Western Blot Analysis

      Total cell lysates (35 μg protein per lane) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore, Cork, Ireland). The membranes were blocked for 1 hour at room temperature with 1% skim milk in phosphate-buffered saline containing 0.1% (v/v) Tween 20 and then incubated with diluted anti-RB1 antibody (BD Biosciences, San Jose, CA; number 554136; dilution 1:1000), anti-TP53 antibody (Leica Biosystems, Buffalo Grove, IL; number NCL-L-p53-DO7; dilution 1:1500), anti-REST antibody (Abcam, Cambridge, UK; number 202962; dilution 1:500), anti-ASCL1 antibody (BD Biosciences; number 556604; dilution 1:250), anti-POU3F1 antibody (Abcam; number 31766; dilution 1:2000), anti-POU3F2 antibody (Merck Millipore; number MABD51, dilution 1:2000), anti-POU3F3 antibody (Abcam; number 106764; dilution 1:1000), or anti-POU3F4 antibody (Abcam; number ab104562; dilution 1:2500) overnight at 4°C. After three washes for 10 minutes with phosphate-buffered saline containing 0.1% (v/v) Tween 20 at room temperature, membranes were incubated at room temperature for 1 hour with a diluted peroxidase-labeled secondary antibody against mouse (GE Healthcare, Buckinghamshire, UK; number NA931V; dilution 1:10000) or rabbit (GE Healthcare; number NA934V; dilution 1:5000) antibodies. The membranes were then washed three times for 10 minutes with phosphate-buffered saline containing 0.1% (v/v) Tween 20 at room temperature, and immunopositive signals were visualized using an EzWestLumi Plus kit (ATTO, Tokyo, Japan; number WSE-7120). Mouse anti–β-actin (Sigma-Aldrich; number A5441; dilution 1:5000) was used as the internal control.

      Cell Growth Analysis

      Cell growth was evaluated by assessment of population doubling levels. Briefly, 1 × 105 cells were seeded on 60-mm dishes. After 72 hours of culture, the cells were collected and counted, and this procedure was repeated three times. The number of cells was calculated using the following formula: final population doubling level = 3.32 [log (harvested cell number) – log (seeded cell number)] + previous population doubling level. Statistical analysis was performed with the paired t test, and differences between values were considered statistically significant at P < 0.05.

      GeneChip Expression Array Analysis

      Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific). Biotinylated complimentary RNA was synthesized by a GeneChip 3′ IVT PLUS Reagent Kit (Thermo Fisher Scientific) from 100 ng of total RNA, according to the manufacturer's instructions. The biotinylated complimentary RNA yield was checked with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific). Following fragmentation, 15 μg of complimentary RNA was hybridized for 16 hours at 45°C on a GeneChip Human Genome U133 Plus 2.0 Array (Thermo Fisher Scientific). The GeneChip array was washed and stained in a GeneChip Fluidics Station 450 (Thermo Fisher Scientific). The GeneChip array was scanned using a GeneChip Scanner 3000 7 G (Thermo Fisher Scientific). Single-array analysis was performed by Microarray Suite version 5.0 with the Thermo Fisher Scientific default settings and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

      Construction and Transfection of Lentiviral Vectors

      cDNAs of the coding regions of ASCL1 and four POU-IIIs (POU3F1, POU3F2, POU3F3, and POU3F4) constructed previously were used for this study.
      • Ishii J.
      • Sato H.
      • Yazawa T.
      • Shishido-Hara Y.
      • Hiramatsu C.
      • Nakatani Y.
      • Kamma H.
      Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.
      ,
      • Sakaeda M.
      • Sato H.
      • Ishii J.
      • Miyata C.
      • Kamma H.
      • Shishido-Hara Y.
      • Shimoyamada H.
      • Fujiwara M.
      • Endo T.
      • Takana R.
      • Kondo H.
      • Goya T.
      • Aoki I.
      • Yazawa T.
      Neural lineage-specific homeoprotein BRN2 is directly involved in TTF1 expression in small-cell lung cancer.
      ,
      • Yazawa T.
      • Sato H.
      • Shimoyamada H.
      • Okudela K.
      • Woo T.
      • Tajiri M.
      • Ogura T.
      • Ogawa N.
      • Suzuki T.
      • Mitsui H.
      • Ishii J.
      • Miyata C.
      • Sakaeda M.
      • Goto K.
      • Kashiwagi K.
      • Masuda M.
      • Takahashi T.
      • Kitamura H.
      Neuroendocrine cancer-specific up-regulating mechanism of insulin-like growth factor binding protein-2 in small cell lung cancer.
      ,
      • Wapinski O.L.
      • Lee Q.Y.
      • Chen A.C.
      • Li R.
      • Corces M.R.
      • Ang C.E.
      • Treutlein B.
      • Xiang C.
      • Baubet V.
      • Suchy F.P.
      • Sankar V.
      • Sim S.
      • Quake S.R.
      • Dahmane N.
      • Wernig M.
      • Chang H.Y.
      Rapid chromatin switch in the direct reprogramming of fibroblasts to neurons.
      For cotransfection of the ASCL1 and POU-III constructs, a T2A sequence was inserted between the ASCL1 and POU-III cDNAs. Each cDNA fragment was ligated to the pLVSIN-EF1α-Puro lentiviral vector (Takara Bio), and the lentiviral vectors were transfected into Lenti-X 293T cells (Takara Bio) together with Lentiviral High Titer Packaging Mix (Takara Bio) using TransIT-293 Reagent (Mirus Bio, Madison, WI). Forty-eight hours after transfection, the culture supernatant containing recombinant lentiviral particles was recovered and filtered. The lentiviral solution was added to the cell culture media of target cells, and the infected cells were positively selected with 1 μg/mL puromycin for 2 weeks.

      RT-PCR and RT-qPCR

      First-strand cDNA was synthesized from the total RNA extracted from cultured cells using the SuperScript First-Strand Synthesis System (Invitrogen, Waltham, MA), according to the manufacturer's instructions. The resulting cDNA was used as a template for RT-PCR and quantitative RT-PCR (RT-qPCR), and the specific primer sets for neuroendocrine marker molecules [chromogranin A (CHGA), synaptophysin (SYP), and CD56 (alternatively named neural cell adhesion molecule 1)], proneural TFs (ASCL1, NEUROD1, INSM1, and POU-IIIs), and a housekeeping gene ribosomal protein S18 are shown in Table 1. Amplification reactions were conducted using GoTaq Green (Promega) and TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio) for RT-PCR, according to the manufacturer's instructions. For RT-qPCR, amplification reactions were performed using TB Green Fast qPCR mix (Takara Bio; number RR430) and Real Time System III (Takara Bio; number TP-970), and the data were obtained from triplicate reactions. The mean and SD of the copy number were normalized to the values for ribosomal protein S18 mRNA.

      Animal and Immunohistochemistry Experiments

      The animal experiments in this study were approved by the Institutional Ethical Review Board (number 1207) and conducted at the Laboratory Animal Research Center of Dokkyo Medical University. Six-week–old nonobese diabetic–severe combined immunodeficiency male mice were purchased from CLEA Japan (Tokyo, Japan). Each mouse was kept at room temperature in a 12-hour light cycle and acclimated to the environment for a week. A total of 1.0 × 107 cells were inoculated into the s.c. layer of the backs of mice. Mice were sacrificed under deep anesthesia when the inoculated tumors grew to 10 mm in diameter. The tumors were fixed with 10% buffered formalin and embedded in paraffin. Tissue sections (4 μm thick) were stained with hematoxylin and eosin or immunostained using anti-CHGA antibody (Abcam; number ab15160; dilution 1:300), anti-SYP antibody (Japan Tanner, Osaka, Japan; number 336A-75; dilution 1:100), anti-CD56 antibody (Leica Biosystems, Wetzlar, Germany; number CD56-NCAM; dilution 1:200), anti-NEUROD1 antibody (Abcam; number ab213725; dilution 1:1000), or anti-INSM1 antibody (Santa Cruz Biotechnology, Dallas, TX; number sc-271408; dilution 1:100) and the Histofine Simplestain MAX-PO system (Nichirei, Tokyo, Japan) after microwave treatment of the tissue sections for antigen retrieval. Diaminobenzidine (Nichirei) was applied for immunostaining, and the nuclei were counterstained with hematoxylin.

      Results

      RB1 and TP53 Gene Editing in A549 Adenocarcinoma Cells

      Alterations in cell growth and gene expression in association with RB1 and TP53 knockout were examined first because inactivation of RB1 and TP53 is found in most lung NECs. It has been speculated that inactivation of both genes is closely associated with lung NEC tumorigenesis. As shown in Figure 1, A549 cell clones with knockout of both RB1 alleles (A549-RB1-KO), both TP53 alleles (A549-TP53-KO), and both RB1 and TP53 alleles (A549-RB1/TP53-DKO) were established. The knockout was confirmed by sequencing the editing regions in which the frameshift mutations were generated in both RB1 and TP53 alleles in each clone (Supplemental Figures S4–S6). Western blot analysis revealed that RB1 and/or TP53 protein was not detected in A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells (Figures 1, A and B). As shown in Figure 1C, significant growth acceleration was not observed in A549-RB1-KO, A549-TP53-KO, or A549-RB1/TP53-DKO cells despite the knockout of tumor suppressor genes. The cell growth of A549-RB1-KO and A549-TP53-KO cells was not significantly altered in comparison with that of A549 cells (P = 0.0801 and P = 0.0554 at day 9, respectively), whereas A549-RB1/TP53-DKO cells showed significantly decreased growth (P = 0.0013 at day 9).
      Figure thumbnail gr1
      Figure 1Establishment of RB1 and/or TP53 knockout A549 cells. A and B: Western blot analysis of RB1 (A) and TP53 (B). The expression of RB1 or TP53 is lost by each gene editing. C: Population doubling levels (PDLs) of A549, A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells. A549-RB1/TP53-DKO cells exhibited significantly decreased growth at day 9 in comparison with A549 cells. D: Genes that are highly expressed in neural cells or associated with epithelial-to-mesenchymal transition were up-regulated in A549-RB1-KO (RB1-KO), A549-TP53-KO (TP53-KO), and A549-RB1/TP53-DKO (RB1/TP53-DKO) cells compared with A549 cells. ∗∗P < 0.01. ACTB, β-actin.
      Next, comprehensive gene expression analysis using A549, A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells was conducted, and alterations in gene expression were based on the levels in A549 cells (Supplemental Tables S1 and S2). Overall, 293, 494, and 305 genes were up-regulated more than threefold in A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells, respectively (Supplemental Table S1). Furthermore, 213, 523, and 295 genes were down-regulated more than threefold in A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells, respectively, versus control cells (Supplemental Table S1). Among the up-regulated genes, genes that were highly expressed in neural cells accounted for 22.5% (66/293 genes) of the altered genes in A549-RB1-KO cells, 5.3% (26/494 genes) of the altered genes in A549-TP53-KO cells, and 22.6% (69/305) of the altered genes in A549-RB1/TP53-DKO cells (Figure 1D). However, proneural TFs (ASCL1, NEUROD1, POU-III, and INSM1) and neuroendocrine markers (CHGA, SYP, and CD56) were not up-regulated despite knockout of RB1 and/or TP53 (Table 2). CDH2, SNAI1, SNAI2, and ZEB2, which are known as EMT-related molecules, were up-regulated by TP53 gene knockout (Figure 1D). These data suggest that genes that are highly expressed in neural cells, and EMT-related molecules are closely regulated by RB1 and TP53. However, the A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells did not undergo transformation into NEC cells.
      Table 2Signal Detection Call of Neuroendocrine-Related Genes in GeneChip Expression Analysis
      GeneA549A549-RB1-KOA549-TP53-KOA549-RB1/TP53-DKO
      ASCL17.1 (A)11.0 (A)13.2 (A)11.7 (A)
      POU3F14.1 (A)5.7 (A)3.2 (A)2.8 (A)
      POU3F22.3 (A)33.0 (A)6.0 (A)5.5 (A)
      POU3F360.5 (A)77.5 (A)51.3 (A)53.0 (A)
      POU3F428.5 (A)13.8 (A)5.2 (A)36.8 (A)
      NEUROD128.6 (A)40.4 (A)5.7 (A)19.1 (A)
      INSM115.8 (A)6.8 (A)39.5 (A)12.4 (A)
      CHGA39.7 (A)49.6 (A)47.9 (A)5.5 (A)
      SYP10.2 (A)20.6 (A)7.1 (A)3.7 (A)
      CD564.1 (A)6.8 (A)6.9 (A)6.7 (A)
      REST1035.9 (P)912.3 (P)1087.7 (P)833.0 (P)
      A, absent call means that the transcripts are not detected; P, present call means that the transcripts are detected.

      Influence of Knockout of the REST Gene on the Transformation into NEC

      REST functions as a negative regulator of neural/neuroendocrine differentiation by silencing neural/neuroendocrine cell-specific genes.
      • Ballas N.
      • Grunseich C.
      • Lu D.D.
      • Speh J.C.
      • Mandel G.
      REST and its correpressors mediate plasticity of neuronal gene chromatin throughout neurogenesis.
      ,
      • Grimes J.A.
      • Nielsen S.J.
      • Battaglioli E.
      • Miska E.A.
      • Speh J.C.
      • Berry D.L.
      • Atouf F.
      • Holdener B.C.
      • Mandel G.
      • Kouzarides T.
      The co-repressor mSin3A is a functional component of the REST-coREST repressor complex.
      It was previously reported that REST expression is severely repressed in lung NEC cells and that REST-knockout conditions are closely associated with CHGA and SYP expression.
      • Kashiwagi K.
      • Ishii J.
      • Sakaeda M.
      • Arimasu Y.
      • Shimoyamada H.
      • Sato H.
      • Miyata C.
      • Kamma H.
      • Aoki I.
      • Yazawa T.
      Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin, and chromogranin A in lung cancer cells.
      In the present study, comprehensive gene expression analysis revealed that the expression levels of REST were not prominently altered by RB1 and/or TP53 gene knockout (Table 2). Therefore, additional knockout of the REST gene in A549-RB1/TP53-DKO cells was performed to investigate the influence on the transformation into NEC. Sequencing analysis confirmed that both REST alleles in A549-RB1/TP53/REST-TKO cells had frameshift mutations (Supplemental Figure S7). Western blot analysis revealed that the REST protein was not detected in A549-RB1/TP53/REST-TKO cells (Figure 2A). REST gene knockout activated the CHGA and SYP genes, as reported previously,
      • Kashiwagi K.
      • Ishii J.
      • Sakaeda M.
      • Arimasu Y.
      • Shimoyamada H.
      • Sato H.
      • Miyata C.
      • Kamma H.
      • Aoki I.
      • Yazawa T.
      Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin, and chromogranin A in lung cancer cells.
      whereas CD56 gene activation was not found in cells with single knockout of RB1 or TP53, double knockout of RB1/TP53, or triple knockout of RB1/TP53/REST (Figure 2B). Furthermore, the proneural TFs ASCL1, POU-III, NEUROD1, and INSM1, which are frequently expressed in lung NECs, were not induced despite knockout of the REST gene in A549-RB1/TP53-DKO cells (Figure 2C). These findings suggest that the REST knockout is necessary for the sufficient expression of neuroendocrine marker genes possessing repressor element 1 sequences in their promoter/enhancer region but that one or more other factors are necessary for the transformation of lung adenocarcinoma into NEC.
      Figure thumbnail gr2
      Figure 2Establishment of RB1, TP53, and REST knockout A549 (A549-RB1/TP53/REST-TKO) cells from A549-RB1/TP53-DKO cells and neuroendocrine marker expression in genetically modified A549 cells. A: Western blot analysis of REST. The expression of REST was lost with its gene editing. B: Quantitative RT-PCR analysis of chromogranin A (CHGA), synaptophysin (SYP), and CD56 expression in A549 cells with genetically modified RB1, TP53, and/or REST genes. CHGA and SYP were expressed with REST gene editing, but CD56 was not. Ribosomal protein S18 (RPS18) was used as a housekeeping gene. C: RT-PCR analysis of the proneural transcription factors ASCL1, POU3F1, POU3F2, POU3F3, POU3F4, NEUROD1, and insulinoma-associated 1 (INSM1). Small-cell lung carcinoma cell lines (Lu-134A and Lu139) and a large-cell neuroendocrine carcinoma cell line (H810) expressed ASCL1, POU3F1, POU3F2, POU3F3, POU3F4, NEUROD1, and INSM1, whereas A549-RB1/TP53-DKO and A549-RB1/TP53/REST-TKO cells did not. Ethidium bromide–stained agarose gel, reverse images. ACTB, β-actin; KO, knockout; Marker, ladder marker.

      Introduction of Proneural TF Transgenes into A549-RB1/TP53/REST-TKO Cells

      Next, proneural TF transgenes were conducted into A549-RB1/TP53/REST-TKO cells to generate transformants with biological characteristics of NEC, because the inactivation of RB1, TP53, and REST was not involved in the induction of proneural TFs (Figure 2C). In this study, ASCL1 and four kinds of POU-IIIs were selected as potent transformation-inducing TFs, as has been reported in previous studies,
      • Ishii J.
      • Sato H.
      • Yazawa T.
      • Shishido-Hara Y.
      • Hiramatsu C.
      • Nakatani Y.
      • Kamma H.
      Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.
      ,
      • Yazawa T.
      • Sato H.
      • Shimoyamada H.
      • Okudela K.
      • Woo T.
      • Tajiri M.
      • Ogura T.
      • Ogawa N.
      • Suzuki T.
      • Mitsui H.
      • Ishii J.
      • Miyata C.
      • Sakaeda M.
      • Goto K.
      • Kashiwagi K.
      • Masuda M.
      • Takahashi T.
      • Kitamura H.
      Neuroendocrine cancer-specific up-regulating mechanism of insulin-like growth factor binding protein-2 in small cell lung cancer.
      ,
      • Linnoila R.I.
      • Sahu A.
      • Miki M.
      • Ball D.W.
      • DeMayo F.J.
      Morphometric analysis of CC10-hASH1 transgenic mouse lung: a model for bronchiolization of alveoli and neuroendocrine carcinoma.
      and 10 kinds of transformants [ASCL1-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1) cells, POU-III–expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-POU3F1, A549-RB1/TP53/REST-TKO-POU3F2, A549-RB1/TP53/REST-TKO-POU3F3, and A549-RB1/TP53/REST-TKO-POU3F4) cells, and both ASCL1- and POU-III–expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1/POU3F1, A549-RB1/TP53/REST-TKO-ASCL1/POU3F2, A549-RB1/TP53/REST-TKO-ASCL1/POU3F3, and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4) cells] were established. The expression of transgene(s) was confirmed by Western blot analyses. Figure 3A shows a representative result, where ASCL1 and/or POU3F4 were expressed at detectable levels. Up-regulation of CHGA, SYP, and CD56 by the ASCL1, POU3F1, POU3F2, and POU3F3 transgenes was limited within fourfold (Figure 3B). However, the expression of CHGA and CD56 was accelerated by cotransfection of the ASCL1 and POU3F4 transgenes (15.5-fold and 401.5-fold, respectively) (Figure 3B), and the expression levels of three neuroendocrine marker molecules were comparable to those in most NEC cell lines (Figure 3C). A complex regulatory network involving many kinds of proneural/neuroendocrine-specific TFs has been demonstrated to function in neural development.
      • Gohlke J.M.
      • Armant O.
      • Parham F.M.
      • Smith M.V.
      • Zimmer C.
      • Castro D.S.
      • Nguyen L.
      • Parker J.S.
      • Gradwohl G.
      • Portier C.J.
      • Guillemot F.
      Characterization of the proneural gene regulatory network during mouse telencephalon development.
      Because NEUROD1 is involved in CD56 expression in NEC and because ASCL1 and INSM1 expression has been reported to show a close association in lung NECs,
      • Ishii J.
      • Sato H.
      • Yazawa T.
      • Shishido-Hara Y.
      • Hiramatsu C.
      • Nakatani Y.
      • Kamma H.
      Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation.
      ,
      • Fujino K.
      • Motooka Y.
      • Hassan W.A.
      • Abdalla M.O.L.
      • Sato Y.
      • Kudoh S.
      • Hasegawa K.
      • Niimori-Kita K.
      • Kobayashi H.
      • Kubota I.
      • Wakimoto J.
      • Suzuki M.
      • Ito T.
      Insulinoma-associated protein 1 is a crucial regulator of neuroendocrine differentiation in lung cancer.
      NEUROD1 and INSM1 expression in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells was compared with that in A549-RB1/TP53/REST-TKO-ASCL1/POU3F2 cells. As shown in Figure 3D, the NEUROD1 gene was activated by forced expression of POU3F4 in A549-RB1/TP53/REST-TKO cells (26.6-fold), and the expression was accelerated by cotransfection of the POU3F4 and ASCL1 transgenes (49.2-fold), whereas the POU3F2 transgene revealed weak induction of NEUROD1 and weak acceleration by ASCL1. Although INSM1 expression was increased 1214.8-fold and 680.0-fold in A549-RB1/TP53/REST-TKO-ASCL1 and A549-RB1/TP53/REST-TKO-ASCL1/POU3F2 cells, respectively, the expression was more strikingly increased (11,133.4-fold) in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. These findings suggest that ASCL1 and POU3F4 cooperatively function not only in regulating neuroendocrine marker expression but also in regulating NEUROD1 and INSM1 expression under REST-inactivated conditions.
      Figure thumbnail gr3
      Figure 3Establishment of ASCL1- and/or POU class 3 homeobox transcription factor (POU-III)–expressing A549-RB1/TP53/REST-TKO cells and their neuroendocrine marker/proneural transcription factor expression. A: Western blot analysis of ASCL1 and POU3F4 in ASCL1- and/or POU3F4-expressing A549-RB1/TP53/REST-TKO cells. The signal for ASCL1 or POU3F4 was detected in the respective gene-transfected A549 cells. The signals of ASCL1 translated from ASCL1-T2A-POU3F4 cDNA inserted into the lentiviral expression vector shifted upward because of the addition of T2A-coding peptides. The asterisk indicates nonspecific signals. B: Relative expression of chromogranin A (CHGA), synaptophysin (SYP), and CD56 in ASCL1- and/or POU-III–expressing A549-RB1/TP53/REST-TKO cells compared with A549-RB1/TP53/REST-TKO-Empty cells. CHGA and SYP were mildly up-regulated by the ASCL1 transgene, whereas CD56 up-regulation was not observed with introduction of the ASCL1 or POU-III transgene. However, the expression of these three neuroendocrine markers was markedly up-regulated by cotransfection of ASCL1 and POU3F4. Data were obtained by quantitative RT-PCR (RT-qPCR), and the expression level of A549-RB1/TP53/REST-TKO-Empty cells was defined as 1. +, Transfected. C: Relative expression of CHGA, SYP, and CD56 in small-cell lung carcinoma (SCLC) cell lines and a large-cell neuroendocrine carcinoma (LCNEC) cell line compared with ASCL1- and POU3F4-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1/POU3F4) cells. The expression levels of neuroendocrine markers in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells were comparable to those in SCLC and LCNEC cell lines. Data were obtained by RT-qPCR, and the expression level of A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells was defined as 1. D: Relative expression of NEUROD1 and insulinoma-associated 1 (INSM1) in ASCL1- and/or POU-III (POU3F2 or POU3F4)–expressing A549-RB1/TP53/REST-TKO cells compared with A549-RB1/TP53/REST-TKO-Empty cells. The expression of NEUROD1 was up-regulated by POU3F4 and accelerated by the addition of an ASCL1 transgene. The expression of INSM1 was induced by ASCL1 and accelerated by the addition of a POU3F4 transgene. The RT-qPCR results were used for analysis. +, Transfection of ASCL1 cDNA. ACTB, β-actin.

      Histology and Immunohistochemistry of Tumors Obtained by S.C. Inoculation of Genetically Modified A549 Cells

      The histology of tumors obtained by s.c. inoculation of genetically modified A549 cells into nonobese diabetic–severe combined immunodeficiency mice is shown in Figure 4. A549 cells proliferated in nest and acinar patterns and possessed rich intracytoplasmic mucin, whereas A549-RB1/TP53-DKO cells focally showed spindle-shaped cytoplasm, suggesting increased EMT. The tumors formed by A549-RB1/TP53/REST-TKO, A549-RB1/TP53/REST-TKO-ASCL1, and A549-RB1/TP53/REST-TKO-POU3F4 cells showed polygonal-shaped cancer cells and a solid growth pattern. Furthermore, necrotic foci were found in the central areas of tumor nests consisting of A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells as LCNEC cells rather than SCLC cells (Supplemental Figure S8).
      Figure thumbnail gr4
      Figure 4Histology of A549, RB1 and TP53 double-knockout A549 (A549-RB1/TP53-DKO), RB1, TP53, and REST triple-knockout A549 (A549-RB1/TP53/REST-TKO), ASCL1-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1), POU3F4-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-POU3F4), and ASCL1- and POU3F4-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1/POU3F4) cells. A549 cells had cuboidal or low columnar shaped cytoplasm with mucin and proliferated in acinar and nests, whereas A549-RB1/TP53-DKO cells had polygonal or spindle-shaped or polygonal cytoplasm. A549-RB1/TP53/REST-TKO, A549-RB1/TP53/REST-TKO-ASCL1, A549-RB1/TP53/REST-TKO-POU3F4, and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells had polygonal cytoplasm and proliferated to form large nests. Necrotic foci were found in the tumor nests of A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. Hematoxylin and eosin staining is shown. Scale bars = 100 μm.
      The immunohistochemistry results are shown in Figure 5. CHGA, SYP, and CD56 signals were not detected in the A549-RB1/TP53-DKO cells. However, A549-RB1/TP53/REST-TKO, A549-RB1/TP53/REST-TKO-ASCL1, A549-RB1/TP53/REST-TKO-POU3F4, and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells showed detectable CHGA and SYP expression by immunostaining. Furthermore, membranous CD56 signals were observed only in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells among the A549-RB1/TP53/REST-TKO series, supporting the data of the in vitro study (Figure 3B). NEUROD1 was immunohistochemically detected in A549-RB1/TP53/REST-TKO-POU3F4 and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. INSM1 was immunohistochemically detected in <50% of A549-RB1/TP53/REST-TKO-ASCL1 cells and <1% of A549-RB1/TP53/REST-TKO-POU3F4 cells, whereas A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells were diffusely positive for INSM1, supporting the findings of the in vitro study (Figure 3D).
      Figure thumbnail gr5
      Figure 5Immunohistochemistry analysis of chromogranin A (CHGA), synaptophysin (SYP), CD56, NEUROD1, and insulinoma-associated 1 (INSM1) in RB1 and TP53 double-knockout A549 (A549-RB1/TP53-DKO), RB1, TP53, and REST triple-knockout A549 (A549-RB1/TP53/REST-TKO), ASCL1-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1), POU3F4-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-POU3F4), and ASCL1- and POU3F4-expressing A549-RB1/TP53/REST-TKO (A549-RB1/TP53/REST-TKO-ASCL1/POU3F4) cells. Positive signals for CHGA and SYP were detected in REST knockout cells (A549-RB1/TP53/REST-TKO, A549-RB1/TP53/REST-TKO-ASCL1, A549-RB1/TP53/REST-TKO-POU3F4, and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells), whereas positive signals for CD56 were found only in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. The signals of NEUROD1 were scattered in A549-RB1/TP53/REST-TKO-POU3F4 and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. INSM1 was diffusely expressed in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells, whereas the level of INSM1 was low in A549-RB1/TP53/REST-TKO-ASCL1 cells and A549-RB1/TP53/REST-TKO-POU3F4. Scale bars = 50 μm.

      Comparison of Neuroendocrine Marker Gene Expression among Genetically Modified A549 Cells

      Finally, to confirm the importance of double knockout of RB1/TP53 genes and inactivation of the REST gene in neuroendocrine transformation, the conditions of neuroendocrine marker expression were compared between A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells and other established transfectants (ASCL1 and POU3F4 transgene in A549-REST-KO cells and ASCL1- and/or POU3F4-expressing A549, A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells). The expression levels of CHGA and SYP in A549-REST-KO-ASCL1/POU3F4 cells were limited to approximately 20% and 60% of those in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells, respectively, and the CD56 induction was not observed (Figure 6). Furthermore, the expression levels of CHGA, SYP, and CD56 in A549, A549-RB1-KO, A549-TP53-KO, and A549-RB1/TP53-DKO cells with the ASCL1 and/or POU3F4 transgene were much lower than those in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells (Figure 6). These findings suggest the importance of simultaneous inactivation of the RB1, TP53, and REST genes in lung adenocarcinoma cells for complete neuroendocrine transformation through proneural TFs.
      Figure thumbnail gr6
      Figure 6Comparison of chromogranin A (CHGA), synaptophysin (SYP), and CD56 expression levels in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4, A549-REST-KO-ASCL1/POU3F4, ASCL1- and/or POU3F4-transfected A549, A549-RB1-KO, A549-TP53-KO, or A549-RB1/TP53-DKO cells. The expression levels of CHGA, SYP, and CD56 in A549-REST-KO-ASCL1/POU3F4-, ASCL1- and/or POU3F4-transfected A549, A549-RB1-KO, A549-TP53-KO, or A549-RB1/TP53-DKO cells were lower than those in A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. Data were obtained by quantitative RT-PCR, and the expression level of A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells was defined as 1. +, Transfected; KO, knockout.

      Discussion

      Almost all lung NECs exhibit inactivation of both the RB1 and TP53 genes, and the loss of function of RB1 and TP53 is implied to be closely associated with the tumorigenesis of lung NECs; however, the precise process has not been clarified. This study revealed that RB1, TP53, and REST inactivation and the expression of proneural TFs ASCL1 and POU3F4 are involved in the transformation from adenocarcinoma to NEC.
      This study demonstrated that biallelic knockout of RB1 or TP53 did not up-regulate cell growth, but RB1/TP53 double-knockout caused significant growth retardation in A549 adenocarcinoma cells. Rb1 knockout does not affect the cell growth of mouse embryonic fibroblasts and that cell growth is accelerated only by triple knockout of Rb1 family genes [Rb1, p107 (Rbl1), and p130 (Rbl2)], suggesting the existence of a compensatory mechanism against growth acceleration signals among RB1 family molecules.
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      Mouse models of human cancer, especially GEMMs, are useful for investigating basic molecular mechanisms underlying cancer initiation, progression, metastasis, and therapy resistance and for establishing novel therapeutic strategies and clinical applications in cancer therapy.
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      These imply that additional gene expression alterations are required for rapid transformation into NEC. This study revealed that knockout of REST in addition to knockout of RB1 and TP53 and forced expression of ASCL1 and POU3F4 are required for A549 adenocarcinoma cells to transform into NEC cells. The ASCL1 transgene in A549 cells has been reported to generate neuroendocrine differentiation.
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      The immature neural/neuroendocrine phenotype is considered as a reason for the aggressiveness of NEC. The supplemental studies showed that the growth activity of A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells was rather decreased compared with that of RB1/TP53/REST-TKO and A549-RB1/TP53-DKO cells (Supplemental Figure S9). The migratory ability of A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 and A549-RB1/TP53/REST-TKO cells was mildly increased in comparison to that of A549-RB1/TP53-DKO cells, although the difference was not statistically significant (Supplemental Figure S10). SCLC possesses an immature neural/neuroendocrine phenotype-linked immune escape mechanism mediated by the deficient major histocompatibility complex expression.
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      Updated overall survival and PD-L1 subgroup analysis of patients with extensive-stage small-cell lung cancer treated with atezolizumab, carboplatin, and etoposide (IMpower133).
      These findings and reports suggest that novel strategies for overcoming immature neural/neuroendocrine phenotype-linked aggressiveness, such as the reverse transformation of NEC cells into non-NEC cells, are necessary for the establishment of effective therapies against NEC.
      In conclusion, this study revealed that the inactivation of RB1/TP53/REST genes is an indispensable condition for the transformation from adenocarcinoma to NEC and that ASCL1 and POU3F4 cooperate as a powerful driving force of complete transformation into NEC. Because inactivation of the REST gene is particularly important for neuroendocrine differentiation/transformation, further investigations focusing on the inactivation mechanism as well as the activating mechanisms of ASCL1 and POU3F4 are necessary to fully understand how lung adenocarcinoma cells transform into NEC cells.

      Acknowledgment

      We thank Sei-ichiro Suzuki for technical assistance.

      Author Contributions

      M.M., H.S.-Y., and T.Y. designed the study; M.M., H.S.-Y., K.K., J.I., C.M.-H., M.I., K.K., T.M., M.O., and S.No. performed experiments; M.M., H.S.-Y., K.K., J.I., C.M.-H., M.I., K.K., T.M., M.O., S.No., Y.S., S.Ni., and T.Y. analyzed the data; M.M., H.S.-Y., K.K., J.I., and T.Y. visualized the data; M.M., H.S.-Y., K.K., J.I., and T.Y. wrote and revised the manuscript; H.S.-Y. and T.Y. acquired funding; H.S.-Y., S.Ni., and T.Y. supervised the study; H.S.-Y. and T.Y. performed project administration; all authors read and approved the final version of the article.

      Supplemental Data

      • Supplemental Figure 1

        Schematic diagram of RB1 gene and its coding protein, guide RNA (gRNA; yellow) and PAM (purple) sequences, and the localization of the epitope recognized by the RB1 antibody.

      • Supplemental Figure 2

        Schematic diagram of TP53 gene and its coding protein, guide RNA (gRNA; yellow) and PAM (purple) sequences, and the localization of the epitope recognized by the TP53 antibody.

      • Supplemental Figure 3

        Schematic diagram of REST gene and it coding protein, guide RNA (gRNA; yellow) and PAM (purple) sequences, and the localization of the epitope recognized by the REST antibody.

      • Supplemental Figure 4

        Frameshift mutations of RB1 gene in A549-RB1-KO, A549-RB1/TP53-DKO, and A549-RB1/TP53/REST-TKO. Nucleotide sequences of the editing region in which the frameshift mutations were generated in RB1 alleles of A549-RB1-KO, A549-RB1/TP53-DKO, and A549-RB1/TP53/REST-TKO. Two mutation patterns, one-base insertion and two-base deletion, were found.

      • Supplemental Figure 5

        Frameshift mutations of TP53 gene in A549-RB1/TP53-DKO and A549-RB1/TP53/REST-TKO. Nucleotide sequences of the editing region in which the frameshift mutations were generated in TP53 alleles of A549-RB1/TP53-DKO and A549-RB1/TP53/REST-TKO. Two mutation patterns, one-base insertion and two-base insertion, were found.

      • Supplemental Figure 6

        Frameshift mutations of TP53 gene in A549-TP53-KO. Nucleotide sequences of the editing region in which the frameshift mutations were generated in TP53 alleles of A549-TP53-KO. Two mutation patterns, 2-base deletion and 168-base deletion plus 19-base insertion, were found.

      • Supplemental Figure 7

        Frameshift mutations of REST gene in A549-RB1/TP53/REST-TKO. Nucleotide sequences of the editing region in which the frameshift mutations were generated in REST alleles of A549-RB1/TP53/REST-TKO. Two mutation patterns, 89-base insertion and 1-base insertion, were found.

      • Supplemental Figure 8

        Histology of xenografts of H810 [large-cell neuroendocrine carcinoma (LCNEC)], A549-RB1/TP53/REST-TKO-ASCL1/POU3F4, and Lu134A [small-cell lung carcinoma (SCLC)] cells. A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells showed morphologic similarity to LCNEC cells but not to SCLC cells. Hematoxylin and eosin staining is shown. Scale bars = 100 μm.

      • Supplemental Figure 9

        Population doubling levels (PDLs) of A549-RB1/TP53-DKO, A549-RB1/TP53/REST-TKO, and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. The cell growth of A549-RB1/TP53/REST-TKO cells was not significantly altered in comparison with that of A549-RB1/TP53/REST-DKO cells (P = 0.8526 at day 9), whereas A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells showed significantly decreased growth in comparison with A549-RB1/TP53-DKO cells and A549-RB1/TP53/REST-TKO cells (P = 0.0332 and P = 0.0208 at day 9, respectively). ∗P < 0.05.

      • Supplemental Figure 10

        A: Migration of A549-RB1/TP53-DKO, A549-RB1/TP53/REST-TKO, and A549-RB1/TP53/REST-TKO-ASCL1/POU3F4 cells. The migratory ability was evaluated by wound healing assay. Wound areas measuring 500 μm in width were generated with a Culture-Insert 2 Well dish (ibidi, Grafelfing, Germany). To eliminate the influence of cell proliferation, the cells were treated with mitomycin C and cultivated in the wells. The phase-contrast microscopic images were taken from the same field at 0 and 6 hours, and cell migration was analyzed using ImageJ software version 1.53f (NIH, Bethesda, MD; https://imagej.nih.gov/ij, last accessed February 4, 2022). B: The wound closure rate was calculated by the following formula: wound closure % = (the wound area at 0 hours − the wound area at 6 hours/the wound area at 0 hours) × 100. Scale bars = 200 μm (A).

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