Defining the KRAS signaling cascade using a genome-edited human bronchial epithelial cell line

• Project/Area Number: 25460395
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Pathological medical chemistry
• Research Institution: Aichi Medical University
• Principal Investigator: Sivasundaram Karnan 愛知医科大学, 医学部, 講師 (30557096)
• Co-Investigator(Kenkyū-buntansha): 小西 裕之 愛知医科大学, 医学部, 教授 (20344335) ; 細川 好孝 愛知医科大学, 医学部, 教授 (60229193)
• Project Period (FY): 2013-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: KRAS / 変異ノックイン / 気管支上皮細胞株 / 気管支上皮 / 遺伝子改変 / 肺癌 / 気道上皮 / ゲノム編集

Outline of Final Research Achievements

The KRAS gene is mutated and constitutively activated in many types of cancers including lung cancer. In this study, we utilized a non-tumorigenic human bronchial epithelial cell line which had undergone targeted knock-in of an oncogenic KRAS mutation to investigate the biological function of the mutant KRAS gene. We found that the mutant KRAS induced an altered cellular morphology similar to cancer cells, conferred the capacity of anchorage-independent cell growth, and augmented cell migration and invasion into Matrigel. The MEK-ERK pathway which promotes cell proliferation was hyperactivated in the mutant KRAS-knocked-in cells. Comprehensive analysis of mRNA and protein expression in the cells revealed the increased expression of some molecules potentially explaining the transformed phenotype seen in the mutant KRAS-knocked-in cells. This study helps us to understand the biological function of mutant KRAS and will assist in the development of medicine for KRAS-mutated cancers.

Research Products

• [Journal Article] Delta40p53 suppresses tumor cell proliferation and induces cellular senescence in hepatocellular carcinoma cells (2017)
  • Author(s): Ota A, Nakao H, Sawada Y, Karnan S, Wahiduzzaman M, Inoue T, Kobayashi Y, Yamamoto T, Ishii N, Ohashi T, Nakade Y, Sato K, Itoh K, Konishi H, Hosokawa Y, Yoneda M
  • Journal Title: J Cell Sci Volume: 130 Issue: 3 Pages: 614-625
  • DOI: 10.1242/jcs.190736
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Inhibition of Nox1 induces apoptosis by attenuating the AKT signaling pathway in oral squamous cell carcinoma cell lines (2016)
  • Author(s): Ito K, Ota A, Ono T, Nakaoka T, Wahiduzzaman M, Karnan S, Konishi H, Furuhashi A, Hayashi T, Yamada Y, Hosokawa Y, and Kazaoka Y
  • Journal Title: Oncol Rep Volume: 36 Issue: 5 Pages: 2991-2998
  • DOI: 10.3892/or.2016.5068
  • Peer Reviewed
• [Journal Article] Efficient AAV-mediated gene targeting using 2A-based promoter-trap system (2016)
  • Author(s): Karnan S, Ota A, Konishi Y, Wahiduzzaman M, Tsuzuki S, Hosokawa Y, and Konishi H
  • Journal Title: Bio-Protoc Volume: 6 Issue: 24
  • DOI: 10.21769/bioprotoc.2058
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide (2016)
  • Author(s): Karnan S, Ota A, Konishi Y, Wahiduzzaman M, Hosokawa Y, and Konishi H
  • Journal Title: Nucleic Acids Research Volume: 44 Issue: 6 Pages: e54-e54
  • DOI: 10.1093/nar/gkv1338
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Overexpression of salivary-type amylase reduces the sensitivity to bortezomib in multiple myeloma cells (2015)
  • Author(s): Mizuno, S., Hanamura, I., Ota, A., Karnan, S., Narita, T., Ri, M., Mizutani, M., Goto, M., Gotou, M., Tsunekawa, N., Shikami, M., Iida, S., Hosokawa, Y., Miwa, H., Ueda, R., Nitta, M. and Takami, A
  • Journal Title: Int J Hematol Volume: 102 Issue: 5 Pages: 569-78
  • DOI: 10.1007/s12185-015-1859-0
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Inhibition of NADPH oxidase 4 induces apoptosis in malignant mesothelioma: Role of reactive oxygen species (2015)
  • Author(s): Tanaka M, Miura Y, Numanami H, Karnan S, Ota A, Konishi H, Hosokawa Y, and Hanyuda M
  • Journal Title: Oncology Reports Volume: 34 Issue: 4 Pages: 1726-1732
  • DOI: 10.3892/or.2015.4155
  • Peer Reviewed
• [Journal Article] Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines (2015)
  • Author(s): Ono T, Ota A, Ito K, Nakaoka T, Karnan S, Konishi H, Furuhashi A, Hayashi T, Yamada Y, Hosokawa Y, Kazaoka Y
  • Journal Title: Oral Dis Volume: - Issue: 4 Pages: 501-511
  • DOI: 10.1111/odi.12310
  • Peer Reviewed
• [Journal Article] High-resolution 400K oligonucleotide array comparative genomic hybridization analysis of neurofibromatosis type 1-associated cutaneous neurofibromas (2015)
  • Author(s): Asai A, Karnan S, Ota A, Takahashi M, Damdindorj L, Konishi Y, Hossain E, Konishi H, Nagata A, Yokoo K, Hosokawa Y
  • Journal Title: Gene Volume: 558 Pages: 220-226
  • Peer Reviewed
• [Journal Article] Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages (2015)
  • Author(s): Hossain E, Ota A, Karnan S, Takahashi M, Mannan SB, Konishi H, Hosokawa Y
  • Journal Title: Mol Cell Biochem Volume: 400 Issue: 1-2 Pages: 29-40
  • DOI: 10.1007/s11010-014-2259-0
  • Peer Reviewed
• [Journal Article] A comparative analysis of constitutive promoters located in adeno-associated viral vectors (2014)
  • Author(s): Damdindorj L, Karnan S, Ota A, Hossain E, Konishi Y, Hosokawa Y, Konishi H
  • Journal Title: PLoS ONE Volume: 9 Issue: 8 Pages: e106472-e106472
  • DOI: 10.1371/journal.pone.0106472
  • Peer Reviewed / Open Access
• [Journal Article] Array-comparative genomic hybridization profiling of immunohistochemical subgroups of diffuse large B-cell lymphoma shows distinct genomic alterations. (2014)
  • Author(s): Guo Y, Takeuchi I, Karnan S, Miyata T, Ohshima K, Seto M.
  • Journal Title: Cancer Sci. Volume: 105 Issue: 4 Pages: 481-489
  • DOI: 10.1111/cas.12378
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Clonal heterogeneity of lymphoid malignancies correlates with poor prognosis. (2014)
  • Author(s): Suguro M, Yoshida N, Umino A, Kato H, Tagawa H, Nakagawa M, Fukuhara N, Karnan S, Takeuchi I, Hocking TD, Arita K, Karube K, Tsuzuki S, Nakamura S, Kinoshita T, Seto M.
  • Journal Title: Cancer Sci. Volume: 105 Issue: 7 Pages: 897-904
  • DOI: 10.1111/cas.12442
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Combined arsenic trioxide-cisplatin treatment enhances apoptosis in oral squamous cell carcinoma cells. (2014)
  • Author(s): Nakaoka T, Ota A, Ono T, Karnan S, Konishi H, Furuhashi A, Ohmura Y, Yamada Y, Hosokawa Y, and Kazaoka Y.
  • Journal Title: Cell Oncol Volume: 37 Issue: 2 Pages: 119-129
  • DOI: 10.1007/s13402-014-0167-7
  • Peer Reviewed
• [Journal Article] Arsenic upregulates the expression of angiotensin II Type I receptor in mouse aortic endothelial cells. (2013)
  • Author(s): Hossain E, Ota A, Takahashi M, Karnan S, Damdindorj L, Konishi Y, Konishi H, and Hosokawa Y.
  • Journal Title: Toxicol Lett Volume: 220 Issue: 1 Pages: 70-75
  • DOI: 10.1016/j.toxlet.2013.04.006
  • Peer Reviewed
• [Journal Article] Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation. (2013)
  • Author(s): Wang GM, Wong HY, Konishi H, Blair BG, Abukhdeir AM, Gustin JP, Rosen DM, Denmeade S, Rasheed Z, Matsui W, Garay JP, Mohseni M, Higgins MJ, Cidado J, Jelovac D, Croessmann S, Cochran R, Karnan S, Konishi Y, Ota A, Hosokawa Y, Argani P, Lauring J, and Park BH.
  • Journal Title: Cancer Res Volume: 73 Issue: 11 Pages: 3248-3261
  • DOI: 10.1158/0008-5472.can-12-1578
  • Peer Reviewed
• [Journal Article] Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells. (2013)
  • Author(s): Hossain E, Ota A, Karnan S, Damdindorj L, Takahashi M, Konishi Y, Konishi H, and Hosokawa Y.
  • Journal Title: Toxicol Appl Pharmacol Volume: 273 Issue: 3 Pages: 651-658
  • DOI: 10.1016/j.taap.2013.10.012
  • Peer Reviewed
• [Presentation] 遺伝子改変・ゲノム編集効率を測定するアッセイ系としてのPIGA遺伝子の利用 (2016)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2016-11-30
• [Presentation] NOX1はAKTシグナルを介して口腔扁平上非癌細胞の生存に寄与する (2016)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 風岡宜暁, 細川好孝
  • Organizer: 第75回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2016-10-06
• [Presentation] 悪性胸膜中皮腫の早期診断の腫瘍マーカーの検討 (2016)
  • Author(s): Sivasundaram Karnan, 太田明伸, Md Wahiduzzaman, 小西裕之, 細川好孝
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台国際センター（宮城・仙台）
  • Year and Date: 2016-09-25
• [Presentation] Combined arsenic trioxide-cisplatin treatment enhances apoptosis in leukemic cell lines (2016)
  • Author(s): Md Wahiduzzaman, Akinobu Ota, Sivasundaram Karnan, Hiroyuki Konishi, Yoshitaka Hosokawa
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台国際センター（宮城・仙台）
  • Year and Date: 2016-09-25
• [Presentation] 肝がん細胞株におけるp53アイソフォームdelta40p53の性状解析 (2016)
  • Author(s): 太田明伸, Md Wahiduzzaman, Sivasundaram Karnan, 小西裕之, 細川好孝
  • Organizer: 第89回日本生化学会大会
  • Place of Presentation: 仙台国際センター（宮城・仙台）
  • Year and Date: 2016-09-25
• [Presentation] CRISPR-Cas9システムによる変異ノックイン ― プロモータートラップ法の比較検討 (2016)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝
  • Organizer: 日本ゲノム編集学会第1回大会
  • Place of Presentation: 広島国際会議場（広島・広島）
  • Year and Date: 2016-09-06
• [Presentation] 400KオリゴアレイCGHを用いたNF1患者の皮膚神経線維腫の解析 (2015)
  • Author(s): Sivasundaram Karnan, 太田明伸, 小西裕之, 細川好孝
  • Organizer: 第38回日本分子生物学会年会第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸国際展示場（兵庫・神戸）
  • Year and Date: 2015-12-01
• [Presentation] 口腔がん細胞株に対するプランバギンのプロオキシダント作用とアポトーシス誘導にはミトコンドリア遺伝子発現系が関与する (2015)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 細川好孝
  • Organizer: 第38回日本分子生物学会年会第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸国際展示場（兵庫・神戸）
  • Year and Date: 2015-12-01
• [Presentation] 癌原性KRAS変異をノックインしたヒト気管支上皮細胞株モデルの樹立と解析 (2015)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 細川好孝
  • Organizer: 第74回日本癌学会学術総会
  • Place of Presentation: 名古屋国際会議場（愛知・名古屋）
  • Year and Date: 2015-10-08
• [Presentation] Delta40p53はHepG2細胞に対して細胞増殖抑制効果を示す (2015)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 細川好孝
  • Organizer: 第74回日本癌学会学術総会
  • Place of Presentation: 名古屋国際会議場（愛知・名古屋）
  • Year and Date: 2015-10-08
• [Presentation] アデノ随伴ウイルスに基づく遺伝子改変ベクターにおいて薬剤耐性遺伝子の発現を制御するプロモーターと改変効率の関係 (2014)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 細川好孝
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] リポポリサッカライドはErk1/2シグナルを介したLOX-1の発現増加によってマクロファージの酸化LDLの取り込みを増強する (2014)
  • Author(s): 太田明伸, Ekhtear Hossain, Sivasundaram Karnan, Shahnewaj Mannan, 小西裕之, 細川好孝
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 国立京都国際会館（京都・京都）
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] プランバギンは口腔扁平上皮癌に対して活性酸素産生を介した細胞増殖抑制効果を示す (2014)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 風岡宜暁, 細川好孝
  • Organizer: 第73回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] NF1関連の皮膚神経線維腫における400KオリゴアレイCGH解析 (2014)
  • Author(s): Sivasundaram Karnan, 太田明伸, 小西裕之, 細川好孝
  • Organizer: 第73回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Arsenic trioxide inhibits interferon-gamma-mediated nitric oxide production in mouse vascular endothelial cells. (2013)
  • Author(s): Hossain E, Ota A, Karnan S, Damdindorj L, Konishi Y, Konishi H, Hosokawa Y.
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: パシフィコ横浜（横浜）
• [Presentation] ヒ素は酸化LDL受容体LOX-1の発現を増加させることによりマウス血管内皮細胞の酸化LDL取り込み能を増強する. (2013)
  • Author(s): 太田明伸, Ekhtear Hossain, Sivasundaram Karnan, Damdindorj Lkhagvasuren, 小西裕子, 小西裕之, 細川好孝.
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: パシフィコ横浜（横浜）
• [Presentation] ヒト細胞株のゲノム編集によるKRAS-PI3Kシグナル経路の解析. (2013)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 細川好孝.
  • Organizer: 第72回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（横浜）