Development of invasion and/or metastasis of pancreatic and lung cancers by controlling S100A4

• Project/Area Number: 23590452
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Experimental pathology
• Research Institution: Tohoku University
• Principal Investigator: HORII Akira 東北大学, 医学(系)研究科(研究院), 教授 (40249983)
• Project Period (FY): 2011 – 2013
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2011: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: S100A4 / 膵癌 / 肺癌 / 神経浸潤 / 薬剤耐性 / 浸潤 / 転移 / メチル化 / ヒストン修飾 / Gemcitabine耐性

Research Abstract

Overexpression of S100A4 is frequently found in pancreatic and lung cancers. Knockdown suppresses cell growth and forced expression promotes cell motility in both of the cancer types. Furthermore, we analyzed primary resected pancreatic cancer tissues immunohistochemically and found positive association between overexpression of S100A4 and tumor cell invasion, particularly to nerves. Microarray analyses after forced expression in S100A4-low expressing cells identified candidate downstream genes and we are now analyzing functions of these genes.
Because overexpression of S100A4 and poor prognoses after gemcitabine treatment was reported in pancreatic cancer, we established 8 cancer cell lines with acquired gemcitabine resistance; these include four pancreatic, one gastric, one colon, one gallbladder, one bile duct cancers. Frequent inactivating mutation in deoxycytidine kinase was observed, and further investigation is on the way.

Research Products

• [Journal Article] Characterization of functional transient receptor potential melastatin 8 (TRPM8) channels in human pancreatic ductal adenocarcinoma cells. (2014)
  • Author(s): Cucu D, Chiritoiu G, Petrescu S, Babes A, Stanica L, Duda DG, Horii A, Dima SO, Popescu I.
  • Journal Title: Pancreas Volume: in press Issue: 5 Pages: 795-800
  • DOI: 10.1097/mpa.0000000000000106
  • Peer Reviewed
• [Journal Article] S100A4 is frequently overexpressed in lung cancer cells and promotes cell growth and cell motility. (2014)
  • Author(s): Chen N, Sato D, Saiki Y, Sunamura M, Fukushige S, Horii A.
  • Journal Title: Biochem Biophys Res Commun Volume: in press Issue: 3 Pages: 459-464
  • DOI: 10.1016/j.bbrc.2014.04.025
  • Peer Reviewed
• [Journal Article] Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers. (2014)
  • Author(s): Nakahata S, Ichikawa T, Maneesaay P, Saito Y, Nagai K, Tamura T, Manachai N, Yamakawa N, Hamasaki M, Kitabayashi I, Arai Y, Kanai Y, Taki T, Abe T, Kiyonari H, Shimoda K, Ohshima K, Horii A, Shima H, Taniwaki M, Yamaguchi R, Morishita K.
  • Journal Title: Nat Commun Volume: in press Issue: 1 Pages: 3393-3393
  • DOI: 10.1038/ncomms4393
  • Peer Reviewed / Open Access
• [Journal Article] Molecular pathology of pancreatic cancer. (2014)
  • Author(s): Saiki Y, Horii A.
  • Journal Title: Pathol Int Volume: 64 Issue: 1 Pages: 10-19
  • DOI: 10.1111/pin.12114
  • Peer Reviewed
• [Journal Article] Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer (2013)
  • Author(s): Yamamura A, et al
  • Journal Title: Biochem Biophys Res Commun Volume: 441(1) Issue: 1 Pages: 102-7
  • DOI: 10.1016/j.bbrc.2013.10.010
  • Peer Reviewed
• [Journal Article] p190A RhoGAP is involved in EGFR pathways and promotes proliferation, invasion and migration in lung adenocarcinoma cells. (2013)
  • Author(s): Notsuda H, Sakurada A, Endo C, Okada Y, Horii A, Shima H, Kondo T.
  • Journal Title: Int J Oncol Volume: 43 Issue: 5 Pages: 1569-1577
  • DOI: 10.3892/ijo.2013.2096
  • Peer Reviewed
• [Journal Article] DNA Methylation in Cancer: A Gene Silencing Mechanism and the Clinical Potential of Its Biomarkers (2013)
  • Author(s): Fukushige S, Horii A.
  • Journal Title: The Tohoku Journal of Experimental Medicine Volume: 229 Issue: 3 Pages: 173-185
  • DOI: 10.1620/tjem.229.173
  • NAID: 10031161717
  • ISSN: 0040-8727, 1349-3329
  • Peer Reviewed
• [Journal Article] The expression of S100A4 in human pancreatic cancer is associated with invasion (2013)
  • Author(s): Tsukamoto N, et al
  • Journal Title: Pancreas Volume: 42(6) Issue: 6 Pages: 1027-33
  • DOI: 10.1097/mpa.0b013e31828804e7
  • Peer Reviewed
• [Journal Article] Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2013)
  • Author(s): Kinoshita H, Imoto S, Suda Y, Ishida M, Watanabe M, Kawai Y, Kitazawa H, Miura K, Horii A, Saito T.
  • Journal Title: Anim Sci J. Volume: 84 Issue: 2 Pages: 150-158
  • DOI: 10.1111/j.1740-0929.2012.01054.x
  • Peer Reviewed
• [Journal Article] New screening methods for probiotics with adhesion properties to sialic acid and sulfate residues in human colonic mucin using the Biacore assay. (2013)
  • Author(s): Huang I-N, Okawara, T, Watanabe M, Kawai Y, Kitazawa H, Ohnuma S, Shibata C, Horii A, Kimura K, Taketomo N, Xiao J, Iwatsuki K, Saito T.
  • Journal Title: J Appl Microbiol Volume: 114 Issue: 3 Pages: 854-860
  • DOI: 10.1111/jam.12063
  • Peer Reviewed
• [Journal Article] The expression of S100A4 in human pancreatic cancer is associated with invasion. (2013)
  • Author(s): Tsukamoto N, Egawa S, Akada M, Abe K, Saiki Y, Kaneko N, Yokoyama S, Shima K, Yamamura A, Motoi F, Abe H, Hayashi H, Ishida K, Moriya T, Tabata T,Sunamura M, Unno M, Horii A, et al.
  • Journal Title: Pancreas Volume: in press
  • Peer Reviewed
• [Journal Article] Road to early detection of pancreatic cancer : Attempts to utilize epigenetic biomarkers (2012)
  • Author(s): Shinichi Fukushige
  • Journal Title: Cancer Lett Volume: (未定)(in press) Issue: 2 Pages: 231-237
  • DOI: 10.1016/j.canlet.2012.03.022
  • Peer Reviewed
• [Journal Article] S100A4, frequently overexpressed in various human cancers, accelerates cell motility in pancreatic cancercells (2012)
  • Author(s): Sekine H, Chen N, Sato K, Saiki Y, Yoshino Y, Umetsu Y, Jin G, Nagase H, Gu Z, Fukushige S, Sunamura M, Horii A.
  • Journal Title: Biochem Biophys Res Commun Volume: 429 Issue: 3-4 Pages: 214-219
  • DOI: 10.1016/j.bbrc.2012.10.048
  • Peer Reviewed
• [Journal Article] An Adhesin-Like Protein, Lam29, from <i>Lactobacillus mucosae</i> ME-340 Binds to Histone H3 and Blood Group Antigens in Human Colonic Mucus (2012)
  • Author(s): Watanabe M, Kinoshita H, Huang IN, Eguchi K, Tsurumi T, Kawai Y, Kitazawa H, Kimura K, Taketomo N, Kikuchi D, Sase T, Miura K, Ogawa H, Shibata C, Horii A, Saito T.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 76 Issue: 9 Pages: 1655-1660
  • DOI: 10.1271/bbb.120150
  • NAID: 130004137955
  • ISSN: 0916-8451, 1347-6947
  • Peer Reviewed
• [Journal Article] Potential utility of eGFP-expressing NOG mice (NOG-EGFP) as a high purity cancer sampling system (2012)
  • Author(s): Shima K, et al
  • Journal Title: J Exp Clin Cancer Res Volume: 31 Issue: 1 Pages: 55-61
  • DOI: 10.1186/1756-9966-31-55
  • URL: http://www.jeccr.com/content/31/1/55
  • Peer Reviewed
• [Journal Article] miR-34a is downregulated in cis-diamminedichloroplatinum treated sinonasal squamous cell carcinoma patients with poor prognosis (2012)
  • Author(s): Ogawa T, Saiki Y, Shiga K, Chen N, Fukushige S, Sunamura M, Nagase H, Hashimoto S, Matsuura K, Saijo S, Kobayashi T, Horii A.
  • Journal Title: Cancer Sci Volume: 103 Issue: 9 Pages: 1737-1743
  • DOI: 10.1111/j.1349-7006.2012.02338.x
  • Peer Reviewed
• [Journal Article] DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells (2012)
  • Author(s): Yuriko Saiki
  • Journal Title: Biochemical and Biophysical Research Communications Volume: (in press) Issue: 1 Pages: 98-104
  • DOI: 10.1016/j.bbrc.2012.03.122
  • Peer Reviewed
• [Journal Article] Methylation of death-associated protein kinase (DAPK) is associated with cetuximab and erlotinib resistance (2012)
  • Author(s): Ogawa T, Liggett TE, Melnikov AA, Monitto CL, Kusuke D, Shiga K, Kobayashi T, Horii A, Chattejee A, Levenson VV, Koch W, Sidransky D, Chang X
  • Journal Title: Cell Cycle Volume: 11 Issue: 8 Pages: 1655-1662
  • DOI: 10.4161/cc.20120
  • Peer Reviewed
• [Journal Article] A human head and neck squamous cell carcinoma cell line with acquired cis-diamminedichloroplatinum-resist ance shows remarkable upregulation of BRCA1 and hypersensitivity to taxane (2011)
  • Author(s): Saiki Y, Ogawa T, Shiga K, Sunamura M, Kobayashi T, Horii A.
  • Journal Title: Int J Otolaryngol Volume: 2011 Pages: 521852-521852
  • DOI: 10.1155/2011/521852
  • Peer Reviewed
• [Journal Article] Identification of epigenetically silenced genes in human pancreatic cancer by a novel method "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array) (2011)
  • Author(s): Hideyuki Shimizu
  • Journal Title: Biochem Biophys Res Commun Volume: 411 Issue: 1 Pages: 162-167
  • DOI: 10.1016/j.bbrc.2011.06.121
  • Peer Reviewed
• [Journal Article] Microarray coupled with methyl-CpG targeted transcriptional activation (MeTA-array) identifies hypermethylated genes containing the stringent criteria of CpG islands at high frequency (2011)
  • Author(s): Yuko Sato
  • Journal Title: Epigenetics Volume: 6 Issue: 6 Pages: 752-759
  • DOI: 10.4161/epi.6.6.15906
  • Peer Reviewed
• [Presentation] IFI27 and NOV, downstream regulated genes by S100A4, are playing important roles in pancreatic carcinogenesis (2014)
  • Author(s): Chen N, Saiki Y, Sekine H, Sunamura M, Fukushige S, Motoi F, Egawa S, Unno M, Horii A
  • Organizer: アメリカ癌学会
  • Place of Presentation: アメリカ・サンディエゴ
• [Presentation] TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation (2014)
  • Author(s): Fukushige S, Mizuguchi Y Horii A
  • Organizer: アメリカ癌学会
  • Place of Presentation: アメリカ・サンディエゴ
• [Presentation] IFI27 and NOV, downstream regulated genes by S100A4, are playing important roles in pancreatic carcinogenesis. (2014)
  • Author(s): Na Chen, Yuriko Saiki, Hitoshi Sekine, Makoto Sunamura, Shinichi Fukushige, Fuyuhiko Motoi, Shinichi Egawa, Michiaki Unno, Akira Horii
  • Organizer: American Association for Cancer Research Annual Meeting
  • Place of Presentation: San Diego、アメリカ合衆国
• [Presentation] TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation. (2014)
  • Author(s): Shinichi Fukushige, Yasuhiko Mizuguchi and Akira Horii
  • Organizer: American Association for Cancer Research Annual Meeting
  • Place of Presentation: San Diego、アメリカ合衆国
• [Presentation] IFI27 and NOV, downstream regulated genes by S100A4, are playing important roles in pancreatic carcinogenesis (2013)
  • Author(s): Chen N, Saiki Y, Sunamura M, Fukushige S, Motoi F, Egawa S, Unno M, Horii A
  • Organizer: 日本癌学会
  • Place of Presentation: 横浜
• [Presentation] Chemosensitivity prediction by microRNA profiling in head and neck and esophageal squamous cell carcinoma (2013)
  • Author(s): Saiki Y, Ogawa T, Nakano T, Chen N, Sunamura M, Horii A
  • Organizer: 日本癌学会
  • Place of Presentation: 横浜
• [Presentation] TET1 hydroxylase activity reactivates the expression of epigenetically silenced genes (2013)
  • Author(s): Fukushige S, Mizuguchi Y, Horii A
  • Organizer: 日本癌学会
  • Place of Presentation: 横浜
• [Presentation] マイクロRNAによる抗がん剤感受性予測 (2013)
  • Author(s): 斎木由利子、中野智太、小川武則、砂村眞琴、永瀬浩喜、堀井明
  • Organizer: 日本癌病態治療研究会
  • Place of Presentation: 東京
• [Presentation] MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer (2013)
  • Author(s): Fukushige S, Horii A
  • Organizer: アメリカ癌学会
  • Place of Presentation: アメリカ・ワシントン
• [Presentation] Suppressed NDRG2 expression correlates with poor prognosis in pancreatic cancer, and suppression mechanism is different from colorectal and gastric cancer (2013)
  • Author(s): Mizuguchi Y, Yamamura A, Miura K, Karasawa H, Morishita K, Abe K, Saiki Y, Fukushige S, Kaneko N, Sase T, Motoi F, Egawa S, Shibata C, Unno M, Sasaki I, Horii A
  • Organizer: 日本癌学会-アメリカ癌学会合同会議
  • Place of Presentation: アメリカ・マウイ島
• [Presentation] IFI27 and NOV, downstream regulated genes by S100A4, are playing important roles in pancreatic carcinogenesis. (2013)
  • Author(s): Na Chen, Yuriko Saiki, Makoto Sunamura, Shinichi Fukushige, Fuyuhiko Motoi, Shinichi Egawa, Michiaki Unno, Akira Horii
  • Organizer: 日本癌学会総会
  • Place of Presentation: 横浜
• [Presentation] Chemosensitivity prediction by microRNA profiling in head and neck and esophageal squamous cell carcinoma (2013)
  • Author(s): Yuriko Saiki, Takenori Ogawa, Tomohiro Nakano, Na Chen, Makoto Sunamura, Akira Horii
  • Organizer: 日本癌学会総会
  • Place of Presentation: 横浜
• [Presentation] TET1 hydroxylase activity reactivates the expression of epigenetically silenced genes. (2013)
  • Author(s): Shinichi Fukushige, Yasuhiko Mizuguchi, Akira Horii
  • Organizer: 日本癌学会総会
  • Place of Presentation: 横浜
• [Presentation] マイクロRNAによる抗がん剤感受性予測 (2013)
  • Author(s): 斎木由利子、中野智太、小川武則、砂村眞琴、永瀬浩喜、堀井明
  • Organizer: 第22回日本癌病態治療研究会
  • Place of Presentation: 東京
• [Presentation] MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer. (2013)
  • Author(s): Shinichi Fukushige, Akira Horii
  • Organizer: American Association for Cancer Research Annual Meeting
  • Place of Presentation: Washington DC、アメリカ合衆国
• [Presentation] Suppressed NDRG2 expression correlates with poor prognosis in pancreatic cancer, and the suppression mechanisms are distinct from that of colorectal and gastric cancers. (2013)
  • Author(s): Mizuguchi Y, Yamamura A, Miura K, Karasawa H, Morishita K, Saiki Y, Fukushige S, Motio F, Egawa S, Shibata C, Unno M, Horii A.
  • Organizer: Ninth AACR-Japanese Cancer Association Joint Conference
  • Place of Presentation: Maui, HI, USA
• [Presentation] MeTA-array, a useful method to identify genes transcriptionally silenced by tumor-specific hypermethylation in cancer. (2013)
  • Author(s): Fukushige S, Horii A.
  • Organizer: AACR Annual Meeting 2013
  • Place of Presentation: Washington DC, USA
• [Presentation] ゲムシタビン耐性ヒト癌細胞株におけるdeoxycytidine kinaseの不活化 (2012)
  • Author(s): 齋木由利子、吉野優樹、福重真一、砂村真琴、石田晶玄、元井冬彦、江川新一、海野倫明、堀井明
  • Organizer: 日本癌学会
  • Place of Presentation: 札幌
• [Presentation] ヒト癌において腫瘍特異的な高度メチル化により転写抑制された遺伝子のMeTA-arrayによる効率的探索法の開発 (2012)
  • Author(s): 福重真一、堀井明
  • Organizer: 日本癌学会
  • Place of Presentation: 札幌
• [Presentation] miR-34a発現低下は鼻副鼻腔癌におけるシスプラチン化学療法の予後規定因子である (2012)
  • Author(s): 小川武則、齋木由利子、志賀清人、松浦一登、加藤健吾、西條茂、小林俊光、福重真一、堀井明
  • Organizer: 日本癌学会
  • Place of Presentation: 札幌
• [Presentation] Road to early detection and curative treatment of pancreatic cancer (2012)
  • Author(s): Horii A
  • Organizer: Academician Nicolae Cajalシンポジウム
  • Place of Presentation: ルーマニア・ブカレスト
• [Presentation] ゲムシタビン耐性ヒト癌細胞株におけるdeosycytidine kinaseの不活化 (2012)
  • Author(s): 斎木由利子、吉野優樹、福重真一、砂村眞琴、石田晶玄、元井冬彦、江川新一、海野倫明、堀井明
  • Organizer: 第71回日本癌学会学術総会
  • Place of Presentation: 札幌
• [Presentation] ヒト癌において腫瘍特異的な高度メチル化により転写抑制された遺伝子のMeta-arrayによる効率的探索法の開発 (2012)
  • Author(s): 福重真一、堀井明
  • Organizer: 第71回日本癌学会学術総会
  • Place of Presentation: 札幌
• [Presentation] miR-34a発現低下は鼻副鼻腔癌におけるシスプラチン化学療法の予後規定因子である (2012)
  • Author(s): 小川武則、斎木由利子、志賀清人、松浦一登、加藤健吾、西條茂、小林利光、福重真一、堀井明
  • Organizer: 第71回日本癌学会学術総会
  • Place of Presentation: 札幌
• [Presentation] Road to early detection and curative treatment of pancreatic cancer. (2012)
  • Author(s): Horii A
  • Organizer: Academician Nicolae Cajal Symposium(招待講演)
  • Place of Presentation: Bucharest, Romania
• [Presentation] ゲムシタビン耐性膵癌細胞株におけるゲムシタビン代謝の解析 (2011)
  • Author(s): 齋木由利子、江川新一、砂村眞琴、海野倫明、堀井明
  • Organizer: 日本癌学会
  • Place of Presentation: 名古屋
• [Presentation] メチルCpG配列を標的とする転写活性化とマイクロアレイの組み合わせはヒトがんにおける低メチル化遺伝子の同定を可能にする (2011)
  • Author(s): 福重真一、陳娜、堀井明
  • Organizer: 日本癌学会
  • Place of Presentation: 名古屋
• [Presentation] NOG-EGFPマウス異種移植モデルを用いた高精度な癌細胞sampling systemの構築を目指して (2011)
  • Author(s): 嶋健太郎、水間正道、大村範幸、林洋毅、中川圭、岡田恭穂、北村洋、元井冬彦、力山敏樹、片寄友、江川新一、石井直人、堀井明、海野倫明
  • Organizer: 日本癌学会
  • Place of Presentation: 名古屋
• [Presentation] New prevention strategy for inflammatory bowel disease using probiotic lactic acid bacteria and/or Bifidobacterium with specific adhesion to human intestine (2011)
  • Author(s): Saito T, Yukishita R, Watanabe M, Katazawa H, Kawai Y, Horii A, Ikezawa F, Yamamura A, Ogawa H, Miura K, Shibata C, Sasaki I
  • Organizer: Lactic Acid Bacteriaシンポジウム
  • Place of Presentation: ニュージーランド・エグモントアーンゼー
• [Presentation] 膵癌のGemcitabine耐性機構の解明 (2011)
  • Author(s): 齋木由利子、江川新一、海野倫明、砂村眞琴、堀井明
  • Organizer: 日本膵臓学会
  • Place of Presentation: 弘前
• [Presentation] Each methyl-CpG binding domain has the ability to target different methylated genes in human cancer (2011)
  • Author(s): Fukushige S, Horii A
  • Organizer: アメリカ癌学会
  • Place of Presentation: アメリカ・オーランド
• [Presentation] New prevention strategy for inflammatory bowel disease using probiotic lactic acid bacteria and/or Bifidobacterium with specific adhesion to human intestine (2011)
  • Author(s): Saito T, Yukishita R, Watanabe M, Katazawa H, Kawai Y, Horii A, Ikezawa F, Yamamura A, Ogawa H, Miura K, Shibata C, Sasaki I.
  • Organizer: The 10th Symposium on Lactic Acid Bacteria
  • Place of Presentation: Egmond aan Zee, the Netherlands
• [Presentation] NOG-EGFPマウス異種移植モデルを用いた高精度な癌細胞sampling systemの構築を目指して (2011)
  • Author(s): 嶋健太郎、水間正道、大村範幸、林洋毅、中川圭、岡田恭穂、北村洋、元井冬彦、力山敏樹、片寄友、江川新一、石井直人、堀井明、海野倫明
  • Organizer: 第70回日本癌学会総会
  • Place of Presentation: 名古屋
• [Presentation] ゲムシタビン耐性膵癌細胞株におけるゲムシタビン代謝の解析 (2011)
  • Author(s): 齋木由利子、江川新一、砂村眞琴、海野倫明、堀井明
  • Organizer: 第70回日本癌学会総会
  • Place of Presentation: 名古屋
• [Presentation] メチルCpG配列を標的とする転写活性化とマイクロアレイの組み合わせはヒトがんにおける低メチル化遺伝子の同定を可能にする (2011)
  • Author(s): 福重真一、陳娜、堀井明
  • Organizer: 第70回日本癌学会総会
  • Place of Presentation: 名古屋
• [Presentation] 膵癌のGemcitabine耐性機構の解明 (2011)
  • Author(s): 齋木由利子、江川新一、海野倫明、砂村眞琴、堀井明
  • Organizer: 第42回日本膵臓学会大会
  • Place of Presentation: 弘前
• [Remarks]
  • URL: http://www.med.tohoku.ac.jp/org/medical/13/index.html
• [Remarks]
  • URL: http://www.molpath.med.tohoku.ac.jp/index.html
• [Remarks] 分子病理学分野
  • URL: http://www.med.tohoku.ac.jp/org/medical/13/index.html
• [Remarks] Department of Molecular Pathology
  • URL: http://www.molpath.med.tohoku.ac.jp/index.html
• [Remarks] 東北大学 大学院医学系研究科・医学部 分子病理学分野
  • URL: http://www.med.tohoku.ac.jp/org/medical/13/index.html
• [Remarks] Tohoku Univ School of Med, Depｔ of Molec Pathol
  • URL: http://www.molpath.med.tohoku.ac.jp/index.html