Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Advanced Life Science　Advanced Transdisciplinary Science　Cellular Dynamics Science

Affiliation (Master)

• Faculty of Advanced Life Science　Advanced Transdisciplinary Science　Cellular Dynamics Science

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Profile and Settings

Profile and Settings

• Name (Japanese)

  ISHIHARA

• Name (Kana)

  Seiichiro

• Name

  201401016805088704

Alternate Names

http://kaken.nii.ac.jp/d/r/10719933.ja.html

Achievement

Research Interests

• 腫瘍微小環境   Mesenchymal stem cell   Mechanobiology   Radiotherapy   Cell migration   Cancer   

Research Areas

• Life sciences / Tumor biology
• Life sciences / Cell biology
• Life sciences / Biophysics / Mechanobiology

Research Experience

• 2017/10 - Today Hokkaido University Assistant professor
• 2015/04 - 2017/09 University of Wisconsin-Madison Postdoctoral fellow
• 2013/07 - 2015/03 Hokkaido University Postdoctoral fellow

Awards

• 2024/09 第10回部局横断シンポジウム研究助成採択・金賞
  
• 2024/01 がん関連三学会Rising Starネットワーキング 優秀賞
   「硬さ」に応答したがん細胞が転写因子ATF5を介して悪性化するメカニズム 

  受賞者: 石原 誠一郎
• 2023/10 第9回部局横断シンポジウム研究助成採択・金賞
   

  受賞者: 石原 誠一郎
• 2020/09 AMED NYAS Interstellar Initiative Outstanding Team Presentation
   Interstellar Initiative 

  受賞者: Seiichiro Ishihara;Simona Giunta;Hagar Labouta
• 2019/11 第5回北海道大学部局横断シンポジウム 口頭発表第1位
   膵臓がん細胞でメカニカルな刺激により活性化する転写因子ATF5 

  受賞者: 石原 誠一郎
• 2019/09 先端モデル動物支援プラットフォーム令和元年度若手支援技術講習会 ベストトーク賞
   転写因子ATF5から迫る膵がん細胞のメカノバイオロジー 

  受賞者: 石原 誠一郎
• 2014/09 平成26年度がん若手研究者ワークショップ 優秀演者賞
   肺がん細胞において転写因子ATF5は放射線耐性・増殖能・浸潤能を上昇させる 

  受賞者: 石原 誠一郎
• 2013/10 The American Society for Cell Biology 2013 Annual Meeting Postdoc Travel Award
   Activating transcription factor 5 promotes radioresistance and invasiveness in lung adenocarcinoma cells 

  受賞者: 石原 誠一郎
• 2010/02 The 6th HPAPS+(Special Edition) 若手優秀講演賞
   Cells settle down on a fluffy bed: a hard floor triggers emergency 

  受賞者: 石原 誠一郎

Published Papers

• Matrix stiffness regulates the triad communication of adipocytes/macrophages/endothelial cells through CXCL13.

  Arthur Choisez, Seiichiro Ishihara, Takuro Ishii, Yidan Xu, Sepideh D Firouzjah, Hisashi Haga, Ryoichi Nagatomi, Joji Kusuyama

  Journal of lipid research 100620 - 100620 2024/08/14 [Refereed]
   

  Adipose tissue remodeling and plasticity are dynamically regulated by the coordinated functions of adipocytes, macrophages, and endothelial cells and extracellular matrix (ECM) that provides stiffness networks in adipose tissue component cells. Inflammation and fibrosis are crucial exogenous factors that dysregulate adipose tissue functions and drastically change the mechanical properties of the ECM. Therefore, communication among the ECM and adipose tissue component cells is necessary to understand the multifaceted functions of adipose tissues. To obtain in vivo stiffness, we utilized genipin as a crosslinker for collagen gels. Meanwhile, we isolated primary adipocytes, macrophages, and endothelial cells from C57BL/6J mice and incubated these cells in the differentiation media on temperature-responsive culture dishes. After the differentiation, these cell sheets were transferred onto genipin-crosslinked collagen gels with varying matrix stiffness. We found that inflammatory gene expressions were induced by hard matrix, whereas anti-inflammatory gene expressions were promoted by soft matrix in all three types of cells. Interestingly, the co-culture experiments of adipocytes, macrophages, and endothelial cells showed that the effects of soft or hard matrix stiffness stimulation on adipocytes were transmitted to the distant adipose tissue component cells, altering their gene expression profiles under normal matrix conditions. Finally, we identified that a hard matrix induces the secretion of CXCL13 from adipocytes, and CXCL13 is one of the important transmitters for stiffness communication with macrophages and endothelial cells. These findings provide insight into the mechano-transmission into distant cells and the application of stiffness control for chronic inflammation in adipose tissues with metabolic dysregulation.
• The EGF/EGFR axis and its downstream signaling pathways regulate the motility and proliferation of cultured oral keratinocytes.

  Ryota Kobayashi, Emi Hoshikawa, Taisuke Saito, Orakarn Suebsamarn, Eriko Naito, Ayako Suzuki, Seiichiro Ishihara, Hisashi Haga, Kei Tomihara, Kenji Izumi

  FEBS open bio 13 (8) 1469 - 1484 2023/08 [Refereed]
   

  We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.
• Stiffness-Modulation of Collagen Gels by Genipin-Crosslinking for Cell Culture.

  Seiichiro Ishihara, Haruna Kurosawa, Hisashi Haga

  Gels (Basel, Switzerland) 9 (2) 2023/02/10 [Refereed]
   

  The stiffness of extracellular matrices (ECMs) is critical for cellular functions. Therefore, modulating the stiffness of ECMs in vitro is necessary to investigate the role of stiffness in cellular phenomena. Collagen gels are widely used for cell culture matrices in vitro. However, modulation of the stiffness in collagen gels for cell culture is challenging owing to the limited knowledge of the method to increase the stiffness while maintaining low cytotoxicity. Here, we established a novel method to modulate collagen gel stiffness from 0.0292 to 12.5 kPa with low cytotoxicity. We prepared collagens with genipin, a low-cytotoxic crosslinker of amines, at different concentrations and successfully modulated the stiffness of the gels. In addition, on 10 mM genipin-mixed collagen gels (approximately 12.5 kPa), H1299 human lung cancer cells showed spreading morphology and nuclear localization of yes-associated protein (YAP), typical phenomena of cells on stiff ECMs. Mouse mesenchymal stromal cells on 10 mM genipin-mixed collagen gels differentiated to vascular smooth muscle cells. On the other hand, the cells on 0 mM genipin-mixed collagen gels (approximately 0.0292 kPa) differentiated to visceral smooth muscle cells. Our new method provides a novel way to prepare stiffness-modulated collagen gels with low cytotoxicity in cell culture.
• Substrate stiffness induces nuclear localization of myosin regulatory light chain to suppress apoptosis.

  Katsuya Onishi, Seiichiro Ishihara, Masayuki Takahashi, Akihiro Sakai, Atsushi Enomoto, Kentaro Suzuki, Hisashi Haga

  FEBS letters 2023/02/01 [Refereed]
   

  Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P-MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P-MRLC. Zipper-interacting protein kinase [ZIPK; also known as death-associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P-MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P-MRLC via ZIPK. 2P-MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction.
• The interferon-β/STAT1 axis drives the collective invasion of skin squamous cell carcinoma with sealed intercellular spaces

  Yuji Kumagai, Junko Nio-Kobayashi, Seiichiro Ishihara, Atsushi Enomoto, Masashi Akiyama, Ryosuke Ichihara, Hisashi Haga

  Oncogenesis 11 (1) 2022/12 [Refereed]
   

  Abstract The process by which cancer cells invade as a cell cluster, known as collective invasion, is associated with metastasis and worse prognosis of cancer patients; therefore, inhibition of collective invasion is considered to improve cancer treatment. However, the cellular characteristics responsible for collective invasion remain largely unknown. Here, we successfully established subclones with various invasive potentials derived from human skin squamous carcinoma cells. The cell cluster of the highly invasive subclone had a hermetically sealed and narrow intercellular space. Interferon-β was localized to the sealed intercellular spaces, leading to collective invasion via the activation of signal transducer and activator of transcription 1 (STAT1). On the other hand, interferon-β was not localized to non-sealed and wide intercellular spaces of the cell cluster of low-invasive subclone with deficient STAT1 activity. In the mixed cell cluster of high- and low-invasive subclones, the high-invasive sub-clonal cells were located at the invasive front of the invasive protrusion, leading to collective invasion by the low-invasive sub-clonal cells. Tissue microarray analysis of human skin squamous cell carcinoma (SCC) also showed enrichment of STAT1 in the invasive front of SCCs. These findings indicate that the intercellular structure controls the potential for collective invasion via STAT1 regulation in SCC.
• Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference.

  Sumire Ishida-Ishihara, Ryota Takada, Kazuya Furusawa, Seiichiro Ishihara, Hisashi Haga

  Scientific reports 12 (1) 20269 - 20269 2022/11/24 [Refereed]
   

  Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.
• Mammaglobin 1 mediates progression of trastuzumab-resistant breast cancer cells through regulation of cyclins and NF-κB.

  Ratih Kusumastuti, Yuji Kumagai, Seiichiro Ishihara, Atsushi Enomoto, Takashi Murakami, Motoaki Yasuda, Hisashi Haga

  FEBS open bio 12 (10) 1797 - 1813 2022/07/29 [Refereed]
   

  Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.
• Vasculature atrophy causes a stiffened microenvironment that augments epidermal stem cell differentiation in aged skin

  Ryo Ichijo, Koichiro Maki, Mio Kabata, Teruasa Murata, Arata Nagasaka, Seiichiro Ishihara, Hisashi Haga, Tetsuya Honda, Taiji Adachi, Takuya Yamamoto, Fumiko Toyoshima

  Nature Aging 2 (7) 592 - 600 2022/07 [Refereed]
  
• Correction: Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics.

  Tadashi Iida, Yasuyuki Mizutani, Nobutoshi Esaki, Suzanne M Ponik, Brian M Burkel, Liang Weng, Keiko Kuwata, Atsushi Masamune, Seiichiro Ishihara, Hisashi Haga, Kunio Kataoka, Shinji Mii, Yukihiro Shiraki, Takuya Ishikawa, Eizaburo Ohno, Hiroki Kawashima, Yoshiki Hirooka, Mitsuhiro Fujishiro, Masahide Takahashi, Atsushi Enomoto

  Oncogene 41 (23) 3302 - 3302 2022/05/04
• Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics.

  Tadashi Iida, Yasuyuki Mizutani, Nobutoshi Esaki, Suzanne M Ponik, Brian M Burkel, Liang Weng, Keiko Kuwata, Atsushi Masamune, Seiichiro Ishihara, Hisashi Haga, Kunio Kataoka, Shinji Mii, Yukihiro Shiraki, Takuya Ishikawa, Eizaburo Ohno, Hiroki Kawashima, Yoshiki Hirooka, Mitsuhiro Fujishiro, Masahide Takahashi, Atsushi Enomoto

  Oncogene 41 (19) 2764 - 2777 2022/05 [Refereed]
   

  Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we showed that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, nonnatural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.
• Matrix Stiffness Contributes to Cancer Progression by Regulating Transcription Factors.

  Seiichiro Ishihara, Hisashi Haga

  Cancers 14 (4) 2022/02/18 [Refereed]
   

  Matrix stiffness is critical for the progression of various types of cancers. In solid cancers such as mammary and pancreatic cancers, tumors often contain abnormally stiff tissues, mainly caused by stiff extracellular matrices due to accumulation, contraction, and crosslinking. Stiff extracellular matrices trigger mechanotransduction, the conversion of mechanical cues such as stiffness of the matrix to biochemical signaling in the cells, and as a result determine the cellular phenotypes of cancer and stromal cells in tumors. Transcription factors are key molecules for these processes, as they respond to matrix stiffness and are crucial for cellular behaviors. The Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) is one of the most studied transcription factors that is regulated by matrix stiffness. The YAP/TAZ are activated by a stiff matrix and promotes malignant phenotypes in cancer and stromal cells, including cancer-associated fibroblasts. In addition, other transcription factors such as β-catenin and nuclear factor kappa B (NF-κB) also play key roles in mechanotransduction in cancer tissues. In this review, the mechanisms of stiffening cancer tissues are introduced, and the transcription factors regulated by matrix stiffness in cancer and stromal cells and their roles in cancer progression are shown.
• Soft surfaces promote astrocytic differentiation of mouse embryonic neural stem cells via dephosphorylation of MRLC in the absence of serum.

  Hiroshi Oyama, Akihiro Nukuda, Seiichiro Ishihara, Hisashi Haga

  Scientific reports 11 (1) 19574 - 19574 2021/10/01 [Refereed]
   

  Astrocytes, which can be obtained from neural stem cells (NSCs) by adding serum and/or recombinant proteins in culture media or by passaging NSCs repeatedly, are expected to be applicable in regenerative medicine for the treatment of neurodegenerative diseases. However, astrocytes obtained using existing methods are costly and have poor quality. The stiffness of culture surfaces has been reported to affect astrocytic differentiation of adult NSCs. However, the influence of surface stiffness on astrocytic differentiation of embryonic NSCs has not yet been reported. In this study, we showed that astrocytic differentiation of embryonic NSCs was increased on soft surfaces (1 kPa and 12 kPa) compared with the NSCs on stiff surfaces (2.8 GPa) in serum-free condition. Furthermore, di-phosphorylated myosin regulatory light chain (PP-MRLC) was decreased in embryonic NSCs cultured on the soft surfaces than the cells on the stiff surfaces. Additionally, astrocytic differentiation of embryonic NSCs was induced by a Ras homolog associated kinase (ROCK) inhibitor, which decreased PP-MRLC in NSCs. These results suggest that decreasing the PP-MRLC of embryonic NSCs on soft surfaces or treating NSCs with a ROCK inhibitor is a good method to prepare astrocytes for application in regenerative medicine.
• Osmotic gradient induces stable dome morphogenesis on extracellular matrix.

  Sumire Ishida-Ishihara, Masakazu Akiyama, Kazuya Furusawa, Isao Naguro, Hiroki Ryuno, Takamichi Sushida, Seiichiro Ishihara, Hisashi Haga

  Journal of cell science 133 (14) 2020/06/23 [Refereed][Not invited]
   

  One of the fundamental processes of morphogenesis is dome formation, but many parts of the mechanisms has been unexplored. Previous in vitro studies showed that osmotic gradient is the driving factor of the dome formation. However, these investigations were performed without extracellular matrix (ECM), which provides structural support to morphogenesis. With the use of ECM, we observed that basal hypertonic stress induced stable domes in vitro that have not been seen in previous studies. These domes developed from the ECM swelling via aquaporin water transport activity. Based on computer simulation, uneven swelling, with a positive feedback between extending cell and enhanced water transport, was a cause for dome formation. These results indicate that osmotic gradient induces dome morphogenesis via both enhanced water transport activity and subsequent ECM swelling.
• Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis.

  Mizutani Y, Kobayashi H, Iida T, Asai N, Masamune A, Hara A, Esaki N, Ushida K, Mii S, Shiraki Y, Ando K, Weng L, Ishihara S, Ponik SM, Conklin MW, Haga H, Nagasaka A, Miyata T, Matsuyama M, Kobayashi T, Fujii T, Yamada S, Yamaguchi J, Wang T, Woods SL, Worthley D, Shimamura T, Fujishiro M, Hirooka Y, Enomoto A, Takahashi M

  Cancer research 79 (20) 5367 - 5381 0008-5472 2019/10 [Refereed][Not invited]
   

  Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.
• Modulation and Characterization of the Double Network Hydrogel Surface-Bulk Transition

  Frauenlob Martin, King Daniel R, Guo Honglei, Ishihara Seiichiro, Tsuda Masumi, Kurokawa Takayuki, Haga Hisashi, Tanaka Shinya, Gong Jian Ping

  MACROMOLECULES 52 (17) 6704 - 6713 0024-9297 2019/09/10 [Refereed][Not invited]
  
• The intercellular expression of type-XVII collagen, laminin-332, and integrin-β1 promote contact following during the collective invasion of a cancer cell population.

  Kumagai Y, Nio-Kobayashi J, Ishida-Ishihara S, Tachibana H, Omori R, Enomoto A, Ishihara S, Haga H

  Biochemical and biophysical research communications 514 (4) 1115 - 1121 0006-291X 2019/07 [Refereed][Not invited]
   

  Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-β1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-β1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-β1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-β1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.
• Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts.

  Yamamoto K, Otomo K, Nemoto T, Ishihara S, Haga H, Nagasaki A, Murakami Y, Takahashi M

  Experimental cell research 376 (1) 67 - 76 0014-4827 2019/03 [Refereed][Not invited]
   

  Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.
• Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

  Ishihara S, Aoki K, Mizutani T, Amano M, Nishimura SI, Haga H

  Cell structure and function 43 (2) 177 - 185 0386-7196 2018 [Refereed][Not invited]
  
• Mechano-Signal Transduction in Mesenchymal Stem Cells Induces Prosaposin Secretion to Drive the Proliferation of Breast Cancer Cells

  Seiichiro Ishihara, David R. Inman, Wan-Ju Li, Suzanne M. Ponik, Patricia J. Keely

  Cancer research 77 (22) 6179 - 6189 0008-5472 2017/11 [Refereed][Not invited]
   

  In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of alpha-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. (C) 2017 AACR.
• Mesenchymal stem cells in breast cancer: Response to chemical and mechanical stimuli

  Seiichiro Ishihara, Suzanne M. Ponik, Hisashi Haga

  Oncoscience 4 (11-12) 158 - 159 2331-4737 2017 [Not refereed][Invited]
  
• An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

  Seiichiro Ishihara, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  Cytotechnology 68 (1) 25 - 32 0920-9069 2016/01 [Refereed][Not invited]
   

  Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-kappa B family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.
• Stiff substrates increase YAP-signaling-mediated matrix metalloproteinase-7 expression

  A. Nukuda, C. Sasaki, S. Ishihara, T. Mizutani, K. Nakamura, T. Ayabe, K. Kawabata, H. Haga

  Oncogenesis 4 e165  2157-9024 2015/09 [Refereed][Not invited]
   

  Abnormally stiff substrates have been shown to trigger cancer progression. However, the detailed molecular mechanisms underlying this trigger are not clear. In this study, we cultured T84 human colorectal cancer cells on plastic dishes to create a stiff substrate or on collagen-I gel to create a soft substrate. The stiff substrate enhanced the expression of matrix metalloproteinase-7 (MMP-7), an indicator of poor prognosis. In addition, we used polyacrylamide gels (2, 67 and 126 kPa) so that the MMP-7 expression on the 126-kPa gel was higher compared with that on the 2-kPa gel. Next, we investigated whether yes-associated protein (YAP) affected the MMP-7 expression. YAP knockdown decreased MMP-7 expression. Treatment with inhibitors of epidermal growth factor receptor (EGFR) and myosin regulatory light chain (MRLC) and integrin-alpha 2 or integrin-beta 1 knockdown downregulated MMP-7 expression. Finally, we demonstrated that YAP, EGFR, integrin-alpha 2 beta 1 and MRLC produced a positive feedback loop that enhanced MMP-7 expression. These findings suggest that stiff substrates enhanced colorectal cancer cell viability by upregulating MMP-7 expression through a positive feedback loop.
• ATF5: development of oncogenic resistance to radiotherapy

  Seiichiro Ishihara, Hisashi Haga

  Aging-US 7 (7) 453 - 454 1945-4589 2015/07 [Not refereed][Invited]
  
• Activating transcription factor 5 enhances radioresistance and malignancy in cancer cells

  Seiichiro Ishihara, Motoaki Yasuda, Akihiro Ishizu, Masayori Ishikawa, Hiroki Shirato, Hisashi Haga

  Oncotarget 6 (7) 4602 - 4614 1949-2553 2015/03 [Refereed][Not invited]
   

  Radiotherapy is effective for treating various types of tumors. However, some cancer cells survive after irradiation and repopulate tumors with highly malignant phenotypes that correlate with poor prognosis. It is not known how cancer cells survive and generate malignant tumors after irradiation. Here, we show that activating transcription factor 5 (ATF5) promotes radioresistance and malignancy in cancer cells after irradiation. In the G1-S phase of the cell cycle, cancer cells express high levels of ATF5, which promotes cell cycle progression and thereby increases radioresistance. Furthermore, ATF5 increases malignant phenotypes, such as cell growth and invasiveness, in cancer cells in vitro and in vivo. We have identified a new mechanism for the regeneration of highly malignant tumors after irradiation and shown that ATF5 plays a key role in the process.
• Compressive Stress Induces Dephosphorylation of the Myosin Regulatory Light Chain via RhoA Phosphorylation by the Adenylyl Cyclase/Protein Kinase A Signaling Pathway

  Kenji Takemoto, Seiichiro Ishihara, Takeomi Mizutai, Kazushige Kawabata, Hisashi Haga

  PLOS ONE 10 (3) e0117937  1932-6203 2015/03 [Refereed][Not invited]
   

  Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression- induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.
• Filamin B Enhances the Invasiveness of Cancer Cells into 3D Collagen Matrices

  Yuta Iguchi, Seiichiro Ishihara, Yoshimi Uchida, Kaori Tajima, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  Cell structure and function 40 (2) 61 - 67 0386-7196 2015 [Refereed][Not invited]
   

  Numerous types of cancer cells migrate into extracellular tissues. This phenomenon is termed invasion, and is associated with poor prognosis in cancer patients. In this study, we demonstrated that filamin B (FLNb), an actin-binding protein, is highly expressed in cancer cell lines that exhibit high invasiveness, with a spindle morphology, into 3D collagen matrices. In addition, we determined that knockdown of FLNb in invasive cancer cells converts cell morphology from spindle-shaped, which is associated with high invasiveness, to round-shaped with low invasiveness. Furthermore, di-phosphorylation of myosin regulatory light chain (MRLC) and phosphorylation of focal adhesion kinase (FAK) are inhibited in FLNb-knockdown cancer cells. These results suggest that FLNb enhances invasion of cancer cells through phosphorylation of MRLC and FAK. Therefore, FLNb may be a new therapeutic target for invasive cancers.
• Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture

  Hiro-taka Masuda, Seiichiro Ishihara, Ichiro Harada, Takeomi Mizutani, Masayori Ishikawa, Kazushige Kawabata, Hisashi Haga

  Biotechniques 56 (4) 172 - 179 0736-6205 2014/04 [Refereed][Not invited]
   

  We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.
• Substrate stiffness regulates temporary NF-kappa B activation via actomyosin contractions

  Seiichiro Ishihara, Motoaki Yasuda, Ichiro Harada, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  EXPERIMENTAL CELL RESEARCH 19 319 (19) 2916 - 2927 0014-4827 2013/11 [Refereed][Not invited]
   

  Physical properties of the extracellular matrix (ECM) can control cellular phenotypes via mechanotransduction, which is the process of translation of mechanical stresses into biochemical signals. While current research is clarifying the relationship between mechanotransduction and cytoskeleton or adhesion complexes, the contribution of transcription factors to mechanotransduction is not well understood. The results of this study revealed that the transcription factor NF-kappa B, a major regulator for immunoreaction and cancer progression, is responsive to substrate stiffness. NF-kappa B activation was temporarily induced in H1299 lung adenocarcinoma cells grown on a stiff substrate but not in cells grown on a soft substrate. Although the activation of NF-kappa B was independent of the activity of integrin beta 1, an ECM-binding protein, the activation was dependent on actomyosin contractions induced by phosphorylation of myosin regulatory light chain (MRLC). Additionally, the inhibition of MRLC phosphorylation by Rho kinase inhibitor Y27632 reduced the activity of NF-kappa B. We also observed substrate-specific morphology of the cells, with cells grown on the soft substrate appearing more rounded and cells grown on the stiff substrate appearing more spread out. Inhibiting NF-kappa B activation caused a reversal of these morphologies on both substrates. These results suggest that substrate stiffness regulates NF-kappa B activity via actomyosin contractions, resulting in morphological changes. (C) 2013 Elsevier Inc. All rights reserved.
• Lung Cancer Cells That Survive Ionizing Radiation Show Increased Integrin alpha 2 beta 1-and EGFR-Dependent Invasiveness

  Xue Li, Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Masayori Ishikawa, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga

  PLOS ONE 8 8 (8) e70905  1932-6203 2013/08 [Refereed][Not invited]
   

  Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as a contributor to the limited benefit of radiotherapy; however, its mechanism is still unclear. We previously showed that subcloned lung adenocarcinoma A549 cells (P cells), which survived 10 Gy IR (IR cells), acquired high invasiveness in vitro. Here, we tried to identify the mechanism by which IR cells increase their invasiveness by examining altered gene expression and signaling pathways in IR cells compared with those in P cells. To simulate the microenvironment in vivo, cells were embedded in a three-dimensional (3D) collagen type I gel, in which the IR cells were elongated, while the P cells were spherical. The integrin expression pattern was surveyed, and expression levels of the integrin alpha 2 and beta 1 subunits were significantly elevated in IR cells. Knockdown of alpha 2 expression or functional blockade of integrin alpha 2 beta 1 resulted in a round morphology of IR cells, and abrogated their invasion in the collagen matrix, suggesting the molecule's essential role in cell spread and invasion in 3D collagen. Epidermal growth factor receptor (EGFR) also presented enhanced expression and activation in IR cells. Treatment with EGFR tyrosine kinase inhibitor, PD168393, decreased the ratio of elongated cells and cell invasiveness. Signaling molecules, including extracellular signal-regulated kinase-1/2 (Erk1/2) and Akt, exhibited higher activation in IR cells. Inhibition of Akt activation by treating with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 decreased IR cell invasion, whereas inhibition of Erk1/2 activation by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 did not. Our results show that integrin alpha 2 beta 1 and EGFR cooperatively promote higher invasiveness of IR-survived lung cancer cells, mediated in part by the PI3K/Akt signaling pathway, and might serve as alternative targets in combination with radiotherapy.
• Irradiation-tolerant lung cancer cells acquire invasive ability dependent on dephosphorylation of the myosin regulatory light chain

  Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga

  FEBS letters 587 (6) 732 - 736 0014-5793 2013/03 [Refereed][Not invited]
   

  Radiotherapy is one of the major treatment modalities for malignancies. However, cells surviving irradiation often display high levels of invasiveness. This study shows that irradiation-tolerant lung adenocarcinoma demonstrates high invasive capability depending on dephosphorylation of the myosin regulatory light chain (MRLC). In a collagen gel overlay condition, low-invasive subclones of lung adenocarcinoma (A549P-3) showed a round morphology and diphosphorylation of MRLC. In contrast, irradiation-tolerant A549P-3 cells (A549P-3IR) displayed high invasiveness and a lower level of MRLC diphosphorylation. In addition, inhibition of MRLC phosphatase activity decreased the invasive activity. These findings suggest that A549P-3IR cells acquire high invasiveness through MRLC dephosphorylation. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
• Integrin beta 1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

  Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Takeshi Nishioka

  Biochemical and biophysical research communications 396 (3) 651 - 655 0006-291X 2010/06 [Refereed][Not invited]
   

  Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta 1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta 1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta 1-dependent phenotype, and integrin beta 1 might be a potentially effective therapeutic target in combination with radiotherapy. (C) 2010 Elsevier Inc. All rights reserved.
• Increased Motility and Invasiveness in Tumor Cells That Survive 10 Gy Irradiation

  Kaori Tsutsumi, Masumi Tsuda, Natsuka Yazawa, Hirotaka Nakamura, Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Rie Yamazaki, Hiroki Shirato, Hideaki Kawaguchi, Takeshi Nishioka, Yusuke Ohba

  Cell structure and function 34 (2) 89 - 96 0386-7196 2009 [Refereed][Not invited]
   

  Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta 1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.

MISC

• 組織の「硬さ」から迫る線維症研究の新展開

  石原 誠一郎, 芳賀 永  実験医学増刊  38-  (12)  185  -190  2020/07  [Not refereed][Invited]
  
• 第2節 ハイドロゲル法

  石原 誠一郎, 芳賀 永  三次元培養における培養手法と周辺技術動向  23  -30  2019/04  [Not refereed][Invited]
  
• 硬い地面を掘り進む！がん硬化と集団浸潤

  石原 誠一郎  実験医学  36-  (19)  3270  -3270  2018/12  [Not refereed][Invited]
  
• Activating transcription factor 5 promotes cancer cell invasion through transcriptional activation of integrins

  A. Nukuda, H. Endoh, S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  26-  2015  [Not refereed][Not invited]
  
• Lung cancer cells after irradiation indicate viability and malignancy dependent on activating transcription factor 5.

  S. Ishihara, M. Yasuda, A. Ishizu, H. Haga  MOLECULAR BIOLOGY OF THE CELL  25-  2014/12  [Not refereed][Not invited]
  
• EGFR and Integrin alpha 2 beta 1 Dependent Invasiveness of Lung Adenocarcinoma Cells that Survived 10 Gy Ionizing Radiation.

  X. Li, S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, T. Nishioka, H. Haga  MOLECULAR BIOLOGY OF THE CELL  23-  2012  [Not refereed][Not invited]
  
• Substrate stiffness regulates NF-kB expression and activity in cancer cells

  S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  22-  2011  [Not refereed][Not invited]
  
• Increased Invasiveness and Non-spheroid Morphology Change of Human Lung Adenocarcinoma Cells That Survived 10 Gy Irradiation

  T. Nishioka, S. Ishihara, Y. Miyai, T. Mizutani, K. Kawabata, H. Shirato, R. Yamazaki, M. Yasuda, H. Haga, K. Kawabata  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  75-  (3)  S540  -S540  2009  [Not refereed][Not invited]
  

Association Memberships

• 米国癌学会   米国細胞生物学会（ASCB）   JAPAN SOCIETY FOR CELL BIOLOGY   THE JAPANESE CANCER ASSOCIATION   

Research Projects

• 硬さ応答性の転写因子ATF5によるがん細胞の悪性化メカニズム

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2027/03 

  Author : 石原 誠一郎
• Mechanobiology of metastasis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/09 -2027/03 

  Author : 石原 誠一郎, 石井 琢郎, 石橋 公二朗, 丹下 正一朗
• 細胞外基質の硬さ変化を指標とした多段階発がんの再定義

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2026/03 

  Author : 石原 誠一郎
• 脳転移におけるメカノレスポンス機構の解明

  金沢大学 がん進展制御研究所 共同研究

  Date (from‐to) : 2024/04 -2025/03
• 生体組織を模倣した新規の三次元細胞培養システム

  公益財団法人JKA：機械振興補助事業

  Date (from‐to) : 2023/04 -2024/03 

  Author : 石原 誠一郎
• 圧縮ストレスがトリガーとなるがん細胞の浸潤能獲得機構

  日本学術振興会：科学研究費補助金 基盤研究（C）

  Date (from‐to) : 2021/04 -2024/03 

  Author : 石原 誠一郎
• メカノバイオロジーから迫るがん転移機構

  金沢大学 がん進展制御研究所：共同研究

  Date (from‐to) : 2020/04 -2024/03 

  Author : 石原 誠一郎, 平田 英周
• 組織の硬さから迫るがんが転移しやすい臓器の共通性

  秋山記念生命科学振興財団：研究助成（奨励）

  Date (from‐to) : 2022/07 -2023/03 

  Author : 石原 誠一郎
• 足場材の硬さの違いを利用した上皮角化・非角化様式解明と培養口腔粘膜作成法への応用

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 泉 健次, 芳賀 永, 石原 誠一郎, 加来 賢, 佐藤 大祐, 鈴木 絢子

   

  今年度は、コロナの影響で魚うろこコラーゲンの入手がままならず、既製品の魚うろこコラーゲンであるセルキャンパスしか手に入らなかったため、コラーゲンゲルの足場材の硬さをランダムに変えての培養口腔粘膜作成はできなかった。その分、セルキャンパスを用いて、コントロールの硬い培養皿面、同表面でカルシウム濃度をあげた分化培地、および、いわゆるコラーゲンゲルの軟性面で培養したケラチノサイトに対し、運動能解析と外注によるマイクロアレイ解析による網羅的遺伝子発現分析（現在までに2サンプル解析終了し、現在もう2サンプル培養しており、外注予定）を実施し、結果がでたら新たに分担者に加わっていただいた凌先生にヒートマップとクラスター図を作成してもらい、ビッグデータ解析をお願いする。 細胞運動能解析では驚いたことに、コラーゲンゲル上の細胞運動能が、硬い培養皿面での細胞運動能より上回っていた。これは、いわゆる癌細胞のメカノバイオロジーと真逆の現象である。さらに、運動能が高いにも関わらず、ケラチノサイトの分化マーカー遺伝子発現が更新していることが示唆され、申請者の以前の基盤Ｂ研究で報告した結果とも相反するデータを得ており、今後考察を加え、メカニズム解明したい。また、コロナの影響で、研究分担者のいる北海道大学にＡＦＭを用いた細胞の硬さ検索ができなかった。
• Development of novel 3D cell culure system for the model of in vivo tissue

  Japan Science and Technology Agency：研究成果展開事業 A-STEPトライアウトタイプ

  Date (from‐to) : 2021/05 -2022/03 

  Author : 石原 誠一郎
• Structural analysis of tumor hotspots, an epithelial tissue-intrinsic novel oncogenic niche

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

  Date (from‐to) : 2018/10 -2022/03 

  Author : Tamori Yoichiro

   

  In the experimental studies using Drosophila wing imaginal epithelia as a model, we found that oncogenic mutant cells break the basement membrane and begin their invasive behavior into the stroma in microenvironments (invasion hotspots) scattered within the epithelial tissue. Analysis using the TCPD program (a program developed in this study to predict structural singularities in tissues) based on fluid analysis revealed that the locations detected as tissue confluence points (saddle points and sink points) in the tissues coincided with these invasion hotspots. These data indicate a novel idea that cancer cell invasion into the stroma starts from structural singularities intrinsic to epithelial tissues. Our experiments using a mouse model also showed that a similar phenomenon might be able to occur in mammalian tissues as well.
• 臓器の硬さからアプローチするがん転移機構

  安田記念医学財団：若手癌研究助成

  Date (from‐to) : 2021/01 -2021/12 

  Author : 石原 誠一郎
• 圧縮ストレスに引き起こされるがん細胞の浸潤能獲得

  公益財団法人 上原記念生命科学財団：研究奨励金

  Date (from‐to) : 2020/03 -2021/04 

  Author : 石原 誠一郎
• Targeted Nanotherapy for Rescuing Endothelial Dysfunction under Biomimetic Conditions of Atherosclerosis

  NYAS/AMED：Interstellar Initiative

  Date (from‐to) : 2020/10 -2021/03 

  Author : 石原 誠一郎, Hagar Labouta, Simona Giunta
• 力学刺激に応答した膵臓がん細胞における遺伝子発現変動パターンの網羅解析

  北海道大学：次世代研究者リーダー育成共同研究助成

  Date (from‐to) : 2020/09 -2021/03 

  Author : 石原 誠一郎, 丹下 正一朗
• 圧縮ストレスが誘導するがん細胞の浸潤能獲得機構

  公益財団法人 アステラス病態代謝研究会：研究助成金

  Date (from‐to) : 2019/12 -2020/03 

  Author : 石原 誠一郎
• 腫瘍組織におけるがん細胞および間質細胞の硬さ応答機構を解明する

  北海道大学：リーダー育成プログラム支援

  Date (from‐to) : 2019/09 -2020/03 

  Author : 石原 誠一郎, 榎本 篤
• 圧縮ストレスがもたらすMMP依存的ながん細胞の浸潤メカニズム

  公益財団法人 北海道科学技術総合振興センター：札幌ライフサイエンス産業活性化事業 研究シーズ発掘補助金（札幌タレント補助金）

  Date (from‐to) : 2019/07 -2020/03 

  Author : 石原 誠一郎
• Cancer metastasis regulated by tissue stiffness

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists

  Date (from‐to) : 2018/04 -2020/03 

  Author : Ishihara Seiichiro

   

  In this study, we investigated whether tissue stiffness is critical for metastasis of cancer cells. First, we prepared sheet of blood vessel endothelial cells on substrates with different stiffness. We also prepared collagen gels of different stiffness by treating with genipin, a cross-linker of collagen. We successfully made collagen gels with 0.02 to 10 kPa stiffness. Next, we performed live imaging to observe metastasis in vitro cell culture model. We observed the penetration of cancer cells in a sheet of blood vessel cells by using confocal laser scanning microscopy. We also found three genes up-regulated in blood vessel cells on stiff substrates.
• 3D morphogenesis of epithelial cell sheets in vitro

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2015/06 -2020/03 

  Author : Haga Hisashi

   

  Three-dimensional morphogenesis of epithelial cells plays pivotal roles in many biological events as it is observed in embryogenesis. Mechanical properties of the extracellular substrate such as viscoelasticity and geometrical constraints are known as crucial factors that affect 3D morphogenesis of epithelial sheets. In this study, we found that epithelial cells (MDCK cells) cultured on a Matrigel crosslinked by Genipin exhibit dome formation by both the swelling of extracellular matrix and aquaporin water transport triggered by the basal hypertonic stress. Computer simulation was conducted and revealed that uneven swelling with a positive feedback between extending cell and enhanced water transport induces the dome formation. Moreover, we succeeded to establish a novel spherical substrate for cell culture by using a gelatin gel capsule. Human dermal fibroblasts cultured on the gel capsule made wrinkle-like structures on the spherical substrate, mimicking the early embryogenesis.
• 硬さ依存的に活性化する新規がん促進性転写因子の同定と機能解析

  公益財団法人 SGH財団：第30回SGHがん研究助成

  Date (from‐to) : 2018/12 -2019/11 

  Author : 石原 誠一郎
• 組織の硬さに応答した間葉系幹細胞が引き起こすがん悪性化

  日本学術振興会：海外特別研究員

  Date (from‐to) : 2018/04 -2019/09 

  Author : 石原 誠一郎
• 間葉系幹細胞のメカノレスポンスにより引き起こされるがん悪性化機構

  公益財団法人 東洋紡バイオテクノロジー研究財団：長期研究助成

  Date (from‐to) : 2017/04 -2018/03 

  Author : 石原 誠一郎
• 放射線治療後に肺がんが再発し悪性化する機構の解明

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2014/04 -2015/03 

  Author : 石原 誠一郎

   

  本研究は当初の計画よりも順調に進められた。本研究では、放射線治療後に肺がんが悪性化する機構の解明を目指した。特に、転写因子Activating transcription factor 5（ATF5）に着目し、この分子が肺がん細胞の放射線耐性・悪性度亢進に寄与するかどうかを調べた。そして主に以下の点を明らかにした。（１）ATF5は肺がん細胞の放射線耐性を増強させる。（２）肺がん細胞におけるATF5の発現および放射線耐性は、細胞周期のG1後期～S期で上昇する。（３）放射線照射後に生き残った肺がん細胞は、ATF5を高発現させる。（４）ATF5は、肺がん細胞の増殖能を亢進させる。（５）ATF5は、肺がん細胞の浸潤能を増強させる。（６）ATF5は、細胞‐基質間接着および細胞内張力を介して肺がん細胞の浸潤能を制御する。上記の通り、ATF5は肺がん細胞の放射線耐性および悪性度を上昇させることが明らかになった。これらの結果は、ATF5の阻害と放射線治療の併用が、肺がん治療に有効であることを示唆するものである。 我々は本研究の成果を学術論文として発表した（Ishihara et al., Oncotarget, 6, 4602-4614）。この論文はOncotarget誌のPriority Research Paperとして大きく紹介された。さらに、本研究の成果は4回の国際・国内学会等で発表された。特に、「平成26年度がん若手研究者ワークショップ」（2014年9月3日～9月6日、長野・蓼科温泉）では、「優秀演者賞」を受賞した。また、「がん研究分野の特性等を踏まえた支援活動公開シンポジウム」においても発表した。 以上の通り、本研究を順調に進めることができ、その成果を発表することができた。
• がん細胞におけるメカノセンシング機構の解明

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows

  Date (from‐to) : 2011 -2012 

  Author : 石原 誠一郎

   

  細胞は基質の硬さを感知し、それに応答して表現型を変化させることが知られている(メカノセンス)。しかし、メカノセンスの詳細なメカニズムは未だ不明である。メカノセンスでは、細胞骨格や細胞接着にかかわる分子が重要な働きをしていることがこれまでの研究で明らかになっている。しかし、基質の硬さと転写因子の関与についてはこれまでほとんど報告されていない。そこで、本研究では炎症反応に深く関与する転写因子NF-kBに着目し、基質の硬さがNF-kBに与える影響を調べた。今年度は、ヒト肺がん細胞H1299において、基質の硬さが転写因子NF-kBにどのような影響を与えるかを調べた。本研究により、(1)硬い基質上のH1299細胞は軟らかい基質上の細胞に比べて高いNF-kBの活性を示すこと、(2)NF-kBの活性化は、H1299細胞において炎症反応を引き起こすこと、(3)硬い基質はミオシン調節軽鎖のリン酸化を亢進させ、それがNF-kBの活性化を誘導すること、(4)転写因子NF-kBの活性化により、H1299細胞は浸潤性の形態を示すことが明らかになった。本研究結果により、硬い基質はがん細胞においてNF-kBの活性化とそれによる炎症反応を引き起こし、ひいてはがんの悪性化をもたらすことが示唆された。本結果をもとに、1回の国際セミナー(2013年1月、シンガポール)と1回の国内学会(2012年9月、札幌)で講演を行った。現在は本研究結果をまとめて、原著論文として投稿準備中である。
• 科学の芽を育む実験教室

  北海道大学：北大元気プロジェクト

  Date (from‐to) : 2006 -2007