リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

リケラボ 全国の大学リポジトリにある学位論文・教授論文を一括検索するならリケラボ論文検索大学・研究所にある論文を検索できる

大学・研究所にある論文を検索できる 「Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

コピーが完了しました

URLをコピーしました

論文の公開元へ論文の公開元へ
書き出し

Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer

Hanada, Keita 京都大学 DOI:10.14989/doctor.k23608

2022.01.24

概要

腫瘍細胞は正常細胞と比較して栄養源としてグルタミンに強く依存しており、特に KRAS 遺伝子変異とグルタミン代謝との関係が近年注目されている。京都大学消化管外科研究室では、これまで KRAS 変異型大腸癌においてグルタミン欠乏に対する耐性機構としてアスパラギン合成酵素(ASNS)による細胞内のアスパラギン合成が重要なこと、ASNS 阻害と L-Asparaginase(L-Asp)の併用によるアスパラギン枯渇はマウス皮下腫瘍モデルで腫瘍増殖を抑制したことを報告した。一方、KRAS 遺伝子変異を有する膵癌では栄養飢餓条件下において Macropinocytosis を亢進させ細胞外のアルブミンを細胞内に取り込むことで細胞増殖を維持する機構が報告されている。本研究では大腸癌における KRAS 遺伝子変異と Macropinocytosis との関連性について検討するとともに、アスパラギン代謝との関連性についても合わせて検討した。

Macropinocytosis の評価法として蛍光デキストランの細胞内への取り込みを Macropinocytosis index(MI)として定量解析したところ、KRAS 変異を有するヒト大腸癌細胞株HCT116 およびDLD-1(KRAS-MT 株)では、その変異アレルを相同組み換え法により欠失させたKRAS 野生型細胞株(KRAS-WT 株)に比べMI は有意に亢進していた。また1%FBS やグルタミン欠乏などの飢餓培養条件下で抑制された細胞増殖能は、KRAS-MT 株では Macropinocytosis を介したアルブミンの細胞内取り込みにより有意に回復したが、KRAS-WT 株ではそのような結果は認めなかった。FRET biosensorを用いた time-lapse imaging により、細胞膜における Rac1 と PIP3 の活性化が KRAS-MT 株の Macropinocytosis に必須なことが分かった。さらに HCT116 のマウス皮下腫瘍モデルでは、Macropinocytosis 阻害剤(EIPA)投与群はコントロール群に比べ腫瘍増殖が有意に抑制された。

次に、KRAS 変異型大腸癌における Macropinocytosis とアスパラギン代謝の関連について検討した。HCT116 の ASNS ノックダウン株ではグルタミン欠乏条件下でコントロール株と比べてMI は有意に亢進した。またグルタミン欠乏条件下で抑制された ASNS ノックダウン株の細胞増殖能は、Macropinocytosis を介したアルブミンの細胞内取り込みによりアスパラギン濃度を上昇させることで有意に回復した。ASNS ノックアウト株(HCT116 およびDLD-1)のマウス皮下腫瘍モデルでは、コントロール群に比べ EIPA 単独投与群またはL-Asp 単独投与群では有意に腫瘍増殖が抑制された。またEIPAとL-Asp の 2 剤投与群では、それぞれの単独投与群よりもさらに有意に腫瘍増殖は抑制され、殆ど増大しなくなった。重要なことに、ASNS がノックアウトされていないempty vector を導入したコントロール株(HCT116 およびDLD-1)のマウス皮下腫瘍モデルではそのような結果は認められなかった。

本研究により KRAS 変異型大腸癌ではグルタミン欠乏下で ASNS を阻害すると Macropinocytosis が誘導され、細胞内アスパラギンの供給源となっていることが明らかとなった。さらに Macropinocytosis 阻害とアスパラギン枯渇の併用療法は著明な腫瘍増殖抑制効果を認めたことから KRAS 変異型大腸癌に対する新規治療になりうることが期待される。

参考文献

[1] C.S. Karapetis, S. Khambata-Ford, D.J. Jonker, C.J. O’Callaghan, D. Tu, N. C. Tebbutt, et al., K-ras mutations and benefit from cetuximab in advanced colorectal cancer, N. Engl. J. Med. 359 (2008) 1757–1765.

[2] L.C. Ye, T.S. Liu, L. Ren, Y. Wei, D.X. Zhu, S.Y. Zai, et al., Randomized controlled trial of cetuximab plus chemotherapy for patients with KRAS wild-type unresectable colorectal liver-limited metastases, J. Clin. Oncol. 31 (2013) 1931–1938.

[3] J. Canon, K. Rex, A.Y. Saiki, C. Mohr, K. Cooke, D. Bagal, et al., The clinical KRAS (G12C) inhibitor AMG 510 drives anti-tumour immunity, Nature 575 (2019) 217–223.

[4] J. Guinney, R. Dienstmann, X. Wang, A. de Reyni`es, A. Schlicker, C. Soneson, et al., The consensus molecular subtypes of colorectal cancer, Nat. Med. 21 (2015) 1350–1356.

[5] E. White, Exploiting the bad eating habits of Ras-driven cancers, Genes Dev. 27 (2013) 2065–2071.

[6] A.C. Kimmelman, Metabolic dependencies in RAS-driven cancers, Clin. Canc. Res. 21 (2015) 1828–1834.

[7] J. Son, C.A. Lyssiotis, H. Ying, X. Wang, S. Hua, M. Ligorio, et al., Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway, Nature 496 (2013) 101–105.

[8] K. Kawada, K. Toda, Y. Sakai, Targeting metabolic reprogramming in KRAS-driven cancers, Int. J. Clin. Oncol. 22 (2017) 651–659.

[9] D.R. Wise, C.B. Thompson, Glutamine addiction: a new therapeutic target in cancer, Trends Biochem. Sci. 35 (2010) 427–433.

[10] Y.D. Bhutia, E. Babu, S. Ramachandran, V. Ganapathy, Amino Acid transporters in cancer and their relevance to "glutamine addiction": novel targets for the design of a new class of anticancer drugs, Canc. Res. 75 (2015) 1782–1788.

[11] E. Bernfeld, D.A. Foster, Glutamine as an essential amino acid for KRas-driven cancer cells, Trends Endocrinol. Metabol. 30 (2019) 357–368.

[12] K. Toda, K. Kawada, M. Iwamoto, S. Inamoto, T. Sasazuki, S. Shirasawa, et al., Metabolic alterations caused by KRAS mutations in colorectal cancer contribute to cell adaptation to glutamine depletion by upregulation of asparagine synthetase, Neoplasia 18 (2016) 654–665.

[13] H. Li, S. Ning, M. Ghandi, G.V. Kryukov, S. Gopal, A. Deik, et al., The landscape of cancer cell line metabolism, Nat. Med. 25 (2019) 850–860.

[14] M.V. Recouvreux, C. Commisso, Macropinocytosis: a metabolic adaptation to nutrient stress in cancer, Front. Endocrinol. 8 (2017) 261.

[15] D. Bar-Sagi, J.R. Feramisco, Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins, Science 233 (1986) 1061–1068.

[16] A. Veithen, P. Cupers, P. Baudhuin, P.J. Courtoy, v-Src induces constitutive macropinocytosis in rat fibroblasts, J. Cell Sci. 109 (1996) 2005–2012.

[17] W. Palm, J. Araki, B. King, R.G. DeMatteo, C.B. Thompson, Critical role for PI3- kinase in regulating the use of proteins as an amino acid source, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E8628–E8636.

[18] W. Palm, Y. Park, K. Wright, N.N. Pavlova, D.A. Tuveson, C.B. Thompson, The utilization of extracellular proteins as nutrients is suppressed by mTORC1, Cell 162 (2015) 259–270.

[19] S.M. Kim, T.T. Nguyen, A. Ravi, P. Kubiniok, B.T. Finicle, V. Jayashankar, et al., PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells, Canc. Discov. 8 (2018) 866–883.

[20] C. Commisso, S.M. Davidson, R.G. Soydaner-Azeloglu, S.J. Parker, J.J. Kamphorst, S. Hackett, et al., Macropinocytosis of protein is an amino acid supply route in Ras- transformed cells, Nature 497 (2013) 633–637.

[21] J.J. Kamphorst, M. Nofal, C. Commisso, S.R. Hackett, W. Lu, E. Grabocka, et al., Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein, Canc. Res. 75 (2015) 544–553.

[22] S.M. Davidson, O. Jonas, M.A. Keibler, H.W. Hou, A. Luengo, J.R. Mayers, et al., Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors, Nat. Med. 23 (2017) 235–241.

[23] L. Seguin, M.F. Camargo, H.I. Wettersten, S. Kato, J.S. Desgrosellier, T. von Schalscha, et al., Galectin-3, a druggable vulnerability for KRAS-addicted cancers, Canc. Discov. 7 (2017) 1464–1479.

[24] H. Tajiri, T. Uruno, T. Shirai, D. Takaya, S. Matsunaga, D. Setoyama, et al., Targeting ras-driven cancer cell survival and invasion through selective inhibition of DOCK1, Cell Rep. 19 (2017) 969–980.

[25] S. Shirasawa, M. Furuse, N. Yokoyama, T. Sasazuki, Altered growth of human colon cancer cell lines disrupted at activated Ki-ras, Science 260 (1993) 85–88.

[26] I. Baba, S. Shirasawa, R. Iwamoto, K. Okumura, T. Tsunoda, M. Nishioka, et al., Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells, Canc. Res. 60 (2000) 6886–6889.

[27] K. Aoki, M. Matsuda, Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors, Nat. Protoc. 4 (2009) 1623–1631.

[28] R. Mizuno, Y. Kamioka, K. Kabashima, M. Imajo, K. Sumiyama, E. Nakasho, et al., In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines, J. Exp. Med. 211 (2014) 1123–1136.

[29] H. Maekawa, H. Miyoshi, T. Yamaura, Y. Itatani, K. Kawada, Y. Sakai, et al., A chemosensitivity study of colorectal cancer using xenografts of patient-derived tumor-initiating cells, Mol. Canc. Therapeut. 17 (2018) 2187–2196.

[30] T. Yamamoto, H. Miyoshi, F. Kakizaki, H. Maekawa, T. Yamaura, T. Morimoto, et al., Chemosensitivity of patient-derived cancer stem cells identifies colorectal cancer patients with potential benefit from FGFR inhibitor therapy, Cancers (Basel) 12 (2020) 2010.

[31] C. Commisso, R.J. Flinn, D. Bar-Sagi, Determining the macropinocytic index of cells through a quantitative image-based assay, Nat. Protoc. 9 (2014) 182–192.

[32] A.I. Ivanov, Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol. Biol. 440 (2008) 15–33.

[33] M. Koivusalo, C. Welch, H. Hayashi, C.C. Scott, M. Kim, T. Alexander, et al., Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling, J. Cell Biol. 188 (2010) 547–563.

[34] H. Miyoshi, T.S. Stappenbeck, In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture, Nat. Protoc. 8 (2013) 2471–2482.

[35] H. Miyoshi, H. Maekawa, F. Kakizaki, T. Yamaura, K. Kawada, Y. Sakai, et al., An improved method for culturing patient-derived colorectal cancer spheroids, Oncotarget 9 (2018) 21950–21964.

[36] T. Yamaura, H. Miyoshi, H. Maekawa, T. Morimoto, T. Yamamoto, F. Kakizaki, et al., Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells, Oncotarget 9 (2018) 37534–37548.

[37] A.J. Ridley, H.F. Paterson, C.L. Johnston, D. Diekmann, A. Hall, The small GTP- binding protein rac regulates growth factor-induced membrane ruffling, Cell 70 (1992) 401–410.

[38] W.K. Chan, P.L. Lorenzi, A. Anishkin, P. Purwaha, D.M. Rogers, S. Sukharev, et al., The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells, Blood 123 (2014) 3596–3606.

[39] U.K. Narta, S.S. Kanwar, W. Azmi, Pharmacological and clinical evaluation of L- asparaginase in the treatment of leukemia, Crit. Rev. Oncol. Hematol. 61 (2007) 208–221.

[40] G. Redelman-Sidi, A. Binyamin, I. Gaeta, W. Palm, C.B. Thompson, P.B. Romesser, et al., The canonical Wnt pathway drives macropinocytosis in cancer, Canc. Res. 78 (2018) 4658–4670.

[41] N. Tejeda-Mun˜oz, L.V. Albrecht, M.H. Bui, E.M. De Robertis, Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins, Proc. Natl. Acad. Sci. U. S. A. 116 (2019) 10402–10411.

[42] E. Michalopoulou, F.R. Auciello, V. Bulusu, D. Strachan, A.D. Campbell, J. Tait- Mulder, et al., Macropinocytosis renders a subset of pancreatic tumor cells resistant to mTOR inhibition, Cell Rep. 30 (2020) 2729–2742.

[43] S.W. Lee, Y. Zhang, M. Jung, N. Cruz, B. Alas, C. Commisso, EGFR-pak signaling selectively regulates glutamine deprivation-induced macropinocytosis, Dev. Cell 50 (2019) 381–392.

[44] J.D. Rabinowitz, E. White, Autophagy and metabolism, Science 330 (2010) 1344–1348.

[45] J.Y. Guo, H.Y. Chen, R. Mathew, J. Fan, A.M. Strohecker, G. Karsli-Uzunbas, et al., Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis, Genes Dev. 25 (2011) 460–470.

[46] C.G. Kinsey, S.A. Camolotto, A.M. Boespflug, K.P. Guillen, M. Foth, A. Truong, et al., Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers, Nat. Med. 25 (2019) 620–627.

[47] K.L. Bryant, C.A. Stalnecker, D. Zeitouni, J.E. Klomp, S. Peng, A.P. Tikunov, et al., Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer, Nat. Med. 25 (2019) 628–640.

[48] J. Zhang, J. Fan, S. Venneti, J.R. Cross, T. Takagi, B. Bhinder, et al., Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion, Mol. Cell 56 (2014) 205–218.

[49] N.N. Pavlova, S. Hui, J.M. Ghergurovich, J. Fan, A.M. Intlekofer, R.M. White, et al., As extracellular glutamine levels decline, asparagine becomes an essential amino acid, Cell Metabol. 27 (2018) 428–438.

[50] A.S. Krall, S. Xu, T.G. Graeber, D. Braas, H.R. Christofk, Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor, Nat. Commun. 7 (2016) 11457.

[51] S.R.V. Knott, E. Wagenblast, S. Khan, S.Y. Kim, M. Soto, M. Wagner, et al., Asparagine bioavailability governs metastasis in a model of breast cancer, Nature 554 (2018) 378–381.

[52] D.M. Gwinn, A.G. Lee, M. Briones-Martin-Del-Campo, C.S. Conn, D.R. Simpson, A. I. Scott, et al., Oncogenic KRAS regulates amino acid homeostasis and asparagine biosynthesis via ATF4 and alters sensitivity to L-asparaginase, Canc. Cell 33 (2018) 91–107.

[53] M.N. Balasubramanian, E.A. Butterworth, M.S. Kilberg, Asparagine synthetase: regulation by cell stress and involvement in tumor biology, Am. J. Physiol. Endocrinol. Metab. 304 (2013) E789–799.

[54] H. Ikeuchi, Y.M. Ahn, T. Otokawa, B. Watanabe, L. Hegazy, J. Hiratake, et al., A sulfoximine-based inhibitor of human asparagine synthetase kills L-asparaginase- resistant leukemia cells, Bioorg. Med. Chem. 20 (2012) 5915–5927.

[55] S. Hettmer, A.C. Schinzel, D. Tchessalova, M. Schneider, C.L. Parker, R.T. Bronson, et al., Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma, Elife 4 (2015), e09436.

[56] C. Hodakoski, B.D. Hopkins, G. Zhang, T. Su, Z. Cheng, R. Morris, et al., Rac- mediated macropinocytosis of extracellular protein promotes glucose independence in non-small cell lung cancer, Cancers (Basel) 11 (2019) 37.

[57] G. Redelman-Sidi, G. Iyer, D.B. Solit, M.S. Glickman, Oncogenic activation of Pak1- dependent pathway of macropinocytosis determines BCG entry into bladder cancer cells, Canc. Res. 73 (2013) 1156–1167.

[58] H.P. Lin, B. Singla, P. Ghoshal, J.L. Faulkner, M. Cherian-Shaw, P.M. O’Connor, et al., Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs, Br. J. Pharmacol. 175 (2018) 3640–3655.

[59] C. Ramirez, A.D. Hauser, E.A. Vucic, D. Bar-Sagi, Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis, Nature 576 (2019) 477–481.

[60] W. Yao, J.L. Rose, W. Wang, S. Seth, H. Jiang, A. Taguchi, et al., Syndecan 1 is a critical mediator of macropinocytosis in pancreatic cancer, Nature 568 (2019) 410–414.

参考文献をもっと見る

全国の大学の
卒論・修論・学位論文

一発検索!

この論文の関連論文を見る