Targeting glutamine metabolism and the focal adhesion kinase additively inhibits the mammalian target of the rapamycin pathway in spheroid cancer stem-like properties of ovarian clear cell carcinoma in vitro

Sato,Masakazu; Kawana,Kei; Adachi,Katsuyuki; Fujimoto,Asaha; Yoshida,Mitsuyo; Nakamura,Hiroe; Nishida,Haruka; Inoue,Tomoko; Taguchi,Ayumi; Ogishima,Juri; Eguchi,Satoko; Yamashita,Aki; Tomio,Kensuke; Wada-Hiraike,Osamu; Oda,Katsutoshi; Nagamatsu,Takeshi; Osuga,Yutaka; Fujii,Tomoyuki

doi:10.3892/ijo.2017.3891

Targeting glutamine metabolism and the focal adhesion kinase additively inhibits the mammalian target of the rapamycin pathway in spheroid cancer stem-like properties of ovarian clear cell carcinoma in vitro

Authors:
- Masakazu Sato
- Kei Kawana
- Katsuyuki Adachi
- Asaha Fujimoto
- Mitsuyo Yoshida
- Hiroe Nakamura
- Haruka Nishida
- Tomoko Inoue
- Ayumi Taguchi
- Juri Ogishima
- Satoko Eguchi
- Aki Yamashita
- Kensuke Tomio
- Osamu Wada-Hiraike
- Katsutoshi Oda
- Takeshi Nagamatsu
- Yutaka Osuga
- Tomoyuki Fujii
View Affiliations

Affiliations: Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan
Published online on: February 23, 2017 https://doi.org/10.3892/ijo.2017.3891
Pages: 1431-1438

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Ovarian cancer is one of the leading causes of death in the world, which is linked to its resistance to chemotherapy. Strategies to overcome chemoresistance have been keenly investigated. Culturing cancer cells in suspension, which results in formation of spheroids, is a more accurate reflection of clinical cancer behavior in vitro than conventional adherent cultures. By performing RNA-seq analysis, we found that the focal adhesion pathway was essential in spheroids. The phosphorylation of focal adhesion kinase (FAK) was increased in spheroids compared to adherent cells, and inhibition of FAK in spheroids resulted in inhibition of the downstream mammalian target of the rapamycin (mTOR) pathway in ovarian clear cell carcinomas. This result also suggested that only using a FAK inhibitor might have limitations because the phosphorylation level of FAK could not be reduced to the level in adherent cells, and it appeared that some combination therapies might be necessary. We previously reported that glutamine and glutamate concentrations were higher in spheroids than adherent cells, and we investigated a synergistic effect targeting glutamine metabolism with FAK inhibition on the mTOR pathway. The combination of AOA, a pan-transaminase inhibitor, and PF 573228, a FAK inhibitor, additively inhibited the mTOR pathway in spheroids from ovarian clear cell carcinomas. Our in vitro study proposed a rationale for the positive and negative effects of using FAK inhibitors in ovarian clear cell carcinomas and suggested that targeting glutamine metabolism could overcome the limitation of FAK inhibitors by additively inhibiting the mTOR pathway.

View Figures

View References

1	Agarwal R and Kaye SB: Ovarian cancer: Strategies for overcoming resistance to chemotherapy. Nat Rev Cancer. 3:502–516. 2003. View Article : Google Scholar : PubMed/NCBI
2	Tancioni I, Uryu S, Sulzmaier FJ, Shah NR, Lawson C, Miller NL, Jean C, Chen XL, Ward KK and Schlaepfer DD: FAK Inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth. Mol Cancer Ther. 13:2050–2061. 2014. View Article : Google Scholar : PubMed/NCBI
3	Friedrich J, Ebner R and Kunz-Schughart LA: Experimental anti-tumor therapy in 3-D: Spheroids - old hat or new challenge? Int J Radiat Biol. 83:849–871. 2007. View Article : Google Scholar : PubMed/NCBI
4	Friedrich J, Seidel C, Ebner R and Kunz-Schughart LA: Spheroid-based drug screen: Considerations and practical approach. Nat Protoc. 4:309–324. 2009. View Article : Google Scholar : PubMed/NCBI
5	Beck HC, Gosau M, Kristensen LP and Morsczeck C: A site-specific phosphorylation of the focal adhesion kinase controls the formation of spheroid cell clusters. Neurochem Res. 39:1199–1205. 2014. View Article : Google Scholar : PubMed/NCBI
6	Heyman L, Kellouche S, Fernandes J, Dutoit S, Poulain L and Carreiras F: Vitronectin and its receptors partly mediate adhesion of ovarian cancer cells to peritoneal mesothelium in vitro. Tumour Biol. 29:231–244. 2008. View Article : Google Scholar : PubMed/NCBI
7	Tanjoni I, Walsh C, Uryu S, Tomar A, Nam JO, Mielgo A, Lim ST, Liang C, Koenig M, Sun C, et al: PND-1186 FAK inhibitor selectively promotes tumor cell apoptosis in three-dimensional environments. Cancer Biol Ther. 9:764–777. 2010. View Article : Google Scholar : PubMed/NCBI
8	Ward KK, Tancioni I, Lawson C, Miller NL, Jean C, Chen XL, Uryu S, Kim J, Tarin D, Stupack DG, et al: Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression. Clin Exp Metastasis. 30:579–594. 2012. View Article : Google Scholar
9	Tancioni I, Miller NL, Uryu S, Lawson C, Jean C, Chen XL, Kleinschmidt EG and Schlaepfer DD: FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth. Breast Cancer Res. 17:472015. View Article : Google Scholar : PubMed/NCBI
10	Sato M, Kawana K, Adachi K, Fujimoto A, Yoshida M, Nakamura H, Nishida H, Inoue T, Taguchi A, Takahashi J, et al: Spheroid cancer stem cells display reprogrammed metabolism and obtain energy by actively running the tricarboxylic acid (TCA) cycle. Oncotarget. 7:33297–33305. 2016.PubMed/NCBI
11	Shield K, Ackland ML, Ahmed N and Rice GE: Multicellular spheroids in ovarian cancer metastases: Biology and pathology. Gynecol Oncol. 113:143–148. 2009. View Article : Google Scholar : PubMed/NCBI
12	Visvader JE and Lindeman GJ: Cancer stem cells in solid tumours: Accumulating evidence and unresolved questions. Nat Rev Cancer. 8:755–768. 2008. View Article : Google Scholar : PubMed/NCBI
13	Alanko J, Mai A, Jacquemet G, Schauer K, Kaukonen R, Saari M, Goud B and Ivaska J: Integrin endosomal signalling suppresses anoikis. Nat Cell Biol. 17:1412–1421. 2015. View Article : Google Scholar : PubMed/NCBI
14	Lark AL, Livasy CA, Calvo B, Caskey L, Moore DT, Yang X and Cance WG: Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses. Clin Cancer Res. 9:215–222. 2003.PubMed/NCBI
15	Sood AK, Coffin JE, Schneider GB, Fletcher MS, DeYoung BR, Gruman LM, Gershenson DM, Schaller MD and Hendrix MJ: Biological significance of focal adhesion kinase in ovarian cancer: Role in migration and invasion. Am J Pathol. 165:1087–1095. 2004. View Article : Google Scholar : PubMed/NCBI
16	Beierle EA, Massoll NA, Hartwich J, Kurenova EV, Golubovskaya VM, Cance WG, McGrady P and London WB: Focal adhesion kinase expression in human neuroblastoma: Immunohistochemical and real-time PCR analyses. Clin Cancer Res. 14:3299–3305. 2008. View Article : Google Scholar : PubMed/NCBI
17	Casanova I, Parreño M, Farré L, Guerrero S, Céspedes MV, Pavon MA, Sancho FJ, Marcuello E, Trias M and Mangues R: Celecoxib induces anoikis in human colon carcinoma cells associated with the deregulation of focal adhesions and nuclear translocation of p130Cas. Int J Cancer. 118:2381–2389. 2006. View Article : Google Scholar
18	Chen CH, Shyu MK, Wang SW, Chou CH, Huang MJ, Lin TC, Chen ST, Lin HH and Huang MC: MUC20 promotes aggressive phenotypes of epithelial ovarian cancer cells via activation of the integrin β1 pathway. Gynecol Oncol. 140:131–137. 2016. View Article : Google Scholar
19	Chen YY, Wang ZX, Chang PA, Li JJ, Pan F, Yang L, Bian ZH, Zou L, He JM and Liang HJ: Knockdown of focal adhesion kinase reverses colon carcinoma multicellular resistance. Cancer Sci. 100:1708–1713. 2009. View Article : Google Scholar : PubMed/NCBI
20	Kong D, Chen F and Sima NI: Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway. Exp Ther Med. 10:1725–1731. 2015.PubMed/NCBI
21	Lark AL, Livasy CA, Dressler L, Moore DT, Millikan RC, Geradts J, Iacocca M, Cowan D, Little D, Craven RJ, et al: High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype. Mod Pathol. 18:1289–1294. 2005. View Article : Google Scholar : PubMed/NCBI
22	Liu W, Bloom DA, Cance WG, Kurenova EV, Golubovskay VM and Hochwald SN: FAK and IGF-IR interact to provide survival signals in human pancreatic adenocarcinoma cells. Carcinogenesis. 29:1096–1107. 2008. View Article : Google Scholar : PubMed/NCBI
23	Madan R, Smolkin MB, Cocker R, Fayyad R and Oktay MH: Focal adhesion proteins as markers of malignant transformation and prognostic indicators in breast carcinoma. Hum Pathol. 37:9–15. 2006. View Article : Google Scholar
24	Rentala S, Chintala R, Guda M, Chintala M, Komarraju AL and Mangamoori LN: Atorvastatin inhibited Rho-associated kinase 1 (ROCK1) and focal adhesion kinase (FAK) mediated adhesion and differentiation of CD133⁺CD44⁺ prostate cancer stem cells. Biochem Biophys Res Commun. 441:586–592. 2013. View Article : Google Scholar : PubMed/NCBI
25	Stone RL, Baggerly KA, Armaiz-Pena GN, Kang Y, Sanguino AM, Thanapprapasr D, Dalton HJ, Bottsford-Miller J, Zand B, Akbani R, et al: Focal adhesion kinase: An alternative focus for anti-angiogenesis therapy in ovarian cancer. Cancer Biol Ther. 15:919–929. 2014. View Article : Google Scholar : PubMed/NCBI
26	Sulzmaier FJ, Jean C and Schlaepfer DD: FAK in cancer: Mechanistic findings and clinical applications. Nat Rev Cancer. 14:598–610. 2014. View Article : Google Scholar : PubMed/NCBI
27	Lee BY, Timpson P, Horvath LG and Daly RJ: FAK signaling in human cancer as a target for therapeutics. Pharmacol Ther. 146:132–149. 2015. View Article : Google Scholar
28	Durán RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E and Hall MN: Glutaminolysis activates Rag-mTORC1 signaling. Mol Cell. 47:349–358. 2012. View Article : Google Scholar : PubMed/NCBI
29	Kassem L and Abdel-Rahman O: Targeting mTOR pathway in gynecological malignancies: Biological rationale and systematic review of published data. Crit Rev Oncol Hematol. 108:1–12. 2016. View Article : Google Scholar : PubMed/NCBI
30	Trapnell C, Pachter L and Salzberg SL: TopHat: Discovering splice junctions with RNA-Seq. Bioinformatics. 25:1105–1111. 2009. View Article : Google Scholar : PubMed/NCBI
31	Langmead B, Trapnell C, Pop M and Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10:R252009. View Article : Google Scholar : PubMed/NCBI
32	Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ and Pachter L: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 28:511–515. 2010. View Article : Google Scholar : PubMed/NCBI
33	Zhang Y, He RQ, Dang YW, Zhang XL, Wang X, Huang SN, Huang WT, Jiang MT, Gan XN, Xie Y, et al: Comprehensive analysis of the long noncoding RNA HOXA11-AS gene interaction regulatory network in NSCLC cells. Cancer Cell Int. 16:892016. View Article : Google Scholar : PubMed/NCBI
34	Fujimoto A, Kawana K, Taguchi A, Adachi K, Sato M, Nakamura H, Ogishima J, Yoshida M, Inoue T, Nishida H, et al: Inhibition of endoplasmic reticulum (ER) stress sensors sensitizes cancer stem-like cells to ER stress-mediated apoptosis. Oncotarget. 7:51854–51864. 2016.PubMed/NCBI
35	Schneider CA, Rasband WS and Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9:671–675. 2012. View Article : Google Scholar : PubMed/NCBI
36	Kang Y, Hu W, Ivan C, Dalton HJ, Miyake T, Pecot CV, Zand B, Liu T, Huang J, Jennings NB, et al: Role of focal adhesion kinase in regulating YB-1-mediated paclitaxel resistance in ovarian cancer. J Natl Cancer Inst. 105:1485–1495. 2013. View Article : Google Scholar : PubMed/NCBI
37	McGrail DJ, Khambhati NN, Qi MX, Patel KS, Ravikumar N, Brandenburg CP and Dawson MR: Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner. Sci Rep. 5:95292015. View Article : Google Scholar : PubMed/NCBI
38	Infante JR, Camidge DR, Mileshkin LR, Chen EX, Hicks RJ, Rischin D, Fingert H, Pierce KJ, Xu H, Roberts WG, et al: Safety, pharmacokinetic, and pharmacodynamic phase I dose-escalation trial of PF-00562271, an inhibitor of focal adhesion kinase, in advanced solid tumors. J Clin Oncol. 30:1527–1533. 2012. View Article : Google Scholar : PubMed/NCBI
39	Schultze A and Fiedler W: Therapeutic potential and limitations of new FAK inhibitors in the treatment of cancer. Expert Opin Investig Drugs. 19:777–788. 2010. View Article : Google Scholar : PubMed/NCBI
40	Walsh C, Tanjoni I, Uryu S, Tomar A, Nam J-O, Luo H, Phillips A, Patel N, Kwok C, McMahon G, et al: Oral delivery of PND-1186 FAK inhibitor decreases tumor growth and spontaneous breast to lung metastasis in pre-clinical models. Cancer Biol Ther. 9:778–790. 2010. View Article : Google Scholar : PubMed/NCBI
41	Mabuchi S, Kuroda H, Takahashi R and Sasano T: The PI3K/AKT/mTOR pathway as a therapeutic target in ovarian cancer. Gynecol Oncol. 137:173–179. 2015. View Article : Google Scholar : PubMed/NCBI
42	Yamamoto D, Sonoda Y, Hasegawa M, Funakoshi-Tago M, Aizu-Yokota E and Kasahara T: FAK overexpression upregulates cyclin D3 and enhances cell proliferation via the PKC and PI3-kinase-Akt pathways. Cell Signal. 15:575–583. 2003. View Article : Google Scholar : PubMed/NCBI
43	Hou P, Kuo CY, Cheng CT, Liou JP, Ann DK and Chen Q: Intermediary metabolite precursor dimethyl-2-ketoglutarate stabilizes hypoxia-inducible factor-1α by inhibiting prolyl-4-hydroxylase PHD2. PLoS One. 9:e1138652014. View Article : Google Scholar
44	Zhao J, Peng L, Luo Z, Cui R and Yan M: Inhibitory effects of dimethyl α-ketoglutarate in hepatic stellate cell activation. Int J Clin Exp Pathol. 8:5471–5477. 2015.PubMed/NCBI
45	Mariño G, Pietrocola F, Kong Y, Eisenberg T, Hill JA, Madeo F and Kroemer G: Dimethyl α-ketoglutarate inhibits maladaptive autophagy in pressure overload-induced cardiomyopathy. Autophagy. 10:930–932. 2014. View Article : Google Scholar
46	Jin L, Alesi GN and Kang S: Glutaminolysis as a target for cancer therapy. Oncogene. 35:3619–3625. 2016. View Article : Google Scholar
47	Yang L, Moss T, Mangala LS, Marini J, Zhao H, Wahlig S, Armaiz-Pena G, Jiang D, Achreja A, Win J, et al: Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer. Mol Syst Biol. 10:7282014. View Article : Google Scholar : PubMed/NCBI
48	Feld FM, Nagel PD, Weissinger SE, Welke C, Stenzinger A, Möller P and Lennerz JK: GOT1/AST1 expression status as a prognostic biomarker in pancreatic ductal adenocarcinoma. Oncotarget. 6:4516–4526. 2015. View Article : Google Scholar : PubMed/NCBI
49	Thornburg JM, Nelson KK, Clem BF, Lane AN, Arumugam S, Simmons A, Eaton JW, Telang S and Chesney J: Targeting aspartate aminotransferase in breast cancer. Breast Cancer Res. 10:R842008. View Article : Google Scholar : PubMed/NCBI
50	Son J, Lyssiotis CA, Ying H, Wang X, Hua S, Ligorio M, Perera RM, Ferrone CR, Mullarky E, Shyh-Chang N, et al: Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature. 496:101–105. 2013. View Article : Google Scholar : PubMed/NCBI
51	Kashiyama T, Oda K, Ikeda Y, Shiose Y, Hirota Y, Inaba K, Makii C, Kurikawa R, Miyasaka A, Koso T, et al: Antitumor activity and induction of TP53-dependent apoptosis toward ovarian clear cell adenocarcinoma by the dual PI3K/mTOR inhibitor DS-7423. PLoS One. 9:e872202014. View Article : Google Scholar : PubMed/NCBI