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. 2019 Jun;38(23):4637-4654.
doi: 10.1038/s41388-019-0747-0. Epub 2019 Feb 11.

MSC-regulated lncRNA MACC1-AS1 Promotes Stemness and Chemoresistance Through Fatty Acid Oxidation in Gastric Cancer

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Free PMC article

MSC-regulated lncRNA MACC1-AS1 Promotes Stemness and Chemoresistance Through Fatty Acid Oxidation in Gastric Cancer

Wanming He et al. Oncogene. .
Free PMC article

Abstract

Chemotherapy is the preferred treatment for advanced stage gastric cancer (GC) patients and chemotherapy resistance is the major obstacle to effective cancer therapy. Increasing evidence suggests that mesenchymal stem cells (MSCs) make important contributions to development of drug resistance. However, the underlying mechanism remains elusive. In this study, we discovered that abundant MSCs in tumor tissues predicted a poor prognosis in GC patients. MSCs promoted stemness and chemoresistance in GC cells through fatty acid oxidation (FAO) in vitro and in vivo. Mechanically, transforming growth factor β1 (TGF-β1) secretion by MSCs activated SMAD2/3 through TGF-β receptors and induced long non-coding RNA (lncRNA) MACC1-AS1 expression in GC cells, which promoted FAO-dependent stemness and chemoresistance through antagonizing miR-145-5p. Moreover, pharmacologic inhibition of FAO with etomoxir (ETX) attenuated MSC-induced FOLFOX regiment resistance in vivo. These results suggest that FAO plays an important role in MSC-mediated stemness and chemotherapy resistance in GC and FAO inhibitors in combination with chemotherapeutic drugs present as a promising strategy to overcome chemoresistance.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Mesenchymal stem cells (MSCs) promote stemness and chemoresistance and predict a poor prognosis in gastric cancer (GC). a Representative images of sphere-formation assay in AGS and MKN45 culture either alone or with MSCs. Scale bar = 500 μm. b Expression levels of stemness-associating genes measured by quantitative real-time polymerase chain reaction (qRT-PCR) in AGS and MKN45 sphere co-culture with or without MSCs. c Colony-formation assay and the quantitative graph of AGS and MKN45 with or without MSCs when treated with 1 μg/mL 5-florouracil and 3 μg/mL oxaliplatin. d Numbers of tumor formation after injection of MKN45 cells with or without MSCs. e Tumor weights derived from either different dosages of MKN45 cells or together with MSCs. Gray dots indicate that no tumor grew at the site of injection. Other color dots indicate individual tumor weights. f The ranges of estimated tumor-initiating cell frequencies evaluated by ELDA web tool (http://bioinf.wehi.edu.au/software/elda) with 95% confidence. g Percentage of double-negative CD29 and CD90 (CD29(−)CD90(−)) and double-positive CD29 and CD90 (CD29(+)CD90(+)) expression in The Cancer Genome Atlas (TCGA) database. h Kaplan–Meier curves of postoperative recurrence of stage I–III GC in CD29(+)CD90(+) patients in TCGA database. i Representative immunohistochemical staining of CD29 and CD90 in stage I–IV GC and normal gastric tissues. Scale bar = 100 μm. j The frequency of CD29(−)CD90(−) and CD29(+)CD90(+) expression in GC categorized by tumor, node metastasis stage (P = 0.000464, χ2 test), tumor invasion (P = 0.047, χ2 test), lymph node metastasis (P = 0.004, χ2 test), distant metastasis (P = 0.017, χ2 test), recurrence (P = 0.000002, χ2 test). kl Kaplan–Meier analysis of disease-free survival (stage I–III GC patients) and overall survival (stage IV GC patients) in response to the co-expression of CD29 and CD90. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 2
Fig. 2
Fatty acid oxidation plays an important role in mesenchymal stem cell (MSC)-induced stemness and chemoresistance. a Expression levels of carnitine palmitoyltransferase 1 (CPT1) and acetyl-coenzyme A synthetase (ACS) in AGS and MKN45 co-culture with MSCs compared to culture alone by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. b qRT-PCR for CPT1 and ACS in AGS and MKN45 sphere culture either alone or with MSCs. ce CPT1 enzyme activity (c), relative fatty β-oxidation rate (d), and ATP levels (e) in AGS and MKN45 cells with or without MSCs. f Expression levels of stemness-associating genes in AGS and MKN45 transfected with siCPT1. g, h qRT-PCR (g) and western blotting (h) for stemness-associating genes in AGS and MKN45 cell culture either alone or with MSCs and with or without 100 μmol/L etomoxir (ETX). i Representative images of sphere-formation assay in AGS and MKN45 cell culture either alone or with MSCs and with or without 100 μmol/L ETX for 7 days. Scale bar = 500 μm. j Colony-formation assay and the quantitative graph of AGS and MKN45 cell culture either alone or with MSCs and with or without 100 μmol/L ETX when treated with 1 μg/mL 5-florouracil and 3 μg/mL oxaliplatin. k ATP level in AGS and MKN45 cell culture either alone or with MSCs and with or without 100 μmol/L ETX for 48 h. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 3
Fig. 3
MACC1-AS1 is induced by mesenchymal stem cell (MSC)-derived TGF-β1 and contributes to stemness and chemoresistance. a, b Expression of MACC1-AS1 in gastric cancer (GC) cells (a) and spheres of GC cells (b) after co-culture with MSCs. c Expression of MACC1-AS1 in the indicated subcutaneous tumor of nude mice, formed by MKN45 cells with or without MSCs. d The score of MACC1-AS1 in CD29(−)CD90(−) and CD29(+)CD90(+) GC tissues. e, f Expression levels of stemness-associating genes was increased in the indicated AGS and MKN45 cells after MACC1-AS1 overexpression by quantitative real-time polymerase chain reaction (e) and western blotting (e) (V vector, M MACC1-AS1 overexpression). g Representative images of sphere-formation assay in AGS and MKN45 cell after overexpressing MACC1-AS1. Scale bar = 500 μm. h Colony-formation assay and the quantitative graph of AGS and MKN45 cells treated with 5-florouracil (1 μg/mL) and oxaliplatin (3 μg/mL) after transfected with MACC1-AS1 compared to vector. i Expression of MACC1-AS1 in AGS and MKN45 incubated with GC medium, <3 kD MSC-CM or >3 kD MSC-CM. j Transforming growth factor (TGF)-β1 concentration in AGS and MKN45 medium, >3 kD MSC-CM and <3 kD MSC-CM measured by enzyme-linked immunosorbent assay. k Expression levels of TGF-β receptors and SMAD family in AGS and MKN45 cells after co-culture with MSCs. l Expression of MACC1-AS1 in AGS and MKN45 cells treated with TGF-β1 (20 μg/mL) for 24 h. m, n Expression of MACC1-AS1 in AGS and MKN45 cells incubated with >3 kD MSC-CM treated with TGF-β1 inhibitor disitertide (m) and TGFβR-I inhibitor LY-364947 (n) for 24 h. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 4
Fig. 4
The role of MACC1-AS1 on stemness and chemoresistance is dependent on fatty acid oxidation. a The top ten BioCyc metabolic pathway enrichment analysis after MACC1-AS1 overexpression. The horizontal axis represented BioCyc pathway annotation. The vertical axis represented –lg (P value). b, c Quantitative real-time polymerase chain reaction (b) and western blotting (c) for the expression levels of carnitine palmitoyltransferase 1 (CPT1) and acetyl-coenzyme A synthetase in AGS and MKN45 cells after stable transfection with MACC1-AS1 (M) or vector (V). d, e Relative fatty β-oxidation rate (d) and ATP levels (e) in AGS and MKN45 after overexpressing MACC1-AS1. f Western blotting for stemness-associating genes in AGS and MKN45 cells after overexpressing MACC1-AS1 and treating with or without 100 μmol/L etomoxir (ETX) for 48 h. g Western blotting for expression levels of CPT1 and stemness-associating genes in AGS and MKN45 cells after overexpressing MACC1-AS1 and siCPT1#1 transfection. h Representative images of sphere-formation assay in AGS and MKN45 after overexpressing MACC1-AS1 with or without 100 μmol/L ETX for 7 days. Scale bar = 500 μm. i Relative ATP levels of AGS and MKN45 cells after overexpressing MACC1-AS1 with or without 100 μmol/L ETX for 48 h. j Growth inhibition by MTT (3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyltetrazolium bromide) assay of AGS and MKN45 cells treated with 5-florouracil and oxaliplatin after overexpressing MACC1-AS1 with or without 100 μmol/L ETX for 48 h. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 5
Fig. 5
miR-145-5p is downstream of MACC1-AS1 to promote stemness and chemoresistance through fatty acid oxidation (FAO). a Schematic diagram of binding sites between MACC1-AS1 and miR-145-5p predicted by LncRNASNP database. b Expression of miR-145-5p in AGS and MKN45 cells after stable transfection with MACC1-AS or vector. c Luciferase activity in 293T cells when MACC1-AS1 wild-type or MUT vector was co-transfected with miR145-5p mimic or negative control (NC). d Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of MACC1-AS1 in the miR-145-5p pull-down complex. e, f qRT-PCR (e) and western blotting (f) for the expression levels of FAO enzymes and stemness-associating genes in AGS and MKN45 cells after overexpression of MACC1-AS1 with or without transfection with miR-145-5p. g, h Relative fatty β-oxidation rate (g) and ATP levels (h) in AGS and MKN45 cells after overexpressing MACC1-AS1 with or without transfection with miR-145-5p. i Growth inhibition by MTT (3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyltetrazolium bromide) assay of AGS and MKN45 cells treated with 5-florouracil and oxaliplatin after overexpressing MACC1-AS1 with or without transfection with miR-145-5p. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 6
Fig. 6
Inhibition of fatty acid oxidation (FAO) attenuates mesenchymal stem cell (MSC)-induced chemoresistance in vivo. a, b Kaplan–Meier analysis of overall survival (OS) (a) and progression-free survival (b) corresponding to the expression of carnitine palmitoyltransferase 1 (CPT1) analyzed by the online Kaplan Meier Plotter database (http://kmplot.com/analysis/). c, d Representative immunohistochemical (IHC) staining of CPT1 (c) and CPT1 score (d) in adjacent noncancerous and gastric cancer (GC) tissues. Scale bar = 500 μm (magnification: ×100, left panel); Scale bar = 100 μm (magnification: ×400, right panel). e CPT1 score in CD29(−)CD90(−) and CD29(+)CD90(+) expression in GC tissues. f, g Kaplan–Meier analysis of disease-free survival (stage I–III GC patients) and OS (stage IV GC patients) corresponding to the expression of CPT1. h Tumor growth of with MKN45 cells (1 × 106 cells) alone (GC) or MKN45 cells treated with FOLFOX regiment weekly (oxaliplatin 6 mg/kg followed 2 h later by 5-florouracil 50 mg/kg and calcium folinatc 90 mg/kg, intraperitoneally (i.p.), GC+FOLFOX) or MKN45 cells in combination with MSCs (5 × 106 cells) treated with FOLFOX regiment (GC+FOLFOX+MSC) or MKN45 cells in combine with MSCs (5 × 106 cells) treated with FOLFOX regiment plus ETX (40 mg/kg, i.p., every other day, GC+FOLFOX+MSC+ETX). Tumor volumes were calculated every 3 days (n = 5). i Representative images of transplanted subcutaneous tumors. j, k Representative images of OCT4, CPT1, ki67, and cleaved caspase 3 in subcutaneous tumors of xenograft nude mice by IHC staining (j) and hematoxylin–eosin staining of the hearts, livers, spleens, lungs, and kidneys in subcutaneous tumors (k). Scale bar = 500 μm (magnification: ×100, left panel); scale bar = 100 μm (magnification: ×400, right panel). l Schematic representation of the pathway that MSCs secreted TGF-β1 and induced lncRNA MACC1-AS1 expression in GC cells, which promoted FAO-dependent stemness and chemoresistance through miR-145-5p. *P < 0.05; **P < 0.01; ***P < 0.001

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References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66:7–30. doi: 10.3322/caac.21332. - DOI - PubMed
    1. Digklia A, Wagner AD. Advanced gastric cancer: current treatment landscape and future perspectives. World J Gastroenterol. 2016;22:2403–14. doi: 10.3748/wjg.v22.i8.2403. - DOI - PMC - PubMed
    1. Davidson M, Okines AF, Starling N. Current and future therapies for advanced gastric cancer. Clin Colorectal Cancer. 2015;14:239–50. doi: 10.1016/j.clcc.2015.05.013. - DOI - PubMed
    1. Ajani JA, D’Amico TA, Almhanna K, Bentrem DJ, Chao J, Das P, et al. Gastric Cancer, Version 3.2016, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2016;14:1286–312. doi: 10.6004/jnccn.2016.0137. - DOI - PubMed
    1. Shitara K. Chemotherapy for advanced gastric cancer: future perspective in Japan. Gastric Cancer. 2017;20:102–10. doi: 10.1007/s10120-016-0648-7. - DOI - PubMed

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