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. 2022 Oct 8;15(1):141.
doi: 10.1186/s13045-022-01359-4.

Cancer-associated fibroblast-specific lncRNA LINC01614 enhances glutamine uptake in lung adenocarcinoma

Tongyan Liu #  1   2 , Chencheng Han #  1 , Panqi Fang #  1 , Zhifei Ma #  1 , Xiaoxiao Wang #  3 , Hao Chen #  1 , Siwei Wang  1 , Fanchen Meng  1 , Cheng Wang  4   5   6 , Erbao Zhang  4   5 , Guozhang Dong  1 , Hongyu Zhu  1 , Wenda Yin  1 , Jie Wang  1   2   7 , Xianglin Zuo  1   2   7 , Mantang Qiu  8 , Jinke Wang  9 , Xu Qian  4   5 , Hongbing Shen  4   5 , Lin Xu  1 , Zhibin Hu  10   11 , Rong Yin  12   13   14   15
Affiliations

Cancer-associated fibroblast-specific lncRNA LINC01614 enhances glutamine uptake in lung adenocarcinoma

Tongyan Liu et al. J Hematol Oncol. .

Abstract

Background: Besides featured glucose consumption, recent studies reveal that cancer cells might prefer "addicting" specific energy substrates from the tumor microenvironment (TME); however, the underlying mechanisms remain unclear.

Methods: Fibroblast-specific long noncoding RNAs were screened using RNA-seq data of our NJLCC cohort, TCGA, and CCLE datasets. The expression and package of LINC01614 into exosomes were identified using flow cytometric sorting, fluorescence in situ hybridization (FISH), and quantitative reverse transcription polymerase chain reaction (RT-PCR). The transfer and functional role of LINC01614 in lung adenocarcinoma (LUAD) and CAFs were investigated using 4-thiouracil-labeled RNA transfer and gain- and loss-of-function approaches. RNA pull-down, RNA immunoprecipitation, dual-luciferase assay, gene expression microarray, and bioinformatics analysis were performed to investigate the underlying mechanisms involved.

Results: We demonstrate that cancer-associated fibroblasts (CAFs) in LUAD primarily enhance the glutamine metabolism of cancer cells. A CAF-specific long noncoding RNA, LINC01614, packaged by CAF-derived exosomes, mediates the enhancement of glutamine uptake in LUAD cells. Mechanistically, LINC01614 directly interacts with ANXA2 and p65 to facilitate the activation of NF-κB, which leads to the upregulation of the glutamine transporters SLC38A2 and SLC7A5 and eventually enhances the glutamine influx of cancer cells. Reciprocally, tumor-derived proinflammatory cytokines upregulate LINC01614 in CAFs, constituting a feedforward loop between CAFs and cancer cells. Blocking exosome-transmitted LINC01614 inhibits glutamine addiction and LUAD growth in vivo. Clinically, LINC01614 expression in CAFs is associated with the glutamine influx and poor prognosis of patients with LUAD.

Conclusion: Our study highlights the therapeutic potential of targeting a CAF-specific lncRNA to inhibit glutamine utilization and cancer progression in LUAD.

Keywords: Cancer-associated fibroblasts; Glutamine; Long noncoding RNA; Metabolic reprograming; Tumor microenvironment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Exosome-packaged RNA from CAFs enhances the glutamine uptake and progression of LUAD cells. A, B Representative transcriptome analysis of the Reactome pathway (A) and GSEA (B) in the A549 cells co-cultured with CAFs compared with those with NFs (n = 3). B Reactome pathway-based GSEA revealed an enrichment of “Metabolism of amino acids and derivatives” in the A549 cells co-cultured with CAFs compared to NFs co-cultured with A549 cells (n = 3). C OCR of A549 cells with indicated treatments (n = 3). D ATP production of A549 cells with indicated treatments (n = 3). E Schematic and heatmap of metabolomic experiments for conditioned media of indicated cells. F–L A549 cells were treated with exosomes or CM of CAFs and then used for the indicated experiments. anti-CD81 antibody was used for depleting exosomes in the CM of CAFs. F OCR of A549 cells with the indicated treatments (n = 3). G ATP production of A549 cells with indicated treatments (n = 3). H Extracellular glutamine levels of A549 cells cultured for 4, 8, and 12 h in 25 mM glucose DMEM (containing 1 mM pyruvate and 4 mM glutamine) with the indicated treatments (n = 3). I 3H-glutamine uptake (n = 3). J Glutamine-derived TCA cycle intermediates in A549 cells with indicated treatments (n = 3). K, L RTCA proliferation, migration (lower), and invasion (upper) assays and ATP production of A549 cells with indicated treatments. M Extracellular levels of glutamine of A549 cells with the indicated treatments (n = 3). N RTCA proliferation assays, migration (lower), and invasion (upper) assays of A549 cells with indicated treatments (n = 3). For C, D and F–N, Means ± s.d. are shown, and independent sample t-tests determined P values. * P < 0.05, ** P < 0.01, *** P < 0.001. UT, cancer cells without any treatment; CM, conditioned medium; Exos, exosomes; GSEA, gene set enrichment analysis; RTCA, Real-time xCELLigence analysis; LUAD, lung adenocarcinoma
Fig. 2
Fig. 2
Intercellular transfer of a CAF-specific lncRNA LINC01614 by exosomes. A Schematic showing the screening strategy of TME-specific lncRNAs. B LncRNAs that are moderately to highly positively correlated with TME cells in the NJLCC cohort (left) and TCGA database (right) (ENSG0000230838 is for LINC01614). C qRT-PCR of LINC01614 (ENSG0000230838) and ENSG00000261327 in CAFs, NFs, MRC5, and LUAD cell lines (n = 3). D Flow cytometric sorting of CAFs from cell suspension of human LUAD tissue. Live/Dead Fixable Viability Dye staining was used to detect dead cells. CD45 was used as an immune cell marker, EpCAM as an epithelial cell marker, fibroblast-specific protein (FSP) and PDGFR β (CD140b) as fibroblast markers. qRT-PCR analysis of LINC01614 and ENSG00000261327 in isolated CAFs and CAF-depleted cells (n = 3). E qRT-PCR of LINC01614 and ENSG00000261327 in the CM of A549 cells treated with RNase (2 μg mL−1) alone or combined with Triton-X-100 (0.1%) for 20 min (n = 3). F qRT-PCR analysis of LINC01614 in CAFs exosomes versus NFs exosomes (n = 3), and A549 cells with indicated treatments versus those cultured alone for 24 h (n = 3). G Relative expressions of LINC01614 in CAF-derived exosomes (left) and the A549 cells co-cultured with the CAFs transfected with shRAB27 (right) were determined by qRT-PCR. H Representative images of α-SMA immunofluorescent staining and LINC01614 fluorescence in situ hybridization staining in LUAD patient tissues (n = 10). I Schema and representative images for measuring RNA transfer from CAFs to A549 cells utilizing the uridine analog EU for fluorescence microscopy (green). CAFs were labeled with EU and co-cultured with Dil lipid-labeled A549 cells for the indicated time. Representative images of EU-positive A549 cells (orange) are shown (n = 3). J Schema for measuring LINC01614 transfer from CAFs to A549 cells. Conditioned medium from CAFs labeled with 4sU was added to A549 cells, 4sU RNA in A549 cells was isolated with streptavidin pull-down. Relative transfer of 4sU RNA to mono-cultured A549 cells the addition of CM collected from 4sU-labeled CAFs. CAFs CM depleted of exosomes with GW4869 is shown as a control for exosome-dependency (n = 3). For all experiments, means ± s.d. are shown, and independent sample t-tests determined P values. *P < 0.05, **P < 0.01, ***P < 0.001. TME tumor microenvironment; NJLCC Nanjing lung cancer cohort; UT cancer cells without any treatment; CM conditioned medium; Exos, exosomes; EU 5-ethynyl uridine; 4sU 4-thiouridine; LUAD lung adenocarcinoma. Source data are provided
Fig. 3
Fig. 3
CAF-exosome-packaged LINC01614 enhances glutamine influx of LUAD cells via upregulating amino acid transporters. A, B, Exosomes isolated from the CM of CAFs transduced with lenti-LINC01614-shRNA or transfected with shRAB27 were added to A549 cells for 48 h. A OCR values of A549 cells were measured (n = 3). B 3H-glutamine uptake, glutamine-derived TCA cycle intermediates, and ATP content of A549 cells with the indicated treatments (n = 3). C, D and G, H A549 cells transfected with pcDNA 3.1-LINC01614. Antisense for LINC01614 was used as the control. C, OCR values of A549 cells (n = 3). D 3H-glutamine uptake, glutamine-derived TCA cycle intermediates, and ATP content of A549 cells with the indicated treatments (n = 3). E–H Quantification of the proliferation (E and G) and migration and invasion (F and H) of A549 cells with indicated treatments assessed by RTCA and Boyden chamber assay with or without Matrigel-coated inserts (n = 3). I–K RTCA proliferation, glutaminase and glutamate content, migration and invasion assays of A549 cells with indicated treatments. L–M Exosomes isolated from CM of CAFs transduced with lenti-LINC01614-shRNA or lenti-LINC01614 were added to A549 cells for 48 h. L qRT-PCR analysis was used to determine the mRNA profile of glutamine transporters in A549 cells with indicated treatments. M–N, Western blotting of SLC38A2 and SLC7A5 expression in A549 cells with indicated treatments. For A–K, means ± s.d. are shown, and independent sample t-tests P values were determined. *P < 0.05, **P < 0.01, ***P < 0.001. UT, cancer cells without treatment; CM, conditioned medium; RTCA, Real-time xCELLigence analysis; Exos, exosomes
Fig. 4
Fig. 4
LINC01614 enhances ANXA2 and p65 interactions and promotes NF-κB activation. A p65 and ANXA2 were pulled down by biotin-labeled LINC01614 but not LINC01614 antisense RNA in whole-cell lysates of A549 cells treated with exosomes from CAFs (n = 3). B, C RIP evaluation of the interaction between ANXA2 (B) and p65 (C) using anti-ANXA2 and anti-p65 antibodies in A549 cells treated with exosomes from CAFs (n = 3). D ANXA2 and LINC01614 were co-precipitated with p65 in whole-cell lysates of A549 cells treated with exosomes from CAFs (n = 3). E IP (immunoprecipitation) analysis for the in vitro interaction of ANXA2 and p65. LINC01614 promoted the binding between recombinant ANXA2 and p65 in vitro (n = 3). F The secondary structure of LINC01614 is shown as predicated by the centroid method (http://rna.tbi.univie.ac.at). RNA pull-down assay for the interactions of sequentially deleted LINC01614 variants with ANXA2 and p65 in ectopic LINC01614 expressed A549 cells (n = 3). Schematic of sequentially deleted LINC01614 variants (left). Representative western blot for ANXA2 and p65 pulled down by LINC01614 variants (right). G nucleotide mutation lacking the stem-loop structure of LINC01614 (973–1775) abolished the interaction between p65 and ANXA2 as revealed by IP analysis. H LINC01614973–1775 enhanced the interaction between p65 and ANXA2, as revealed by IP analysis. I GSEA revealed enrichment of NF-κB target genes in the exosome-packaged LINC01614 treated A549 cells. J NF-κB activity of CAF-exosome-treated A549 cells, examined by luciferase reporter assay (n = 3). K Western blot analysis of the nuclear factor NF-κB p65 subunit following nuclear fractionation of exosome treated A549 cells. L Immunofluorescent p65 staining showing nuclear translocation in A549 cells with indicated treatments (n = 3). M NF-κB activity of A549 transduced with lenti-LINC01614, determined by luciferase reporter assay (n = 3). N Western blot analysis of the nuclear factor NF-κB p65 subunit following nuclear fractionation of A549 cells transduced with LINC01614. Loading controls, GAPDH (cytoplasmic fractions), and H3 (nuclear fractions) (n = 3). O Immunofluorescent p65 staining showing its nuclear translocation in A549 cells with indicated treatments (n = 3). For B, C, J, and M means ± s.d. are shown, and independent sample t-tests determined P values. *P < 0.05, **P < 0.01, ***P < 0.001. UT cancer cells without any treatment; CM conditioned medium; Exos exosomes; GESA gene set enrichment analysis; LUAD lung adenocarcinoma
Fig. 5
Fig. 5
LINC01614 promotes ANXA2-dependent p65 phosphorylation and the transcription of SLC38A2 and SLC7A5. A, D Exosomes isolated from the CM of CAFs transduced with lenti-LINC01614-shRNA were added into A549 cells for 48 h. Exosomes from shctrl CAFs-treated A549 cells were used as controls. A, B Western blotting for total and phosphorylated IKK and IκBα in A549 cells with indicated treatments. C A549 cells were transduced without or with lenti-LINC01614 and pretreated with an inhibitor of NF-κB nuclear translocation (JSH-23) or IKK inhibitor (BAY 11-7-82). Immunofluorescent p65 staining showing its nuclear translocation in A549 cells (n = 3). D Expression of ANXA2, total, Ser276, and Ser536 phosphorylation of p65 in A549 cells with indicated treatments (n = 3). E Overexpressing LINC01614 or ANXA2 promoted Ser276 phosphorylation of p65 in A549 cells (n = 3). F Knockdown of ANXA2 abrogated the effects of LINC01614 on Ser276 phosphorylation (n = 3). G Representative immunofluorescent images of p65 nuclear translocation in A549 cells with indicated treatments. Scale bars, 50 μm. H NF-κB activity of A549 cells with indicated treatments. I qRT-PCR analysis of SLC38A2 and SLC7A5 in A549 cells with indicated treatments. J A conserved NF-κB binding element at the promoters of SLC38A2 and SLC7A5 were predicated by JASPAR (n = 3). K ChIP-PCR analysis for NF-κB occupancy at the promoters of SLC38A2 and SLC7A5 in A549 cells (n = 3). L Luciferase reporter assays of the transduced A549 cells transfected with reporter plasmids containing the SLC38A2 and SLC7A5 promoter, respectively. Wild type: -2000–0 construct; mutant: -2000–0 constructed with a point mutation at the NF-κB binding site. Transduced A549 cells transfected with a blank pGL3 plasmid used as a negative control (n = 3). M Graphic for ENCODE database of p65 ChIP-seq. For H, I and K, L, means ± s.d. are shown, and independent sample t-tests determined P values. *P < 0.05, **P < 0.01, ***P < 0.001. UT cancer cells without any treatment; CM conditioned medium; Exos, exosomes; IP immunoprecipitation; LUAD lung adenocarcinoma
Fig. 6
Fig. 6
CAFs release LINC01614 in exosomes to enhance glutamine uptake and progression of LUAD in vivo. A Schematic diagram of xenograft and subcutaneous tumorigenicity in mice. B–E, Nude mice were subcutaneously xenografted with A549 cells (1 × 107 cells) and treated intratumorally with CAF-derived exosomes (0.5 μg kg−1) every 3 d for 2 weeks (n = 6 mice per group). B Images of tumor engraftment in nude mice. C, D Tumor weight and growth curves. E Representative H&E staining and IHC staining for Ki67, SLC38A2, and SLC7A5. F NCG (NOD prkdc−/−IL-2Rg−/−) mice were xenografted with A549-luc cells (5 × 106 cells) through tail vein injection and treated intravenously with CAF-derived exosomes once a week for 4 weeks (n = 6 mice per group). Representative bioluminescent images for lung metastasis. Scanning was performed 8 weeks after tumor implantation. G Schematic diagram of implantation of cancer cells in zebrafish embryos (Fli1:EGFR). A549 cells were labeled with Dil implanted into the perivitelline space of each zebrafish. After 24 h, the exosomes (5 ng) isolated from CAFs transduced without (shctrl) or with shRNA for LINC01614 (shLINC01614) were injected into the (Fli1:EGFR) zebrafish embryos. Dissemination of A549 was monitored. H Representative images of Dil-red-labeled A549 cells in zebrafish embryos with indicated treatments. I Representative confocal microscopy images of the dissemination of implanted DiD-labeled A549 cells in zebrafish embryos (Fli1:EGFP) injected with CAF-derived exosomes (5 ng) (n = 15). The approach schema is illustrated. J Quantification of total numbers of disseminated and metastatic cells in the primary tumor surroundings (upper) and the trunk regions (lower) of zebrafish. For D, J means ± s.d. are shown, and independent sample t-tests determined P values. *P < 0.05, **P < 0.01, ***P < 0.001. Exos exosomes, LUAD lung adenocarcinoma
Fig. 7
Fig. 7
LINC01614 correlates with glutamine transporters and poor survival in patients with LUAD. A Representative ISH for LINC01614 and IHC for α-SMA in a TMA cohort containing 78 paired LUAD tissues and adjacent normal tissues. Scale bars, 500 μm (×100 magnification), 100 μm (×400 magnification) (n = 78). B–D The CISH results of the TMA. B The expression of LINC01614 was upregulated in the α-SMA High expression (high CAFs infiltration) group (≥ 6, median). C, D The expression level of LINC01614 in LUAD tissues was positively correlated with the T stage and TNM stage in the TMA cohort. E Higher expression (≥ 6, median) of LINC01614 in LUAD tissues was associated with poor prognosis in the TMA cohort. F Univariate and multivariate Cox regression analyses indicated that high expression of LINC01614 in LUAD tissues was an independent prognostic factor for poor survival (n = 78). G Representative H&E staining, ISH for LINC01614, and IHC for α-SMA, SLC38A2, and SLC7A5 in LUAD and adjacent normal tissues (n = 10). H Correlation between LINC01614 expression in CAFs and SLC38A2 in tumor cells from the same patients (n = 15). I Correlation between LINC01614 expression in CAFs and SLC7A5 in tumor cells from the same patients (n = 15). J Graphical illustration of the interaction between CAFs and LUAD cells. For B–D, the mean ± s.d. are shown, and Student’s t-test determined P values. For H, I Spearman correlation analysis was performed. **P < 0.01, ***P < 0.001, ****P < 0.001. LUAD, lung adenocarcinoma
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