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LINC01615 maintains cell survival in adaptation to nutrient starvation through the pentose phosphate pathway and modulates chemosensitivity in colorectal cancer

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Abstract

Numerous mechanisms involved in promoting cancer cell survival under nutrient starvation have been described. Long noncoding RNAs (lncRNAs) have emerged as critical players in colorectal cancer (CRC) progression, but the role of lncRNAs in the progression of CRC under nutrient starvation has not been well clarified. Here, we identified a lncRNA, LINC01615, that was significantly upregulated in response to serum starvation. LINC01615 can contribute to the adaptation of CRC cells to serum-deprived conditions and enhance cell survival under similar conditions. LINC01615 activated the pentose phosphate pathway (PPP) under serum starvation, manifested as decreased ROS production and enhanced nucleotide and lipid synthesis. Glucose-6-phosphate dehydrogenase (G6PD) is a key rate-limiting enzyme of the PPP, and LINC01615 promoted G6PD expression by competitively binding with hnRNPA1 and facilitating G6PD pre-mRNA splicing. Moreover, we also found that serum starvation led to METTL3 degradation by inducing autophagy, which further increased the stability and level of LINC01615 in a m6A-dependent manner. LINC01615 knockdown combined with oxaliplatin achieved remarkable antitumor effects in PDO and PDX models. Collectively, our results demonstrated a novel adaptive survival mechanism permitting tumor cells to survive under limiting nutrient supplies and provided a potential therapeutic target for CRC.

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Availability of data and materials

The data generated in this study are available upon request from the corresponding author.

Abbreviations

CRC:

Colorectal cancer

PPP:

Pentose phosphate pathway

G6PD:

G6P dehydrogenase

lncRNAs:

Long noncoding RNAs

ROS:

Reactive oxygen species

3-MA:

3-Methyladenine

Baf-A1:

Bafilomycin

shRNA:

Short hairpin RNA

siRNA:

Short interfering RNA

qRT–PCR:

Real-time quantitative reverse transcription PCR

WB:

Western blot

ChIRP-MS:

Chromatin isolation by RNA purification-mass spectrometry

RIP:

RNA immunoprecipitation

PDO:

Patient-derived organoid

PDX:

Patient-derived xenograft

GSEA:

Gene set enrichment analysis

OS:

Overall survival

DFS:

Disease-free survival

ceRNA:

Competing endogenous RNA

Co-IP:

Co-immunoprecipitation

IHC:

Immunohistochemistry

6-AN:

6-Aminonicotinamide

NAC:

N-Acetyl-L-cysteine

RGG:

Arg-Gly-Gly tripeptide repeats

CCK-8:

Cell counting kit-8

NMR:

Nuclear magnetic resonance

FISH:

Fluorescence in situ hybridization

meRIP:

Methylated RNA immunoprecipitation

TEM:

Transmission electron microscopy

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Acknowledgements

We thank all individuals who participated in this work.

Funding

This research was supported by grants from the National Natural Science Foundation of China (No.82072729, No.81602568, No.81773130), China Postdoctoral Science Foundation (No.2018M643009), the Natural Science Foundation of Jiangsu (BK20211606), the National Natural Science Foundation of Hunan Province (No. 2019JJ50906) and Xuzhou Key R&D Program (KC20064).

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Author notes

  1. Yi Zhang, Lei Xu and Zeqiang Ren contributed equally to this work.

Authors and Affiliations

  1. Department of General Surgery, Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, China

    Yi Zhang, Lei Xu, Zeqiang Ren, Jun Song, Pengbo Zhang, Chong Zhang, Shuai Gong, Nai Wu, Xiuzhong Zhang & Changwei Lin

  2. Institute of Digestive Diseases, Xuzhou Medical University, Xuzhou, 221000, China

    Yi Zhang & Lei Xu

  3. Department of Endocrinology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, China

    Xin Liu

  4. Department of Gastrointestinal Surgery, The Third Xiangya Hospital of Central South University, Central South University, Changsha, 410013, Hunan, China

    Chanbin Xie, Zhixing Lu, Min Ma, Yi Zhang, Yifei Chen & Changwei Lin

  5. Department of Otolaryngology and Head Neck Surgery, Affiliated Changsha Hospital of Hunan Normal University, Changsha, China

    Yifei Chen

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  1. Yi Zhang
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  2. Lei Xu
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Contributions

YZ and LX conceived and designed the experiment, performed experiment, and wrote the manuscript. ZR, XL, JS, PZ, CZ, SG, NW, and XZ performed experiment, collected data and analyzed data. CX, ZL, MM, YZ, and YC interpreted data and constructed figures. CL conceptualized the study, designed the experiment, interpreted data and reviewed the manuscript. All authors read and approved the final version of the manuscript. YZ and CL have accessed and verified the data, and CL was responsible for the decision to submit the manuscript.

Corresponding author

Correspondence to Changwei Lin.

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The authors declare no potential conflicts of interest.

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This study was performed according to the ethical standards of Declaration of Helsinki and was approved by the ethics committee of the Xuzhou Medical University (XYFY2019-KL221-01). All animal experiments were approved by the Animal Care Committee of Xuzhou Medical University.

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Supplementary Information

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Supplementary file1 (XLSX 15 kb)

18_2022_4675_MOESM2_ESM.tif

Supplementary file2 Supplementary Figure 1 (A) Venn diagram showing the numbers of overlapping lncRNAs between the differentially downregulated lncRNAs in HCT116 and DLD1 cells after serum-free medium treatment. (B) Kaplan–Meier analysis of the OS rate in CRC patients in the TCGA database with high or low expression of CTD-2147F2.1. (C) The non-coding nature of LINC01615 was predicted by coding-potential analysis (PhyloCSF). (D) The relative expression of LINC01615 in various types of tumor tissues and corresponding peritumor tissues based on data obtained from TCGA database. (E) The relative expression of LINC01615 in tumor tissue and corresponding peritumor tissue based on data obtained from the GEO database (GSE115856). (F) qRT-PCR showed the expression of LINC01615 in normal intestinal epithelial cell line FHC and CRC cell lines (DLD1, HT-29, SW620, SW480, LoVo and HT29). (G) qRT-PCR examined the expression of LINC01615 after knockdown in DLD1 and HCT116 cells. (H) qRT-PCR examined the expression of LINC01615 after overexpression in LoVo cells. (I) CCK-8 assay showed the effects of LINC01615 knockdown on proliferation in HCT116 cells without serum supplementation. (J) Colony formation assays were performed to examine the effects of LINC01615 knockdown on proliferation in HCT116 cells without serum supplementation. (K) Statistical analysis of colony formation numbers in (J). (L-M) CCK-8 assay showed the effect of LINC01615 knockdown on proliferation in DLD1 and HCT116 cells under normal conditions. (N-O) Images of flow cytometry analysis showing the effects of LINC01615 knockdown and LINC01615 overexpression on the apoptotic rate in DLD1, HCT116 and LoVo cells without serum supplementation. (P) Flow cytometry data presented as histograms showing the effects of LINC01615 knockdown on the apoptotic rate in HCT116 cells without serum supplementation. (Q) WB was used to examine the effects of LINC01615 knockdown on the expression of cleaved caspase 3, caspase 7, caspase 9 and BCL-2 in HCT116 cells without serum supplementation. GAPDH was used as a loading control. Data are shown as the mean ± S.E. * p<0.05, ** p<0.01. (TIF 6432 kb)

18_2022_4675_MOESM3_ESM.tif

Supplementary file3 Supplementary Figure 2 (A) HCT116 cells were transfected with LINC01615 shRNA or control shRNA. Then, the cells were cultured in medium containing [2-13C] glucose and without serum supplementation. Oxidative PPP flux was measured based on the rate of glucose consumption and the ratio of 13C incorporated into carbon 2 (indicating glycolysis) and carbon 3 (indicating PPP) of lactate by nuclear magnetic resonance (NMR) spectroscopy. (B) NADP+/NADPH ratios were measured in the cells from (A). (C) ROS levels were measured in the cells from (A). (D) Images of flow cytometry analysis showing the effects of LINC01615 knockdown and LINC01615 overexpression on the ROS level in DLD1 and LoVo cells without serum supplementation. (E) CCK-8 assay showed the effects of LINC01615 inhibition on proliferation in HCT116 cells treated with or without NAC under serum-free starvation conditions. (F) Images of flow cytometry analysis showing the effects of LINC01615 inhibition on the apoptotic rate in DLD1 and HCT116 cells treated with or without NAC under serum-free starvation conditions. (G) Flow cytometry data presented as histograms show the effects of LINC01615 inhibition on the apoptotic rate in HCT116 cells treated with or without NAC under serum-free starvation conditions. (H) WB was used to examine the effects of LINC01615 inhibition on cleaved caspase 3, caspase 7, caspase 9 and BCL-2 expression in HCT116 cells treated with or without NAC under serum-free starvation conditions. (I-J) CCK-8 assay showed the effects of LINC01615 inhibition on proliferation in DLD1 and HCT116 cells treated with or without R5P under serum-free starvation conditions. (K) Statistical analysis of EdU (+) cells in (Figure 3M). (L) EdU staining assay showed the effect of LINC01615 inhibition on nucleotide synthesis in HCT116 cells treated with or without R5P under serum-free starvation conditions. (M) Statistical analysis of EdU (+) cells in (L). (N-O) CCK-8 assay showed the effects of LINC01615 inhibition on proliferation in DLD1 and HCT116 cells treated with or without NADPH under serum-free starvation conditions. (P) Oil red O staining assay showed the effect of LINC01615 inhibition on lipid synthesis in HCT116 cells treated with or without NADPH under serum-free starvation conditions. Data are shown as the mean ± S.E. * p<0.05, ** p<0.01. (TIF 5630 kb)

18_2022_4675_MOESM4_ESM.tif

Supplementary file4 Supplementary Figure 3 (A) The top-scoring genes recurring in the PPP gene sets, including G6PD. (B) The statistical analysis of G6PD expression in 275 colon cancer tissues and 41 normal tissues; paired t-test. In the boxplots, the middle line depicts the median and the whiskers are the min to max range. (C) The correlation between LINC01615 and G6PD expression in 275 colon cancer tissues. (D) The expression of G6PD in normal tissues and CRC tissues was assessed by IHC. (E-F) Statistical analysis of EdU (+) cells in (Figure 4I-J). (G) Antibodies against hnRNPA1 were used for RIP, followed by LINC01615 qRT–PCR in DLD1 and LoVo cells. (H) Antibodies against hnRNPA1 were used for RIP, followed by LINC01615 qRT–PCR, in DLD1 cells with or without serum supplementation. (I) Antibodies against hnRNPA1 were used for RIP, followed by G6PD pre-mRNA qRT–PCR in DLD1 and LoVo cells. (J) Antibodies against hnRNPA1 were used for RIP, followed by G6PD pre-mRNA qRT–PCR in LoVo cells transfected with LINC01615 overexpression plasmid or control vector. (K-L) WB was used to examine the effects of hnRNPA1 knockdown and overexpression on the expression of hnRNPA1 in DLD1, HCT116 and LoVo cells. (M) Antibodies against Flag were used for RIP, followed by G6PD pre-mRNA qRT–PCR in DLD1 cells. (N) Colony formation assay was conducted to examine the effects of hnRNPA1 truncated plasmids with or without RRM2 domain on proliferation in DLD1 cells which overexpressing LINC01615 and without serum supplementation. (O) Statistical analysis of colony formation numbers in (N). (P) Images of flow cytometry analysis showing the effects of hnRNPA1 truncated plasmid with or without RRM2 domain on the apoptotic rate in DLD1 cells overexpressing LINC01615 under serum-free starvation conditions. (Q) Flow cytometry data presented as histograms showing the effects of hnRNPA1 truncated plasmid with or without RRM2 domain on the apoptotic rate in DLD1 cells overexpressing LINC01615 and without serum supplementation. Data are the means ± SD (n = 3 independent experiments), * p<0.05, ** p<0.01. (TIF 7126 kb)

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Supplementary file5 Supplementary Figure 4 (A) SRAMP (http://www.cuilab.cn/sramp/) prediction revealed several m6A sites distributed in LINC01615. (B) WB analysis of the expression of METTL3, METTL14 and FTO in DLD1 cells cultured with or without serum supplementation. (C-D) WB was used to examine the effects of METTL3 knockdown and overexpression on the expression of METTL3 in DLD1 and LoVo cells. (E) qRT–PCR analysis of the expression of LINC01615 in LoVo cells transfected with METTL3 overexpression plasmid or control vector. (F) qRT–PCR analysis of LINC01615 expression in LoVo cells cultured with or without serum supplementation and transfected with METTL3 overexpression plasmid or control vector. (G) The correlation between METTL3 and LINC01615 expression in colon tissues. (H) Statistical analysis of IHC staining scores in (Figure 6D). (I) meRIP-qPCR showing the m6A modification enrichment of LINC01615 in LoVo cells after METTL3 overexpression. (J) qRT–PCR analysis of the stability of LINC01615 in LoVo cells with or without METTL3 overexpression. Data are the means ± SD (n = 3 independent experiments), * p<0.05, ** p<0.01. (TIF 2502 kb)

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Supplementary file6 Supplementary Figure 5 (A) WB analysis of METTL3 protein stability in LoVo cells cultured with or without serum supplementation for different times. (B) Transmission electron microscopy images of autophagic vacuoles in DLD1 and LoVo cells under normal or serum-free starvation conditions. Arrows indicate autophagosomes; scale bar, 500 nm. (C) Representative confocal microscopy images of LC3B puncta distribution in LoVo cells under normal or serum-free starvation conditions and treated with or without Baf A1 (1000× magnification). (D-E) Statistical analysis of fluorescence puncta of autolysosome (free red) and autophagosome (yellow) in (Figure 6H) and (C). (F) WB analysis of ATG5, METTL3 and LC3B expression after ATG5 knockdown in DLD1 cells without serum supplementation. (G) The expression of LINC01615 after 3-MA, ATG5 knockdown and Baf A1 treatment in DLD1 cells without serum supplementation. (H) WB analysis of METTL3 and LC3B expression in LoVo cells treated with or without Baf-A1 under serum-free starvation conditions. (I) Co-IP analysis was performed with specific antibodies against HA and Flag in 293T cells under normal or serum-free starvation conditions after transfection of HA-METTL3 and Flag-LC3B plasmids. WB analysis of the interaction between METTL3 and LC3B. (J) Representative confocal microscopy images of METTL3, LC3B and LAMP1 distribution in DLD1 cells under serum-free starvation conditions (1000× magnification). Data are the means ± SD (n = 3 independent experiments), * p<0.05, ** p<0.01. (TIF 11211 kb)

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Supplementary file7 Supplementary Figure 6 (A) qRT-PCR was used to detect the expression of LINC01615 in two donor patients’ tumor tissues. (B) PDXs images of control group, sh-LINC01615 group, oxaliplatin group, and sh-LINC01615 combined with oxaliplatin group. (C) The expression levels of LINC01615 in PDX tumors of control group, sh-LINC01615 group, oxaliplatin group, and sh-LINC01615 combined with oxaliplatin group through qRT–PCR. (D) Statistical analysis of IHC staining scores in (Figure 7F). (E) WB analysis of LC3B and METTL3 expression in sh-Ctrl and sh-LINC01615 group treated with or without oxaliplatin. Data are the means ± SD (n = 3 independent experiments), * p<0.05, ** p<0.01. (TIF 3063 kb)

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Zhang, Y., Xu, L., Ren, Z. et al. LINC01615 maintains cell survival in adaptation to nutrient starvation through the pentose phosphate pathway and modulates chemosensitivity in colorectal cancer. Cell. Mol. Life Sci. 80, 20 (2023). https://doi.org/10.1007/s00018-022-04675-7

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