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. 2024 Jun 17;18(1):67.
doi: 10.1186/s40246-024-00635-3.

Growth characteristics of HCT116 xenografts lacking asparagine synthetase vary according to sex

Oladimeji Aladelokun  1 , Lingeng Lu  2 , Jie Zheng  1 , Hong Yan  1 , Abhishek Jain  1 , Joanna Gibson  3 , Sajid A Khan  4 , Caroline H Johnson  5
Affiliations

Growth characteristics of HCT116 xenografts lacking asparagine synthetase vary according to sex

Oladimeji Aladelokun et al. Hum Genomics. .

Abstract

Background: Sex-related differences in colorectal (CRC) incidence and mortality are well-documented. However, the impact of sex on metabolic pathways that drive cancer growth is not well understood. High expression of asparagine synthetase (ASNS) is associated with inferior survival for female CRC patients only. Here, we used a CRISPR/Cas9 technology to generate HCT116 ASNS-/- and HCT 116 ASNS+/+ cancer cell lines. We examine the effects of ASNS deletion on tumor growth and the subsequent rewiring of metabolic pathways in male and female Rag2/IL2RG mice.

Results: ASNS loss reduces cancer burden in male and female tumor-bearing mice (40% reduction, q < 0.05), triggers metabolic reprogramming including gluconeogenesis, but confers a survival improvement (30 days median survival, q < 0.05) in female tumor-bearing mice alone. Transcriptomic analyses revealed upregulation of G-protein coupled estrogen receptor (GPER1) in tumors from male and female mice with HCT116 ASNS-/- xenograft. Estradiol activates GPER1 in vitro in the presence of ASNS and suppresses tumor growth.

Conclusions: Our study indicates that inferior survival for female CRC patients with high ASNS may be due to metabolic reprogramming that sustains tumor growth. These findings have translational relevance as ASNS/GPER1 signaling could be a future therapeutic target to improve the survival of female CRC patients.

Keywords: ASNS; GPER1; Colorectal cancer; Metabolism; Sex differences.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Loss of asparagine synthetase (ASNS) prevents compact spheroid formation in asparagine (Asn) adjusted media and inhibits tumorigenesis in vivo. A. ASNS transcript levels in human normal-adjacent colon tissues (n = 51) and colorectal adenocarcinoma tissues (n = 591) from TCGA (COAD) database. B. Selective targeting of ASNS exon 5 in HCT116 cancer cell line. ASNS−/− cell line was generated via CRISPR-Cas9 mediated deletion of exon 5. Mock-transfection of HCT116 wild type ASNS cells with Cas9. Western blot analysis confirmed the knock-out of ASNS showing reduced protein expression. qPCR revealed a significant reduced number of ASNS transcript in the CRISPR-Cas9 edited cell line compared to the ASNS+/+, thus confirming a knock-out efficiency. C. HCT116 ASNS+/+ and HCT116 ASNS−/− cell lines were cultured in DMEM and RPMI media. Representative images of spheroids derived from ASNS+/+ pool and a CRISPR-Cas9 edited clones grown under asparagine depleted conditions (0 mM Asn) and Asn supplemented conditions (4 mM Asn). Spheroid image captured 7 days after seeding using 4× objective lens of an inverted microscope. Note the structural differences and”fuzziness” around the edges in the ASNS+/+ cell lines. D Experimental design of tumorigenicity experiment using Rag2/IL2RG (R2G2) immunocompromised mice, n = 5 mice per group. Female R2G2 immunodeficient mice were inoculated with ten million cells each of CRISPR- edited ASNS-/- and the control ASNS+/+ lines. Day 0 represents the day when cell inoculation was performed. E Tumor kinetics reveal a significant difference (p < 0.05) in tumor volume of HCT116 ASNS−/− mice compared to HCT116 ASNS+/+ mice at day 20 post-implantation. F Representative images of mice tumors from each group. Note that tumor size was relatively smaller in the HCT116 ASNS−/− mice compared to the HCT116 ASNS+/+ mice. G Average tumor volume at day 20 post implantation. H Hematoxylin and eosin staining of tumors showing mitotic activity index in the HCT116 ASNS+/+ and HCT116 ASNS−/− tumor-bearing female mice. I Reduced Ki67 immunostaining in the HCT116 ASNS−/− tumors compared to HCT116 ASNS+/+ tumors. J Quantification of mitotic activity index in the HCT116 ASNS+/+ and HCT116 ASNS−/− tumors. K Proliferative index for HCT116 ASNS+/+ and HCT116 ASNS−/− indicated by Ki67 reactivity. Green box indicates mitotic figures. Representative hematoxylin and eosin staining of tumors derived from HCT116 ASNS+/+ and HCT116 ASNS−/− mice under 100 × total magnification. Mitotic figures (MFs) in the HCT116 ASNS+/+ and HCT116 ASNS−/− tumors under 400× total magnification. Extraction of ASNS expression dataset from TCGA database was performed using R studio and individual scattered dot plot represents expression Log 2 abundance of ASNS. Individual scattered plots for gene expression analysis of HCT116 ASNS+/+ and HCT116 ASNS−/− cells (Fig. 1B), are n = 6 replicates per genotype. Individual scattered plots for spheroid experiments are mean ± SEM of at least 10 spheroids/group. Statistical significance indicated by *p < 0.05, ***p < 0.001 and ****p < 0.0001 using two-tailed Student’s t- test
Fig. 2
Fig. 2
Loss of ASNS extends tumor-specific survival and triggers metabolic reprogramming in male and female R2G2 mice (Study 2). A Study design (n = 10 mice per group): R2G2 mice were inoculated with HCT116 ASNS+/+ and HCT116 ASNS−/− cells lines. Day 0 represents the day when inoculation was performed. In Group 1, male R2G2 mice received 10 million ASNS+/+ cells injected subcutaneously (s.c). Group 2 represents male mice that were implanted with 10 million HCT116 ASNS homozygous knockout cells. In Group 3, female R2G2 mice were implanted with HCT116 ASNS+/+ cells. Group 4 female R2G2 mice were implanted with HCT116 ASNS−/− cells lines. B Tumor kinetics after cell lines implantation. Tumorigenicity experiment showing a sustained growth kinetics in the four groups after subcutaneous implantation of cell lines. C Tumor volume at 18 days post-tumor implantation. D Kaplan Meier curve indicates that loss of ASNS dramatically extends survival of female R2G2 mice by 11 days when compared to the ASNS+/+ male. E Representative images of colorectal cancer tissue microarray (TMA) from ASNS tumors showing immunohistochemical staining of Ki67, cyclin D1 and pHH3. Hematoxylin and eosin stained-sections are also included. 100× magnification (Ki67, Cyclin D1 and H&E) and 400× magnification (phospho-histone H3, pHH3). Quantitation of Ki67 and pHH3 staining in 4 groups (n = 5 mice per group). F Untargeted metabolomics revealed sex differences in transsulfuration and urea cycle metabolism in tumor metabolome. Box and whiskers plots display median value (center line) and whiskers showing the minimum and maximum value. Violin plots of transformed metabolite levels were generated using Graphpad prism. HCT116 ASNS female+/+ n = 10, HCT116 ASNS−/− female n = 8, HCT116 ASNS+/+ Male n = 10, HCT116 ASNS−/− male n = 10. Statistical significance difference was determined using two-way ANOVA with mixed-effects model for the growth kinetics in B, and mean tumor volumes (day 18) in C. Two-way ANOVA with mixed-effects model was used to accommodate the maximum tumor volume reached in several of the HCT116 ASNS+/+ male mice starting day 9 post tumor implantation. Chi-square Fisher’s exact test was used to estimate the difference in proportion of positively stained cells (E-F). Data in the bar plot are presented as mean ± SEM. For tumor growth kinetics and metabolomics data, p value was adjusted for multiple comparisons using FDR (Benjamini Hochberg) correction method. * denotes q < 0.05, and ** denotes q < 0.01
Fig. 3
Fig. 3
Transcriptomic profiling of HCT116 cell-line derived tumors identifies genes related to ASNS in females. A Principal component analysis (PCA) shows a unique separation of ASNS−/− samples from the ASNS+/+ control using normalized count (n = 4 mice/genotype). B Heatmap reveals the top 12 differentially expressed genes using a fold change cut off = 2 after adjusting for multiple comparisons. C Validation of gene expression using RT-qPCR for asparagine synthetase (ASNS), RNA sequencing revealed the downregulation of the glutamate aspartate transporter 1 (SLC1A3). D Ingenuity pathway analysis (IPA) revealed metabolic pathways impacted by ASNS signaling disruption. E. Analysis of top differentially expressed genes revealed that the key metabolic genes Protein Kinase AMP-Activated Catalytic Subunit Alpha 2 (AMPKa2) and Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) were significantly (p < 0.05) downregulated in the female ASNS−/− tumors. F Gene set enrichment analysis (GSEA) revealed GPCR ligand binding enriched in the HCT116 ASNS+/+.with a normalized enrichment score (NES) of − 1.8 and a q value of 0.16 G. Relative mRNA expression of GPER1 in the HCT116 ASNS+/+ female and HCT116 ASNS−/− female tumors. H. Correlation between ASNS and GPER1 using RNA Seq count value. PCA was conducted using normalized log count of annotated genes to generate plot on Qlucore v3.8. Normalization was set to Mean = 0 and variance = 1. q value was set as 0.1. Heatmap showing DEGs from DESEQ2 on R studio. (HCT116 ASNS+/+, n = 4 and HCT116 ASNS−/−, n = 4). Individual dot plot represents mean ± SEM with p value < 0.05 considered to be significant using two-sided t-test. * represents p < 0.05 and **denotes p < 0.01. An outlier in the ASNS−/− group was excluded from the TNFRSF9 dot plot due to the non-detectability of TNFRSF9 in 75% of tumor samples in the ASNS−/− group. For all qPCR experiments, an outlier in HCT116 ASNS−/− group was also excluded due to the non-detectability of RNA transcripts in one sample (CT value > 36). Canonical pathways were identified using a –log (p-value) score cutoff of 1.4 and Fisher’s Exact Test on the IPA program. Linear regression plot was generated using R studio. GSEA was conducted using normalized count data and an FDR q value cut off < 25%
Fig. 4
Fig. 4
Relationship between asparagine synthetase (ASNS) and G-protein coupled estrogen receptor (GPER) signaling. A Western blot indicating ASNS and GPER1 expression in estradiol (E2)-treated ASNS+/+ cell lines. RPMI 1640 containing 0.4 mM of asparagine and supplemented with 4mM glutamine was used for in vitro experiments. G1, a GPER agonist, serves as a positive control for the western blot experiment. B Quantitation of GPER1 levels in E2-treated ASNS+/+ cell line. C Quantitation of ASNS levels in in E2- treated ASNS+/+ cell line n = 3 replicates per condition D Estradiol treatment of HCT116 ASNS+/+ cells under normal nutrient condition. Cells were treated with media supplemented E2 (10 µM). E Estradiol treatment of HCT116 ASNS−/− cells under sufficient nutrient condition. Individual dot plots represent mean ± SEM. Statistical significance was determined using student t-test with *indicating p < 0.05, **indicating p < 0.01, *** indicating p < 0.001 and **** indicating p < 0.0001 after multiple comparison (Benjamini Hochberg’s FDR method). ANOVA was used to compare differences in mean values of the E2, G1 and control groups (B and C). Quantification of band intensity was conducted using Image J software
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