AADAC protects colorectal cancer liver colonization from ferroptosis through SLC7A11-dependent inhibition of lipid peroxidation

Lipid peroxidation restrains CRC liver colonization

Oxidative stress caused by lipid peroxidation poses great threats to metastatic tumor cells in distant organs. To evaluate whether CRC cells experience higher oxidative stress when colonizing in the liver, we tested the GSH/GSSG ratio in paired PTs and LMs from 10 CRLM patients. LMs showed a lower GSH/GSSG ratio than PTs (p = 0.0077) (Fig. 1A). In particular, GSH levels were significantly lower in LMs than in PTs (p = 0.0028), while GSSG levels showed no significant difference (p = 0.3144) (Fig. 1B, C). We then tested the MDA concentration to examine the lipid peroxidation level, and LMs also exhibited higher MDA concentrations than PTs (p = 0.0262) (Fig. 1D).

Fig. 1figure 1

Lipid peroxidation restrains colorectal liver colonization. A-C Relative GSH/GSSG ratio (A), GSH level (B), and GSSG level (C) in paired primary tumors (PTs) and liver metastases (LMs) from CRLM patients. D Relative MDA levels in paired PTs and LMs from CRLM patients. E Schematic diagram of orthotopic CRLM mouse model establishment and subsequent sample arrangement. F–H Relative GSH/GSSG ratio (F), GSH level (G), and GSSG level (H) in paired PTs and LMs from the orthotopic CRLM mouse model. I Relative MDA levels in paired PTs and LMs from the orthotopic CRLM mouse model. J Representative bright field images, HE staining of LMs (scale bar, 1 mm), quantification of PT size and LM number derived from orthotopic CRLM mouse models treated with liproxstatin-1 or vehicle. K Schematic diagram of splenic injection CRC liver colonization mouse model establishment. L Representative bright field images, HE staining (scale bar, 5 mm), and average tumor area derived from the CRC liver colonization mouse model treated with liproxstatin-1 or vehicle. Significance was calculated by two-tailed ratio t test (A-D, F-I, right of J and L). p value < 0.001 (***), p value < 0.01 (**), p value < 0.05 (*), ns (not significant)

To further verify the differences in the GSH/GSSG ratio and MDA concentration between PTs and LMs, we next established an in vivo orthotopic CRC model with spontaneous liver metastasis in BALB/c nude mice (Fig. 1E). After cecal planting, liver metastasis subsequently developed in 7 weeks, and the GSH/GSSG ratio and MDA concentration in PTs and LMs were measured. Consistent with clinical samples, a significantly lower GSH/GSSG ratio (p = 0.0039), lower GSH level (p = 0.0411), and higher MDA concentrations (p = 0.0425) were observed in LMs than in PTs, and no significant difference was observed in the GSSG level (p = 0.1433) (Fig. 1F-I), thereby recapitulating our observed profile of oxidative stress (GSH/GSSG ratio and MDA) in CRLM patient samples. Together, the above results suggested that CRC cells disseminated in the liver experienced higher levels of lipid peroxidation than those in the primary tumor.

Then we investigated the functional effect of lipid peroxidation during CRC liver metastasis in two CRLM mouse models, the abovementioned orthotopic CRLM mouse model and a CRC liver colonization mouse model by splenic injection. After CRC cell implantation, the mice were treated with vehicle or liproxstatin-1 (a lipid peroxidation inhibitor) daily by intraperitoneal injection. In orthotopic CRLM mice, liproxstatin-1 significantly increased the tumor volume of LMs (p < 0.0001) but had little effect on growth of PTs (p = 0.6733) (Fig. 1J). In the CRC liver colonization mouse model, liproxstatin-1 also significantly increased the tumor volume of LMs (p < 0.0001) (Fig. 1K, L). The above results suggested that inhibition of lipid peroxidation markedly promoted CRC liver colonization in different mouse models.

AADAC is upregulated in CRC liver metastases and associated with poor prognosis

During the process of lipid peroxidation, polyunsaturated fatty acids (PUFAs) are first biosynthesized by enzymes such as acyl-CoA synthetase long chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3), then they are converted into hydroperoxides by a series of lipid enzymes and cause damage to the cellular lipid bilayer [26]. This indicates that certain lipid metabolism-related genes may play a role in promoting metastatic CRC survival through elevated oxidative stress in the liver. Hence, we performed RNA-seq on paired PTs and LMs from 3 CRLM patients, and paired differential analysis between PTs and LMs revealed a set of differentially expressed genes (DEGs). Among 243 upregulated genes in LMs, we identified 13 genes involved in lipid metabolism (Supplementary Fig. 1A, B), and enrichment analysis showed that these genes played roles in several lipid metabolic processes (Fig. 2A) (Supplementary Fig. 1C, D). Among the above 13 genes, 5 were most frequently enriched in lipid metabolic processes, which contained 3 secretary factors (FGF21, APOB, and APOC2). Among the 5 genes, AADAC was a lipid enzyme overexpressed more than 4 times in LMs compared to PTs, indicating its potential role in lipid peroxidation mentioned above (Fig. 2B). Then, the expression of AADAC was compared between paired PTs and LMs from 3 independent gene expression profiling datasets (GSE81582, GSE14297, GSE49355), and the results confirmed that AADAC was significantly upregulated in LMs compared with its counterpart in PTs (Fig. 2C).

Fig. 2figure 2

AADAC is upregulated in CRC liver metastases and associated with poor prognosis. A GO analysis of the 13 most enriched lipid metabolism-related genes upregulated in LMs. B Fold change, p value and FDR of the 5 most enriched genes involved in lipid metabolic processes. C Bioinformatic analysis comparing AADAC expression levels between paired PTs and LMs from CRLM patients in 3 GEO datasets: GSE81582 (n = 19), GSE14297 (n = 18), and GSE49355 (n = 20). D Relative AADAC mRNA levels in paired PTs and LMs from CRLM patients (n = 19). E Protein expression of AADAC in paired PTs and LMs from CRLM patients (n = 10). F IHC staining of AADAC expression in paired PTs and LMs from CRLM patients (n = 157). The upper scale bar represents 500 μm. The lower scale bar represents 100 μm. G-H Relative mRNA (G) and protein (H) levels of AADAC in paired PTs and LMs from an orthotopic CRLM mouse model (n = 4). I Kaplan–Meier plots of OS and RFS after hepatectomy in CRLM patients stratified by AADAC expression in LMs and PTs. Significance was calculated by a two-tailed ratio t test (C-D, right of F, G). p value < 0.001 (***), p value < 0.01 (**), p value < 0,05 (*), ns (not significant)

We then collected another set of paired PT and LM samples from 19 CRLM patients in our center and carried out qRT-PCR and immunoblotting to measure the expression of AADAC. Consistently, both the mRNA and protein levels of AADAC in LMs were significantly higher than those in PTs (qRT-PCR p = 0.0003) (Fig. 2D, E) (Supplementary Fig. 1E). AADAC expression evaluated by IHC in a CRLM TMA cohort also confirmed the significant upregulation of AADAC in LMs compared with paired PTs (n = 157, p < 0.0001) (Fig. 2F). Finally, AADAC mRNA and protein expression was also compared in PTs and LMs from an orthotopic CRLM mouse model. Consistently, LMs indeed showed higher expression of AADAC than PTs (qRT-PCR p < 0.0017) (Fig. 2G, H).

Survival analysis of the CRLM TMA cohort showed that CRLM patients with higher AADAC expression (higher IHC score) in LMs showed significantly shorter OS (p = 0.015, HR = 1.782) after hepatectomy, while its association with RFS (p = 0.96, HR = 0.989) was not significant. In contrast, the expression of AADAC in PTs had no impact on either OS (p = 0.25, HR = 0.665) or RFS (p = 0.13, HR = 0.627). Survival analyses in colorectal cancer (p = 0.911, HR = 0.98) and colon cancer (p = 0.794, HR = 1.05) from the TCGA cohort also showed that the expression of AADAC in primary tumors had no association with OS (Fig. 2I) (Supplementary Fig. 1F, G). In addition, although associations between AADAC expression level in LMs and number of LMs, size of LMs, and clinical risk scores (CRS) evaluated by Pearson’s χ2 tests were not significant, patients with higher AADAC expression in LMs still had a larger proportion to develop larger number (52.1%), greater size (70.6%), and higher CRS (61.3%) than those with lower AADAC expression (Supplementary Table 1). Spearman correlation analysis showed that the regional lymph node involvement stage (N classification) of PTs was positively correlated with the AADAC expression level in LMs (LM-AADAC) (p = 0.042) (Supplementary Table 2). Moreover, univariate Cox regression analyses showed that LM-AADAC (p = 0.015, HR = 1.782), N classification (p = 0.009, HR = 1.220), number of liver metastases (p = 0.037, HR = 1.668), metastases in bilateral liver lobes (p = 0.018, HR = 1.735), and recurrence states (p = 0.004, HR = 2.082) were significantly related to OS (Supplementary Table 3), while only LM-AADAC (p = 0.007, HR = 1.976), N classification (p = 0.032, HR = 1.183), and recurrence states (p < 0.005, HR = 2.611) were significantly related to OS in further multivariate Cox regression analyses (Supplementary Table 4). Altogether, these data suggested that AADAC was upregulated in LMs, and that AADAC overexpression in LMs was closely related to poor prognosis.

AADAC promotes CRC proliferation and liver colonization

To investigate the oncogenic role of AADAC, we knocked down and overexpressed AADAC in the CRC cell lines HCT116 and SW480, respectively (Fig. 3A). In functional assays, deletion of AADAC in HCT116 cells dramatically impaired the proliferation (p < 0.001) and colony formation ability (p < 0.001), while ectopic overexpression of AADAC in SW480 cells pronouncedly enhanced proliferation (p < 0.001) and colony formation ability (p < 0.001) (Fig. 3B, C). Consistently, the Edu staining assay confirmed that compared to their control cells, AADAC-oe (ADOE) SW480 cells were significantly more active in DNA replication (p < 0.001), while sh-AADAC HCT116 cells showed decreased DNA replication activity (p < 0.001) (Fig. 3D). In the CRC liver colonization model, mice injected with sh-AADAC HCT116 cells exhibited a markedly decreased number of liver colonies compared to those injected with shNC cells (p < 0.001) (Fig. 3E, F). These results indicated that AADAC played a critical role in CRC proliferation and liver colonization.

Fig. 3figure 3

AADAC promotes CRC proliferation and liver colonization. A Protein expression of AADAC in sh-AADAC HCT116 cells and ADOE SW480 cells. B The proliferation ability of sh-AADAC and ADOE cells. C The colony formation ability of sh-AADAC and ADOE cells. D Representative fluorescent images and relative quantification of EdU staining in sh-AADAC and ADOE cells. E Representative images and HE staining (scale bar, 1 mm) derived from CRC liver colonization mouse model with shNC and sh-AADAC cells. F Quantification of liver colonies in (E). Data are shown as the mean ± SD. Significance was calculated by two-way ANOVA (B) and two-tailed ratio t test (C-F). p value < 0.001 (***), p value < 0.01 (**), p value < 0,05 (*), ns (not significant)

AADAC reduces lipid peroxidation

To determine whether AADAC is involved in regulating lipid peroxidation, we conducted untargeted lipidomic analysis of shNC and sh-AADAC HCT116 cells. Among the 32 most upregulated lipid species, phosphatidylethanolamines (PEs) emerged as one of the most enriched lipid species. Further profiling of these upregulated PEs showed that PUFA-containing lipids including oxygenated PEs, which play indispensable roles in lipid peroxidation as mentioned before, were significantly upregulated in sh-AADAC HCT116 cells (Fig. 4A, B). Consistently, a significant increase in lipid reactive oxygen species (ROS) was observed in sh-AADAC cells compared to shNC cells, confirming the role of AADAC in reducing lipid peroxidation (Fig. 4C). It has been reported that lipid droplets (LDs) act as a form of storage for excess PUFAs in lipid peroxidation [27]. Compared to control cells, we observed significantly increased cellular LDs in sh-AADAC HCT116 cells and decreased LDs in ADOE cells (Fig. 4D). This indicated that AADAC not only reduced the accumulation of PUFA-containing PEs, but also restrained the formation of LDs.

Fig. 4figure 4

AADAC reduces lipid peroxidation. A Significantly enriched lipid species detected by untargeted lipidomic analysis in sh-AADAC HCT116 cells compared to shNC cells. B Heatmap of PUFA-containing PEs significantly enriched in sh-AADAC HCT116 cells. C Representative ROS levels in shNC and sh-AADAC HCT116 cells. D Representative images and quantification of lipid droplets in control, sh-AADAC, and AADAC-oe (ADOE) HCT116 cells (scale, 100 μm). E Venn diagram of significantly enriched and proferroptotic lipid species among 84 upregulated lipids in sh-AADAC cells. F Fold change, p value, and VIP value of 5 proferroptotic lipid species in (E). Fold change > 1.5; FDR-related p value < 0.05; VIP value of lipidomic analysis > 1. G Relative cellular MDA levels in shNC and sh-AADAC HCT116 cells treated with 15 μM erastin. H Relative levels of GSH, GSSG, and the GSH/GSSG ratio in sh-AADAC HCT116 cells. Data are shown as the mean ± SD. Significance was calculated by a two-tailed ratio t test (right of C, left of D, G-H). p value < 0.001 (***), p value < 0.01 (**), p value < 0,05 (*), ns (not significant)

Notably, dysfunctional ROS scavenging and lipid peroxidation play indispensable roles in ferroptosis. Among the 84 most enriched lipid species, including PEs and other fatty acids (FAs), we noted that 5 species were reported to promote ferroptosis [27, 28], and only 4 out of the 5 species remained statistically enriched after further assessment (variable importance in the projection (VIP) value of lipidomic analyses > 1; p value < 0.05; fold change > 1.5) (Fig. 4E, F). Furthermore, we observed higher MDA levels in sh-AADAC HCT116 cells (p < 0.001) (Fig. 4G) and reduced MDA levels in ADOE SW480 cells (p < 0.01) (Supplementary Fig. 2A). Consistently, deletion of AADAC resulted in a significant decrease in GSH levels (p < 0.001) and the GSH/GSSG ratio (p < 0.01) (Fig. 4H). As expected, overexpression of AADAC reversed the above effects on GSH levels (p < 0.001) and the GSH/GSSG ratio (p < 0.001), further confirming the anti-lipid peroxidation role of AADAC (Supplementary Fig. 2B).

AADAC upregulates SLC7A11 to suppress ferroptosis

To investigate the effect of AADAC on the sensitivity of cells to ferroptosis, we administered graded concentrations of erastin, an agonist that specifically induces ferroptosis, to sh-AADAC and shNC HCT116 cells. Compared to shNC HCT116 cells, remarkably attenuated cell viability was observed in sh-AADAC cells. In contrast, overexpression of AADAC in SW480 cells partially abrogated the ferroptotic cell death at different concentrations of erastin. (Fig. 5A).

Fig. 5figure 5

AADAC upregulates SLC7A11 to suppress ferroptosis. A Cell viability of sh-AADAC HCT116 and ADOE SW480 cells treated with graded concentrations of erastin. B Protein expression of ferroptosis-related genes in sh-AADAC HCT116 cells and ADOE SW480 cells. C Protein expression of SLC7A11 in control, ADOE, and ADOE + sh-SLC7A11 (ADSL) SW480 cells. D Relative levels of GSH, GSSG, and the GSH/GSSG ratio in control, ADOE, and ADSL SW480 cells. E Relative MDA levels in control, ADOE, and ADSL SW480 cells treated with 15 μM erastin. F-G Representative images, HE staining (scale bar, 1 mm) (F) and quantification (G) of liver colonies from CRC liver colonization mice model with different SW480 cells (control, ADOE, and ADSL). H Representative IHC staining of AADAC and SLC7A11 expression in the LMs of CRLM patients (left scale bar, 500 μm; right scale bar, 100 μm). I Pearson correlation between IHC H scores of AADAC and SLC7A11 expression in LMs. Data are shown as the mean ± SD. Significance was calculated by two-way ANOVA and two-tailed ratio t test (A, D, E, G). p value < 0.001 (***), p value < 0.01 (**), p value < 0,05 (*), ns (not significant)

The sensitivity of cells to ferroptosis is determined by various factors, namely, GSH biosynthesis, iron metabolism, PUFA metabolism, and so on. To unveil the mechanism by which AADAC protects CRC liver metastasis against ferroptosis, we next evaluated the expression of ferroptosis-related genes in sh-AADAC and ADOE CRC cell lines. Among these genes, SLC7A11, a vital ferroptosis regulator that is involved in cysteine-glutamate metabolism, exhibited a decrease in sh-AADAC, and an upregulation in ADOE cell lines. No significant changes were observed in other genes, such as ACSL4, GPX4, HO-1 and TFR2 (Fig. 5B). To verify whether SLC7A11 mediated the anti-ferroptosis effect of AADAC, we first constructed ADOE + sh-SLC7A11 (ADSL) SW480 cells (Fig. 5C). Overexpression of AADAC ameliorated oxidative stress as it caused a significant increase in the GSH/GSSG ratio (p < 0.001) and reduced MDA levels (p < 0.001), and further depletion of SLC7A11 abrogated this effect (Fig. 5E).

To further determine whether SLC7A11 mediated the promoting effect of AADAC on CRC liver colonization, a CRC liver colonization model was used to compare the colonization formation ability between Ctrl (AADAC-control), ADOE and ADSL SW480 cells. The results showed that mice injected with ADOE SW480 cells developed more liver colonies than those injected with Ctrl cells (p < 0.001), and depletion of SLC7A11 in ADOE cells potently reduced metastatic colonies (p < 0.001) (Fig. 5F, G). In IHC analysis of clinical CRLM samples, the expression of AADAC was positively correlated with that of SLC7A11 (p < 0.0001) (Fig. 5H, I). Collectively, this evidence revealed that AADAC upregulated SLC7A11 to suppress ferroptosis, thus promoting CRC liver colonization.

AADAC upregulates SLC7A11 by activating NRF2

To further understand the underlying mechanism by which AADAC upregulated SLC7A11, we analyzed canonical SLC7A11 upstream regulators. Immunoblotting showed that the expression of NRF2 was markedly downregulated after deletion of AADAC, and in contrast, overexpression of AADAC led to a significant increase in NRF2 expression. The expression of other potential regulators, such as ATF4 and AKT, did not show remarkable changes (Fig. 6A, B). Then we treated sh-AADAC cells with TBHQ, a metabolite that induces NRF2 activation, and found that TBHQ treatment significantly rescued SLC7A11 expression in sh-AADAC cells (Fig. 6B). These results indicated that NRF2 mediated the regulatory role of AADAC on SLC7A11 expression (Fig. 7).

Fig. 6figure 6

AADAC upregulates SLC7A11 by activating NRF2. A Protein expression of SLC7A11 upstream genes in sh-AADAC cells. B Protein expression of SLC7A11 in shNC, sh-AADAC and sh-AADAC + 10 μM TBHQ HCT116 cells. C Relative MDA levels in shNC, sh-AADAC, and sh-AADAC + 10 μM TBHQ HCT116 cells treated with 15 μM erastin, and in SW480 cells (control, ADOE, ADOE + ML385) treated with 15 μM erastin. D Relative GSH level, GSSG level and GSH/GSSG ratio of HCT116 cells (shNC, shNC + 10 μM TBHQ, and sh-AADAC + 10 μM TBHQ) and SW480 cells (control, control + ML385, ADOE + ML385). E Relative levels of lipid ROS in HCT116 and HT29 cells pretreated with or without 10 μM TBHQ for 1 h, and with 15 μM erastin for 48 h. F Data are shown as the mean ± SD. Significance was calculated by a two-tailed ratio t test (C, D, F). p value < 0.001 (***), p value < 0.01 (**), p value < 0,05 (*), ns (not significant)

Fig. 7figure 7

Schematic diagram of AADAC inhibition of lipid peroxidation and ferroptosis via NRF2/SLC7A11 axis

To verify whether NRF2 mediated the suppressive role of AADAC in lipid peroxidation and ferroptosis, we treated sh-AADAC cells with TBHQ and ML385, an NRF2 inhibitor. TBHQ treatment significantly reduced MDA levels and increased the GSH/GSSG ratio of sh-AADAC cells (Fig. 6C, D), while ML385 treatment significantly increased MDA level and reduced GSH/GSSG ratio in ADOE cells (Fig. 6C, D). Consistently, depletion of AADAC in HCT116 and HT29 cells caused significant increase in cellular lipid ROS, indicating failure to protect cells from erastin-induced cell death, while administration of TBHQ efficiently rescued this effect (Fig. 6E-F). Taken together, these results demonstrated that AADAC protected CRC liver colonization from ferroptosis through the NRF2-SLC7A11 axis (Fig. 7).

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