CAF secreted miR-522 suppresses ferroptosis and promotes acquired chemo-resistance in gastric cancer

Abstract

Background: Ferroptosis is a novel mode of non-apoptotic cell death induced by build-up of toxic lipid peroxides (lipid-ROS) in an iron dependent manner. Cancer-associated fibroblasts (CAFs) support tumor progression and drug resistance by secreting various bioactive substances, including exosomes. Yet, the role of CAFs in regulating lipid metabolism as well as ferroptosis of cancer cells is still unexplored and remains enigmatic.

Methods: Ferroptosis-related genes in gastric cancer (GC) were screened by using mass spectrum; exosomes were isolated by ultra-centrifugation and CAF secreted miRNAs were determined by RT-qPCR. Erastin was used to induce ferroptosis, and ferroptosis levels were evaluated by measuring lipid-ROS, cell viability and mitochondrial membrane potential.

Results: Here, we provide clinical evidence to show that arachidonate lipoxygenase 15 (ALOX15) is closely related with lipid-ROS production in gastric cancer, and that exosome-miR-522 serves as a potential inhibitor of ALOX15. By using primary stromal cells and cancer cells, we prove that exosome-miR-522 is mainly derived from CAFs in tumor microenvironment. Moreover, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was found to mediate miR-522 packing into exosomes, and ubiquitin-specific protease 7 (USP7) stabilizes hnRNPA1 through de-ubiquitination. Importantly, cisplatin and paclitaxel promote miR-522 secretion from CAFs by activating USP7/hnRNPA1 axis, leading to ALOX15 suppression and decreased lipid-ROS accumulation in cancer cells, and ultimately result in decreased chemo-sensitivity.

Conclusions: The present study demonstrates that CAFs secrete exosomal miR-522 to inhibit ferroptosis in cancer cells by targeting ALOX15 and blocking lipid-ROS accumulation. The intercellular pathway, comprising USP7, hnRNPA1, exo-miR-522 and ALOX15, reveals new mechanism of acquired chemo-resistance in GC.

Keywords: Cancer-associated fibroblasts; Exosomes; Ferroptosis; GC; miR-522.

Fig. 1

ALOX15 is related to ferroptosis in gastric cancer. a. Mass spectrum analysis of gene expression changes in gastric tumor tissues (n = 45). The heatmap depicts the relative protein abundance in tumor tissues (T) and paired para-carcinoma tissues (P). b. Validation of ALOX15 dys-regulation in GC by using western blotting analysis (n = 12). c. Quantitative analysis of (b) (n = 12). d. Relative levels of ALOX15 mRNA in gastric tumor tissues (n = 12). e. Analysis of ALOX15 distribution in tumor tissues by using IHC (n = 12). f. High expression of ALOX15 predicts poor overall survival in GC. Patients were divided into ALOX15 high group (n = 77) and ALOX15 low group (n = 86) base on the average value of ALOX15 protein levels. g. Relative levels of lipid-ROS levels in gastric tumor tissues (n = 12). h. ALOX15 is positively co-related with Lipid-ROS production (n = 12). ** indicates p < 0.01

Fig. 2

Ferroptosis-related exo-miR-522 mainly derived from CAFs. a. Immunofluorescence staining for α-SMA, FAP, and FSP1 expression of NFs and CAFs (scale bar, 20 μm). b. Western blot analysis of α-SMA, FAP, and FSP1 protein levels in six paired NFs and CAFs (n = 6). c. Comparison of miR-522 levels in primary NFs, tumor cells (TCs) and CAFs (n = 12). d. TEM image of exosomes isolated from primary NFs, TCs and CAFs (scale bar, 100 nm). e. Western blotting analysis of CD63, Alix, and Tsg101 in exosomes. f. Relative levels of miR-522 in the exosomes described above (n = 12). g. The levels of exo-miR-522 derived from CAFs is negatively linked with ALOX15 expression (n = 12). h. CAF exo-miR-522 is negatively related with lipid-ROS levels (n = 12). ** indicates p < 0.01 and *** indicates p < 0.001

Fig. 3

CAFs secrete exo-miR-522 to suppress ferroptosis of GC cells. a. TEM images of exosomes isolated from primary NFs, TCs and CAFs (scale bar, 100 nm). b. WB analysis of exosomal markers in the exosomes isolated from GC cell lines and CAFs. c. Quantification of miR-522 in both cells and exosomes (n = 3). d. Confocal microscopy image of the internalization of fluorescently labeled CAF exosomes in SGC7901 cells and MKN45 cells. Scale bars, 50 μm. e. Effects of CAF-secreted miR-522 on the expression of ALOX15 in GC cells (n = 3). f. Quantitative analysis of (e) (n = 3). g. Relative levels of ALOX15 mRNAs in GC cells treated as described above (n = 3). h-k. Exo-miR-522 derived from CAFs suppresses erastin-induced ferroptosis in GC cells. CAF exo-miR-522 inhibits lipid-ROS accumulation (h), decreases erastin-induced cell death (i) and reduces abnormal increase of MMP (j, k). ** indicates p < 0.01 and *** indicates p < 0.001

Fig. 4

USP7 promotes miR-522 secretion by regulating deubiquitination of hnRNPA1 in CAFs. a. IHC analysis of USP7, hnRNPA1 and ALOX15 by using serial sections in paired para-carcinoma tissues and tumor tissues (n = 12). b. WB analysis of hnRNPA1 and USP7 in 12 paired CAFs and NFs (n = 12). c-d. Quantitative analysis of hnRNPA1 (c) and USP7 (d) described in (b) (n = 12). e-g. Analysis of the correlation between USP7, hnRNPA1 and exo-miR-522 in CAFs. HnRNPA1 is positively related with exo-miR-522 (e), and USP7 also showed positive relevance with both exo-miR-522 (f) and hnRNPA1 (g) (n = 12). h. WB analysis of hnRNPA1 and USP7 in CAFs treated with hnRNPA1 overexpression plasmids (OE.hnRNPA1), hnRNPA1 siRNA (si.hnRNPA1), USP7 overexpression plasmids (OE.USP7) and USP7 siRNA (si. USP7) respectively (n = 3). i. Quantitative analysis of (g) (n = 12). j. USP7/hnRNPA1 promotes miR-522 secretion (n = 3). k. Immuno-precipitation shows the direct interaction between USP7 and hnRNPA1 in CAFs (n = 3). l. WB analysis of hnRNPA1 ubiquitination in CAFs treated with OE.USP7 or si. USP7 (n = 3). ** indicates p < 0.01

Fig. 5

Direct evidence for hnRNPA1 mediated miR-522 packaging into CAF exosomes. a. RBPDB analysis of the specific interaction between miR-522 and RBP motifs (threshold 0.5). b. WB analysis of hnRNPA1, PABPC1 and ACO1 expression in CAFs transfected with corresponding siRNAs (n = 3). c. Relative levels of miR-522 in CAFs transfected with siRNAs (n = 3). d. Quantification of miR-522 in CAF exosomes treated as described above (n = 3). e. Detection of hnRNPA1 protein in the samples derived from miR-522 pull downs performed in CAFs and CAF exosomes (n = 3). f. Schematic diagram for the cell co-culture model of CAFs and SGC7901 cells. h. Capture of CAF-exosome delivered Cys-miR-522 by SGC7901 cells co-cultured with CAFs (n = 3). i. Quantitative analysis of (h) (n = 3). j. Capture of CAF-exosome delivered Cys-miR-522 by MKN45 cells co-cultured with CAFs (n = 3). k. Quantitative analysis of (J) (n = 3). ** indicates p < 0.01; *** indicates p < 0.001

Fig. 6

Chemo-toxicity up-regulates USP7/hnRNPA1 and promotes miR-522 secretion from CAFs. a-b The effects of cisplatin (a) and paclitaxel (b) on cell viability of CAFs (n = 3). c. The expression of USP7 and hnRNPA1 in CAFs treated with sublethal doses of cisplatin (0.8 μg/ml) and paclitaxel (100 nmol/L) (n = 3). d. Quantitative analysis of (c) (n = 3). e. Chemo-toxicity reduces ubiquitination of hnRNPA1 (n = 3). f. Quantitative analysis of (e) (n = 3). g. Effects of cisplatin and paclitaxel on the expression and secretion of miR-522 (n = 3). h-i. Effects of exosomes derived from chemo-toxicity treated CAFs on ALOX15 expression in GC cells. The ALOX15 protein was determined by WB assay (h) and was quantified by gray analysis (i) (n = 3), and ALOX15 mRNA was checked by qRT-PCR (j) (n = 3). k. Exosomes of chemo-toxicity treated CAFs inhibit erastin induced lipid-ROS production in GC cells (n = 3). l. Exosomes of chemo-toxicity treated CAFs inhibit erastin induced cell death of GC cells (n = 3). m. Exosomes of chemo-toxicity treated CAFs contribute to acquired chemo-resistance of GC cells (n = 3). * indicates p < 0.05 and ** indicates p < 0.01

Fig. 7

In vivo role of USP7/hnRNPA1/exo-miR-522 pathway in regulating ferroptosis and chemo-sensitivity of gastric tumors. a. Schematic description of the experimental design used to establish the animal model. b. Images of tumors in each group (n = 6). c. Alterations of tumor diameters in each group (n = 6). d. Weight measurements of the tumors described above (n = 6). e. Relative levels of lipid-ROS in tumors (n = 6). f. WB analysis of USP7 and hnRNPA1 in primary CAFs and ALOX15 in tumor tissues (n = 6). g. Quantitative analysis of (f). h. IHC analysis of ALOX15 distribution in tumor tissues (n = 6). i. Relative levels of ALOX15 in tumor tissues (n = 6). j. TEM images of exosomes isolated from mouse serum. k. Relative levels of miR-522 in serum exosomes (n = 6). l-m. Quantification of ferroptosis marker (l) and apoptosis marker (m) (n = 6). ** indicates p < 0.01

Fig. 8

Effects of USP7/hnRNPA1/exo-miR-522 pathway on the survival of tumor-implanted mice. a-b. Kaplan–Meier curves of mice in the saline groups (a) and cisplatin groups (b) (n = 6). c. A proposed model illustrating the role of CAF-derived exosomal miR-522 in regulating ferroptosis in GC cells. ** indicates p < 0.01