Tyrosine phosphatase SHP2 promoted the progression of CRC via modulating the PI3K/BRD4/TFEB signaling induced ferroptosis

Abstract

Objective: To elucidate the mechanism by which tyrosine phosphatase SHP2 protects CRC through modulation of TFEB-mediated ferritinophagy, thereby suppressing ROS and ferroptosis.

Methods: SW480 and SW620 cells, in the logarithmic growth phase, were treated with or without the SHP2 inhibitor PHPS1, the activator Trichomide A, EGF, or MMP inhibitors, and randomly assigned to four groups. Additionally, SW480 cells in the logarithmic phase underwent treatments with EGF, the ferroptosis inducer erastin, Trichomide A, or the curcumin analog C1, forming seven groups. Cell migration assessment in these groups employed scratch and Transwell assays. Protein expression analysis of total SHP2, total PI3K, p-SHP2, p-PI3K, p-TFEB, TFEB, SQSTM1, LC3, LAMP2, NCOA4, FTH1, GPX4, NOX4, and ACSL4 in the seven SW480 groups was conducted through Western blot and immunofluorescence. Apoptosis analysis was performed on these seven groups, while gene co-expression analysis utilized bioinformatics. SW480 and CCD-841CoN cells were categorized into four groups, undergoing treatment with saline, EGFR-OE lentivirus, SHP2-KD lentivirus, or SHP2-OE lentivirus. Western blot analysis in SW480 cells detected EGFR, total SHP2, p-SHP2, GPX4, and ACSL4 proteins, and tumor volume observations were conducted in a nude mouse xenograft model. Western blot also evaluated total SHP2, p-SHP2, GPX4, and ACSL4 protein expression in CCD-841CoN cells.

Results: Bioinformatics analysis revealed correlations between EGFR and SHP2, SHP2 and PIK3CA, SHP2 and MAPK1, BRK4 and HIF1A, HIF1A and NCOA4, as well as TFEB and FTH1. Scratch and Transwell assays showed that SHP2 diminishes the migratory capacity of SW480 and SW620 cells. Western blot and immunofluorescence demonstrated that EGFR activation of SHP2 markedly elevated p-TFEB levels while reducing TFEB protein expression. EGF stimulation enhanced the expression of FTH1, GPX4, NOX4, and ACSL4. Combined stimulation with EGF and SHP2 further amplified the expression of p-SHP2, p-TFEB, and NCOA4 while reducing TFEB, SQSTM1, LC3, and LAMP2. Erastin augmented FTH1, GPX4, NOX4, and ACSL4 expression while decreasing p-SHP2, p-TFEB, TFEB, SQSTM1, LC3, LAMP2, and NCOA4. TFEB activation suppressed p-SHP2, p-TFEB, NCOA4, FTH1, and GPX4 expression, while promoting TFEB, SQSTM1, LC3, LAMP2, NOX4, and ACSL4 expression. Apoptosis assays indicated that SHP2 activation decelerated apoptosis in SW480 cells, whereas erastin under EGF stimulation accelerated apoptosis, as did TFEB activation. Western blot results in SW480 cells displayed that overexpression of EGFR or SHP2 significantly increased total SHP2, p-SHP2, and GPX4 expression while decreasing ACSL4 levels. SHP2 knockdown decreased total SHP2, p-SHP2, and GPX4 expression, with an increase in ACSL4 expression. In CCD-841CoN cells, overexpression of EGFR or SHP2 resulted in a decrease in p-SHP2 and an increase in total SHP2, more pronounced with SHP2 overexpression, while GPX4 and ACSL4 levels remained stable. SHP2 knockdown led to reduced EGFR, total SHP2, p-SHP2, and GPX4 expression, without a significant impact on ACSL4 levels. The nude mouse xenograft model demonstrated that EGFR overexpression significantly increased tumor size, whereas SHP2 overexpression markedly decreased tumor volume. SHP2 knockdown resulted in significantly larger tumors.

Conclusion: SHP2 advances CRC progression by modulating TFEB-mediated ferritinophagy, suppressing ROS and ferroptosis. Targeting SHP2 presents a promising therapeutic strategy for CRC.

Keywords: CRC; Ferritinophagy; Ferroptosis; SHP2; TFEB.

Fig. 1

Bioinformatics analysis of pathways and differential gene correlations in colorectal cancer. A Boxplot of raw data from the GSE100179 dataset. B Boxplot of normalized data from the GSE100179 dataset. C Volcano plot of differential genes in the GSE100179 dataset. D Heatmap of differential genes in the GSE100179 dataset. E Bar plot of GO enrichment analysis for upregulated genes. F Chord diagram of GO enrichment analysis for upregulated genes. G Bar plot of GO enrichment analysis for downregulated genes. H Chord diagram of GO enrichment analysis for downregulated genes. I Bubble chart of KEGG enrichment analysis for differential genes. J Correlation scatter plot between EGFR and PTPN11. K Correlation scatter plot between PTPN11 and PIK3CA. L Correlation scatter plot between PTPN11 and MAPK1. M Correlation scatter plot between BRK4 and HIF1A. N Correlation scatter plot between HIF1A and NCOA4. O Correlation scatter plot between TFEB and FTH1

Fig. 2

EGFR activation results in elevated p-TFEB expression and suppression of the TFEB protein. A Protein expression levels of p-SHP2, p-PI3K, BRD4, p-TFEB, TFEB, Cathepsin L, and Cathepsin B in each group. B Compared to Group I, all proteins in Group II are upregulated. In Group III, p-SHP2, p-PI3K, BRD4, and p-TFEB activities are decreased compared to Group II, whereas TFEB, Cathepsin L, and Cathepsin B activities are increased, showing significant differences. In Group IV, p-SHP2, p-PI3K, BRD4, and p-TFEB activities are elevated compared to Group II, while TFEB, Cathepsin L, and Cathepsin B activities are reduced, denoting significant differences. (*P < 0.05; **P < 0.01)

Fig. 3

A Scratch assay indicates that SHP2 downregulates the migratory capacity of SW480 and SW620 cells. B Transwell assay demonstrates that SHP2 attenuates the migratory capacity of SW480 and SW620 cells. (*P < 0.05; **P < 0.01)

Fig. 4

A Protein expression levels of p-SHP2, p-TFEB, TFEB, SQSTM1, LC3, LAMP2, and NCOA4 in each group. Combined EGF and SHP2 stimulation augments the expression of p-SHP2, p-TFEB, and NCOA4 while inhibiting TFEB, SQSTM1, LC3, and LAMP2 expression. Erastin inhibits the expression of p-SHP2, p-TFEB, TFEB, SQSTM1, LC3, LAMP2, and NCOA4. TFEB activation suppresses p-SHP2, p-TFEB, and NCOA4 expression while promoting TFEB, SQSTM1, LC3, and LAMP2 expression. B SW480 cell apoptosis assay reveals that SHP2 activation decelerates apoptosis, whereas erastin under EGF stimulation accelerates apoptosis, and TFEB activation further accelerates apoptosis. (*P < 0.05; **P < 0.01)

Fig. 5

A Protein expression levels of FTH1, GPX4, NOX4, and ACSL4 in each group. Western blot results indicate that EGF and erastin stimulation enhance ferroptosis-related protein expression. TFEB activation inhibits FTH1 and GPX4 expression while promoting NOX4 and ACSL4 expression. B Immunofluorescence results demonstrate that EGF and erastin stimulation augment GPX4 and NOX4 expression, while TFEB activation inhibits GPX4 expression and enhances NOX4 expression. (*P < 0.05; **P < 0.01)

Fig. 6

A Western blot results in SW480 cells show that in Group ①, without stimulation, total SHP2, p-SHP2, and GPX4 expression levels are low, while ACSL4 expression is moderate. In Group ②, EGFR overexpression significantly elevates total SHP2, p-SHP2, and GPX4 expression, whereas ACSL4 expression decreases. In Group ③, SHP2 knockdown markedly decreases total SHP2, p-SHP2, and GPX4 expression, while ACSL4 expression increases. In Group ④, SHP2 overexpression significantly enhances total SHP2, p-SHP2, and GPX4 expression while reducing ACSL4 expression. (*P < 0.05; **P < 0.01) B Western blot results in CCD-841CoN cells show that in Group ⑤, without stimulation, EGFR, total SHP2, p-SHP2, GPX4, and ACSL4 expression levels are low. In Group ⑥, EGFR overexpression decreases EGFR and p-SHP2 expression but increases total SHP2 expression, with no significant changes in GPX4 and ACSL4 levels. In Group ⑦, SHP2 knockdown increases EGFR and p-SHP2 expression, decreases total SHP2 expression, and significantly lowers GPX4 expression, with no significant changes in ACSL4. In Group ⑧, SHP2 overexpression reduces EGFR and p-SHP2 expression, increases total SHP2 expression, but causes no significant changes in GPX4 and ACSL4 expression. (*P < 0.05; **P < 0.01). C Tumor growth experiments in nude mice demonstrate that EGFR overexpression significantly increases tumor volume, while SHP2 knockdown also enlarges tumor size. SHP2 overexpression, however, reduces tumor volume. Compared to Group ①, tumor volumes in Groups ②, ③, and ④ are significantly larger. In contrast, Group ③ (with SHP2 inhibition) exhibits larger tumors than Group ② (with EGFR overexpression), while Group ④ (with SHP2 overexpression) shows a significant reduction in tumor size. (*P < 0.05; **P < 0.01)

Fig. 7

Upon EGFR stimulation, SHP2 promotes p-TFEB via PI3K/BRD4, indirectly inhibiting TFEB. Subsequently, TFEB promotes LAMP2 and LC3 in lysosome synthesis and SQSTM1 in ferritinophagy, enhancing NCOA4’s interaction with FTH1, GPX4, NOX4, and ACSL4, which ultimately leads to iron overload and triggers ferroptosis